The ferric complexes of citrate, ETDA and NTA: their nature and the ligand exchange reaction with human transferrin

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content TH E FERRIC C O M PLEX ES OF CITRATE, EDTA A N D HTAl THEIR N A T U R E A N D T H E LIGAND E X C H A N G E REACTION W ITH H O M A N TRANSFERRIN by George Winston Bates A D is se rta tio n Presented te the FA CU LTY OF TH E G R A D U A T E SC H O O L UNIVERSITY OF SO U TH E R N CALIFORNIA In P a r tia l F u lfillm en t o f the Requirements fo r the Degree D O C T O R OF PHILOSOPHY (Bieohem istzy) January 1967 UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA S 0 0 0 7 This dissertation, written by under the direction of ffL&....Dissertation Com mittee, and approved by all its members, has been presented to and accepted by the Graduate School, in partial fulfillment of requirements for the degree of D O C T O R OF P H IL O S O P H Y Dean Date ?.f.!??uary^J967. ION COMMITTEE Chairman ACCTOWLEDGEMEHTS I t l a w ith a ap ao lal jay th a t I taka th is oppor tu n ity ta aip raaa ay ap p reciatio n ta some af the paapla whe have le n t t h e i r support darin g ay graduate c a re e r. I vauld lik e te thank the F aculty o f the D epart- se n t o f B iochem istry, in p& rtioular Dr. John M e h i , Dr. Arran F lu h arty , and the nanbera a f my d ia a a rta tia n com m ittee, Dr. Thomas F ife and Dr. Sam A lle rte n . in e sp e c ia lly fond o ffe r o f thanks goes to Carolyn B illupe and E leanor Jamea. T heir ooapetent support and o o rd la l frie n d sh ip w ill lang be remembered. The a p p re c ia tio n due m y p aren ts can hardly be over estim ated . T heir a ss ista n c e has taken many forma and has always made the ed u catio n al prooesa more m eaningful. Ta my b rid e , Jedy, I am ea p e o ia lly g r a te f u l. Her c h e erfu l p e rso n a lity and confidence hare o ften been a source o f encouragement. I would lik e to thank the U. S. Fublio H ealth Service f o r providing me v lth P re-d o o teral T raining Grant su p p o rt. S pecial thanks goes to th e John A. H artford Foundation, In c . fo r t h e i r generous support o f th is and o th er re se a rc h p ro je c ts in our la b o ra to rie s . F in a lly , I acknowledge the guidance o f ny rsse a rc h d ir e c to r , Dr. Paul Saltm an. During the y ears th a t I have bean asso c ia te d w ith him I have gained in s ig h t In to many asp e cts s f biochem ical theory ana tech n iq u e. Equally im portant to me, however, has been the opportunity te understand h is p h ilo so p h ies o f solenoe and o f the r e la tio n ship o f the academ ician to th e oommunity. --G.W.B. ii TABLE OP CONTENTS Pag* LIST O P TA B U SS . ........................................................................... r i LIST OP PIGURES................................................................................. v i i LIST O P ABBREVIATIONS................................................................... z Chapter I . INTRODUCTION........................................................................ 1 Some A spects o f Iro n Metabolism T ran sitio n Metal Ions and C helation Mechanisms o f Ligand Exehange Reactions The Polym erisation o f P e rric Complexes Metal P ro tein s T ra n sferrin H is to ric a l Physioohemical P ro p erties The Nature o f the Binding S ite I I . DISCUSSION O P TH E PR O B LEM S A N D G E N E R A L EXPERIM ENTAL APPROACH................................................... 17 I I I . EXPERIM ENTAL M ET H O D S............................................................20 B uffer P reparation o f the P rotein P reparation o f P e rric ED TA P reparation o f P erric C itra te P reparation o f P e rric N TA P rep aratio n o f the PBrric T ra n sferrin Complex Spectrophotom etric Methods Treatment o f K inetic Data D ialy sis S tudies i l l Chapter Page IV . PERRIC CITRATE: ITS NATURE AND THE LIGAND E X C H A N G E REACTION W ITH TRANSFERRIN................. 27 S tudies a t a l i l , C itra te sIro n Ratio T itra tio n S tudies S peotral S tudies D ialy sis Experiments D ialy sis P a tte rn a s a A m otion o f C oncentration Is o la tio n o f a Polymeric F ractio n and D eterm ination o f M olecular Weight Disoueslon F e rric C itra te a t V ariable C itra te to Iro n R atios T itra tio n S tudies S peotral S tudies D ialy sis Experiments Proton R elaxation as a Function of Added C itra te D iscussion R esults and D iscussions! The T ransfer o f Iron fro a C itra te to T ran sferrin F e rric C itra te a t a 111 R atio Time Course o f the Reaction Study o f the Rate Dependence Study o f the I n i t i a l Absorbency The R e a c tlrlty o f F errio C itra te as a Function o f the GH/Pe Added The B ffeot o f Excess C itra te in the Reaction o f F e rric C itra te w ith T ran sferrin The I n i t i a l Absorbency a t Various C itra te to Iro n R atios The Rate as a Function o f the C itra te to Iro n R atio K inetic Study o f the R eaction o f F errio D io ltre te w ith T ra n sferrin The E ffeo t o f Bicarbonate Ion D iscussions V. FERRIC VTA: ITS N A T U R E A N D T H E LIGAN D E X C H A N G E REACTION W ITH TRANSFERRIN................. 96 i r Chapter Page The Nature o f P e rric NTAt Re s u ite and D iscussions R esults* The T ransfer o f Iro n ( I I I ) fro n H TA to T ra n sferrin D isoussion VI. PERRIC EDTAt ITS N A T U R E A N D TH E LIG A N D E X C H A N G E REACTION W ITH TRANSPERRIN...................... 100 R esults and D isousslonst The Nature o f P b rrlc B D T A R esults and D isousslonst The T ransfer of Iron ( I I I ) fro n EDTA to T ra n sferrin Tine Course o f the R eaction I n i t i a l Absorbenoy S tudies Bate Dependence S tudies The C a ta ly tic E ffe c t o f Bicarbonate Proposal o f Reaction Meohanlsn V II. THE R E M O V A L OP IRON ( I I I ) P R O M TRANSPERRIN V III. G E N E R A L DISCUSSION LITERATURE CITED IX. S U M M A R Y B Y CHELA TIN G A G E N T S 138 1^9 155 158 T LIST OF TABLES Table Page 1 . The T raction o f the Iro n Comprising the Slowly B ialyslng Component o f th e F e rric C itra te S eries as a Jun ctio n o f Added Base Equiva le n ts per Mole o f I r o n ....................................................36 2. The Bapidly D ialyting F ractio n o f a S eries of F errio C itra te S olutions as a Function o f the C itra te to Iro n B atio . ................................. 51 3* The I n i t i a l Bate o f the Second Phase o f the Beaotion o f F e -c itra te a t a 1*1 B atio , w ith T ra n sfe rrin .............................................................................65 If. The B eaction o f F e -o ltra te a t a r a tio o f 25 C itra te s p er Iro n w ith Varying C oncentration o f T ra n sfe rrin ......................... 91 5* Comparison o f the I n i t i a l Absorbency in the B eaction o f F e rric C itra te w ith T ran sferrin in the Presence o f B icarbonate Ion w ith Combined I n i t i a l Absorbency and the In te r mediate Phase in the Absence o f Bicarbonate a t Various C itra te to Iron B a tio s........................... 93 ▼i LIST Of fIGUBE8 fig u re Page 1 . The T itra tio n Curve o f f e - c i t r a t e , Superimposed on the Absorbenoy a t 270 im . . . . . . . . . 29 2. The Speotra of th e f e - o ltr a te S e rie s Described In fig* 1 ........................................................................ 32 3 . The Tine Course o f E q u ilib ratio n Across a D ialysis Membrane fo r a S eries o f f e - c itr a te S olutions • • • • • • • • • ................................. 35 *t. The D ialysis f a t t e n s fo r a S eries o f f e - o ltr a te S o lu tio n s............................................. 39 5. The Superimposed P otentiom etric T itra tio n Curve and Spectrophotom etrio T itra tio n a t 280 nu f o r f e - o ltr a te In th e Presence o f Excess C itr a te ..................................... **5 6 . The V isible and U ltra v io le t Speotra fo r th e f e - o ltr a te S olutions Described In fig* 5 and the T e x t . .................... **7 7. The D ialysis P a t t e n fo r a S eries o f f e - c itr a te S olutions o f V ariable C itra te to Iron R a tio s. 50 8 . The Speotra fo r th e S eries o f f e - o ltr a te S olutions D escribed In f i g . 7 ...................... 53 9 . The Tine Course o f T ransfer o f Iron ( I I I ) from C itra te to T ra n sferrin as Determined S peetro- pho tome t r i o a l l y ............................................... 58 10. Sem llogarithm ie R epresentation of the T ransfer o f Iron ( I I I ) from C itra te to T ra n sfe rrin • • 61 11. The I n i t i a l Bate o f the Seoond Phase of the T ransfer o f Iron ( I I I ) from C itra te to Trans- — - fu n ctio n o f th e f e - c itr a te C oncentration 63 ▼11 Figure Page 12* The I n i t i a l Absorbenoy ae a Punetion o f F e -c itra te C o n c e n tra tio n ....................................... 68 13 • I n i t i a l Abeorbeney ae a Function o f Trane- f e r r ln C o n c e n tra tio n ............................................... 70 l*f« The I n i t i a l Bate o f the T ranefer o f Iron ( I I I ) fro n C itra te to T ran sferrin a s a Function of the Base E quivalents p er Mole o f Iro n , Added to the F e -c itra te P rio r to B eaction. • • • • 7* f 15* The I n i t i a l Absorbenoy in the Beaotion o f F e -c itra te and T ra n sferrin ae a Funotlon o f the Base E quivalents p er Mole o f Iron Used in th e P rep aratio n o f th e F e -c itra te • • • • 76 16. The Tine Course o f the B eaction o f F e -c itra te and T ra n sfe rrin , a t Various C itra te to Iro n B a tlo s.............................................. 80 17. The I n i t i a l Absorbency in the B eactions o f T ra n sferrin w ith F e -c itra te a t V ariable C itra te to Iro n B atios ....................................... 83 18. The I n i t i a l Bate o f T ransfer o f Iron fro n F e -c itra te to T ra n sferrin as a Function o f C itra te to Iro n B a t i o s ................................. 86 19* The Bate o f T ran sfer o f Iro n fro n F e(C lt)o to T ra n sferrin a s a Function o f th e Concentra tio n of F e -o ltra te a t a Molar B atio of 25*1 C itra te : Iro n ............................................. 89 20. The B ia ly sls P a tte rn o f a 1:1 P rep aratio n o f Fe-EDTA.................................................................................103 21* A B eer's Lav P lo t fo r Pe-EDTA Prepared a t a Mole 1:1 B a t i o ................................................................ 1 0 6 22. The Tine Course o f th e T ransfer o f Iron ( I I I ) fro n EOTA to T r a n s f e r r in ................................. • 109 23* A F ir s t Order P resen ta tio n o f th e Tine Course o f the T ran sfer o f Iron ( I I I ) fro n ED TA to T ra n sfe rrin . ................................................................112 2k. V isib le 8peotra a t Various Tines in the Trans f e r o f Iro n ( I I I ) fro n ED TA to T ra n sferrin • 115 ▼iii Figure 25. 2 6 * 27. 28 * 29. 30. 31. 32. 33. 3**. 35. 36. The I n i t i a l Absorbenoy a t 520 au in the Beao- tlo n of Fe-EDTA w ith T ra n sfe rrin as a Func tio n of th e C oncentration o f Fe-EDTA . . . . The I n i t i a l Absorbenoy a t 520 au in the Beac tio n of T ra n sfe rrin and Fe-EDTA a s a Func tio n o f th e Binding E quivalents o f th e P ro te in ............................................................................. A Doable R eciprocal P lo t o f th e Data Pre sented in F ig . 26....................................................... The Observed F i r s t Order Bate C onstant Obtained f o r a S eries o f Fe-EDTA S olutions as a fu n ctio n o f the C oncen tratio n s. • • • • The I n i t i a l Bate o f the T ra n sfer o f Iro n ( I I I ) from ED TA to T r a n s f e r r in .......................... • • • A Doable B eciproeal P lo t o f th e Data Shown in F ig . 29............................................................................. The I n i t i a l Bate o f the T ran sfer o f Iron ( I I I ) fro n EDTA to T ra n sfe rrin as a Function of the I n i t i a l Absorbenoy a t *f70 a u ...................... The Tins Course o f the Removal of Iro n fro a T ra n sfe rrin by Various C helating A gents. • • The F rac tio n o f E quilibrium A ttain ed as a Function o f T im e .............................................. Bate o f B eaoral o f Iron fro a Iro n T ra n sferrin by ED TA as a Function o f the EDTA Concen tr a tio n . • • • • • • ............................................... The I n i t i a l Bate o f the Benov&l of Iro n fro a T ra n sfe rrin ae a Function o f Iron Trans f e r r in C o n c e n tra tio n ............................................... The Tlae Coarse o f the T ra n sfer o f Iro n ( I I I ) fro a EDTA. H T A and C itra te to the P rotein T ra n sfe rrin .................................................................... Page 117 120 122 125 128 130 133 l*fO l*fr2 1^5 1**7 151 ix LIST 07 ABBREVIATIONS EDTA — E th y lsn sd ia a ln ststra a c e tio acid N TA — N itr ilo tr ia o s tio aold t r i e — trlshydrozym e thylaminone thane hopes — N-2-hydroxyethylpiperaaine-N*-2-ethane su lfo n ic acid CHAPTER I i i t r o u j c t i o i Spas Aapeota o f I r t n Metabolism The p h y sio le g ie a l ro le o f iro n has in trig u e d nan • r e r th e c e n tu rie s e f reeorded h is to r y . The a d m in istra tio n o f Ire n s a l t s f e r the treatm ent o f Iro n deficien cy syn dromes d a te s hack to th e M editerranean c iv ilis a tio n s s f 1500 B.C. (1 ) . At p re se n t, sev eral im portant biochem ical systems have been c h a ra c te rise d in which iro n i s an in d is pensable component. A m ajor p o rtio n o f the iro n in th e human system i s found in the p ro te in s myoglobin and hemo g lo b in which are re sp o n sib le fo r the storage and tra n sp o rt o f oxygen. In a d d itio n , Ire n i s a c tiv e in the oyteohrones o f the e le o tre n tra n s p o rt ehaln and has been im plicated a t the c a ta ly tic s i t e o f se v e ra l im portant enzymes. Biohorn (2) has o u tlin e d some o f the p ro p e rtie s e f iro n th a t make i t e s p e c ia lly u se fu l and v e r s a tile in bio chem ical system s. 1 . Ire n re p re se n ts a co n cen tratio n o f p o sitiv e charge whloh w ill a t t r a c t and d is to r t oovalent 1 2 bonds and b rin g re a o ta n ts together* 2* Ira n banda in an o etah ad ral c o n fig u ra tio n , vhloh can influenoe the stereochem istry o f the re a c tio n by the o rie n ta tio n o f re a c tin g mole c u le s. 3 . Ira n has two sta b le o x id atio n s ta te s w ith w idely varying redox p o te n tia ls , thus i t i s Id e a lly su ite d f a r e le c tro n tra n s fe r re a c tio n s . *f. Both fe rro u s and f e r r io iro n oan e x h ib it d i f fe re n t e le c tro n c o n fig u ratio n s depending on the lig an d s to vhloh they are attao h ed . This le a d s to an a b i l i ty to f o r a ooaplexes o f vary ing s ta b ility * S tu d ies o a rrle d out by S altaan (3) have given in s ig h t in to the aeohanlsa by which iro n i s assim i la te d by the organism* D ietary Ire n , e ith e r in the fe rro u s o r f e r r io form , beoeaes ceaplexed te a s u ita b le le v molecu l a r w eight c h e la tin g ag e n t. The complex then d iffu s e s acro ss the in te s tin a l wall* The tr a n s in te s tin a l flu x follow s passive k in e tic s and dees n o t involve membrane bound o a rrie r* Ferrous iro n , vhloh i s spontaneously o x i dised te th e f e r r io form in the presence o f oxygen i s oxid ised during some stag e o f the tra n sp o rt* A fter the complex has passsd th s gut w a ll, i t e n te rs in to the bleed stream where th e iro n i s given up te tr a n s f e r r in , the iro n tra n s p o rt p ro te in o f th e bleed* The s u b s e q u e n t tra n s p o rt and d ep o sitio n o f the iro n a t ap eo ifle re c e p to r s ite s la o a rrie d out by th e p ro te in . I t hao been shewn Of, 5) th a t tra n s f e r r in i s able to d istin g u ish between natu re and developing red o e lls and w ill p re fe re n tia lly re le a se i t s iro n to the l a t t e r . Other s it e s o f Iro n d ep o sitio n are the sto rag e tis s u e s vhloh include the l i v e r , kidney and sp leen , where f e r r i t i n i s the binding p ro te in . Experiments w ith Iso la te d gut loops fro n the r a t have shown th a t th e r a te o f tra n s in te e tin a l flu x i s dependent on the o h elate which i s used te eonplex the Ire n ( 3 ) . Of those c h e la te s te s te d , N T A and EDTA led to the h ig h est ra te o f tra n s p o rt. The f e r r ic conplexes e f c i t r a t e and fru c to se were slow er in orossing the gut w a ll. The u t i li s a t i o n of iro n by the organism was stu d ied u sing ra d io a ctiv e 59jg as a tr a c e r . In two separate ex p e rin en ts, Ire n bound to e ith e r N TA o r ED TA was in je c te d in to a lig a te d gut loop of a liv e r a t . The uptake o f the iro n was allowed to proceed fo r two h o u rs. A fter th is period o f tin e , the a n la a l was s a c rific e d and the various organs oxamined fo r ra d io a c tiv e iro n . I t was found th a t over 90% of the iro n which was oonplexed to N T A was tra n s fe rre d to the bleed o r iro n storage tis s u e s . Only e f the EDTA Ire n was u t i li s e d in th is manner. The remainder was found in the kidney o r b lad d er. I t i s c le a r th a t although the fe rrio ion oomplexes o f ED TA and N TA have s im ila r r a te s o f tra n s p o rt across the in te s tin a l w a ll, the Iro n fro n the l a t t e r i s acre re a d ily a v a ila b le fo r m eta- b o lic u s e s . T ra n sitio n Metal lens and O holatlon The tr a n s itio n n e ta ls form io n s w ith u n f ille d d o r b ita ls which give r is e to the p ro p e rtie s o f sp e o tra , magnetic s u s c e p tib ility and r e a c tiv ity . W erner's c l a s s i c a l th e o ry , which was published in 1891, la y s the ground work fo r our p resen t knowledge o f tr a n s itio n n e ta l chemis tr y (6 ). In ad d itio n to th e binding e f counterions by e le c tr o s ta tic bonding, the tr a n s itio n m etal ions a re able to bind lig an d s v ia coordinate covalent bonding. The co ordinate covalent bond d if f e r s from the usual covalent bond only in th a t both e le c tro n s o f th e bond are fu rn ish ed by the same atom, which i s c a lle d the lig a n d . C helating agents a re those m olecules which are ab le to sim ultaneously bind a m etal ion w ith two o r more lig an d groups. Two requirem ents fo r a c h e la tin g agent a re apparent: (1) I t must possess two o r more atoms which a re ab le to form coordinate covalent bonds, th a t i s co n tain in g an unshar ed p a ir o f e le o tro n s . (2) The molecule must have the proper s te r io requirem ents fo r rin g form ation (7 ). In b io lo g ic a l systems numerous m etabolites are known which are ab le to a c t as o h elatln g ag e n ts. These include the amine a c id s , d i and tric a rb o x y lic a c id s , 5 carbohydrates and nitrogenous bases* P ro tein s are ab le to bind m etal ions by the side chains o f c e rta in o f the amino acids* By the proper s te r ic d is p o s itio n o f the sid e chains^ p ro te in s a re able to fo rn extrem ely e f f ic ie n t c h e la tin g s ite s * Meehan* bmb o f H fpnd Bxfthwiga R ea ctio n s Ligand exchange re a c tio n s involve the s u b s titu tio n o f one lig an d fo r an o th er about a c e n tra l m etal ion* These re a c tio n s may involve simple anions o r m ultid en tate che la tin g agents* Basolo and Pearson (8 ) have presented an e x c e lle n t d iscu ssio n and review o f th e mechanism o f lig an d exchange re a c tio n s . Following th e term inology fa m ilia r from organic chem istry, the s u b s titu tio n re a c tio n s a re divided in to two c a te g o rie s , Sg 1 and Sg 2* The ra te lim itin g ste p in the Sg 1 mechanism i s the d is s o c ia tio n e f the lig an d from th e m etal ien* Following th is i s a ra p id a tta c k by the n u o leep h ilio agent on th e m etal ion and the subsequent form ation o f the new cemplex* The Sg 2 mechan ism inv o lv es a d lre o t n u c le o p h lllc a tta c k on the m etal ie n by the a tta c k in g lig an d and the displacem ent o f the leaving group on the opposite face o f the ion* I t i s suggested th a t s u b s titu tio n re a c tio n s involving octahedral oomplexes g en e rally preoeed v ia an Sg 1 mechanism, due p rim a rily te the e ffe c tiv e sh ie ld in g o f th e m etal ie n by the s ix lig an d groups* Margerum, a t m l. (9* 10) have stu d ied m etal exchange re a c tio n s f o r v ario u s ions and c h e la tin g agents* The r a te U n itin g ste p in the tra n s f e r o f ED TA fro n sin e to copper appears to be a h a lf unwrapping o f th e ch e la te noleoule fro n the sin e ien* The fre e lig an d s o f the ED TA ra p id ly oonplex the oopper to fe rn a tern a ry complex in whioh both le n s are bound to th e chelate* A fter th e d is s o c ia tio n e f th e s in e , the rem aining lig an d s o f ED TA are able to coordinate th e copper and the re a c tio n i s c o n p le te . Chelate exchange re a c tio n s in general probably p re - oeed through step v ise nechanlsma in which the leav in g che l a t e p a r tia lly u n fo ld s before the a tta c k in g ch e la te i s able te a c t (8)* The amount of unwrapping th a t i s necessary te allow n u o leo p h lllc a tta c k depends on the s te r lc requirements e f th e n u cleo p h llic agent and the degree to which the le a v in g c h e la te encompasses and sh ie ld s the metal ien* I t has been found (8) th a t m u ltid en tate c h e la te exchange re a c tio n s are e fte n cataly sed by simple lig a n d s. I t i s believ ed th a t th ese re a o t ra p id ly w ith the p a r tia lly exposed m etal ion a f t e r th e f i r s t unwrapping ste p and h in d er the rev erse re a o tie n whioh i s the re c le sln g o f the ch elate about the m etal io n . The work in the f ie ld e f o h elate exchange re a o tle n e i s s t i l l f a ir ly lim ited * This i s e sp e c ia lly tru e in the case where one o f the o h elatee i s a p ro te in aoleoule* This 7 type o f re a c tio n ie ne doubt o f importance in th e biosyn th e s is and ao tio n e f the m etal p ro te in s . The Polym erization e f J o rrio Complexes The well-known phenomenon of the h y d ro ly sis and subsequent p re c ip ita tio n e f f e r r ic s a l t s suggests the aggregation o f f e r r i c ie n v lth the e re n tu a l form ation e f s o ls . Hedstrom (11) on the b a s is of p eten tiem etrlo stu d ie s proposed th a t the i n i t i a l produot o f h y d ro ly sis o f iro n > f+ ( I I I ) i s a dim eric sp e c ie s, [Fe2(0H)2] * ^urth*? evidence f o r the foxmation o f a d in e r was presented by Malay and Selwood (1 2 ), who found the iro n to be diam agnetio, in d i c a tin g a spin p a irin g in te ra c tio n o f th e f e r r ic io n s. B ecently S p iro , e t a l . (13) hare Iso la te d and c h a ra c te rise d a high m olecular weight polymer o f hydrolysed iro n ( I I I ) . The em plrloal form ula was determined to be [fe(OH)z (N0^)^.z ] , where z i s between 2.3 and 2.5* The m olecular weight was determined from T elo city sedim entation d ata as l A z 10^. The authors suggest the polymer i s formed by the b rid g in g o f f e r r io ions by hydrozy o r ozo b rid g e s. E lectro n microscopy and v is c o s ity d a ta re v eal th a t the polymer e z is ts as is o la te d spheres w ith a d is c re e t s ite range n ear 70 3L B ecently S altnan (3) has disoussed the polym erisation o f f e r r ic fru c to s e . I t had been shown ( A ) th a t f e r r ic fru o to se displayed a low magnetic sus c e p ti b il ity and d id n o t have an ESB sp e c tra c h a ra c te r is tic o f f e r r io io n . This in d ic a te d a stro n g e leo tro n lo i n t e r a c tio n between the f e r r io io n s, a s would be expected f o r d in e rs o r p o ljn e rs o f ir o n . On the b a sis o f gel f i l t r a tio n , eq u ilib riu m d ia ly s is and u ltra o e n trlfu g e s tu d ie s , i t was d etem ln ed th a t th e f e r r io fru o to se e x is ts both a s a polymer and as low m olecular w eight complexes. Although f e r r ic c i t r a t e has been in v e stig a te d by a number o f la b o ra to r ie s , some o f the r e s u lts are c o n flic tin g and th e n atu re o f t h i s complex i s f a r from d e a r . Bob t e l sky and Jordan (15) have stu d ied f e r r ic o ltr a te by p o ten tlo m etric and conductonetric means. They suggested th a t in the presence o f excess c i t r a t e the most sta b le specie was an Ve2C itra te ^ oomplex. The ex isten ce o f an Ve3 C itr a te 2 vas proposed in so lu tio n s o f exoess f e r r io ion. The v a lid ity o f th e i r work has been o r ltlo ls e d by Imnford and Qulnan (1 6 ). The l a t t e r a u th o rs, on the b a sis o f the epeetrophotom etric techniques o f Vosburg and Cooper (1 7 ), rep o rted th a t the most sta b le complex o f the f e r r ic c it r a te i s a l x l sp e c ie . T heir stu d ie s were c a rrie d out a t a low pH. They did n o t dem onstrate the ex isten ce o f fe r r io e i t r a t e complexes a t o th e r r a ti o s . Varner and Weber (18) also proposed a lx l stoichio m etry. By extending t h e i r pH t i t r a t i o n to the n e u tr a lity reg io n they dem onstrated th a t the hydroxyl proton of the e i t r a t e becomes io n ised upon the binding o f the iro n . They suggested th a t the f e r r io ion i s bound to e i t r a t e by two carboxyl groups and the hydroxyl oxygen. Gustafson and M artell (19) have proposed, on the b a s is o f p o te n tlo a e trle t i t r a t i o n s , dim eric sp ecies fo r both Pe-HTA and Pe-EDTA. T heir stu d ie s were c a rrie d out w ith a H I mole r a ti o o f ch elate to iron* They suggest a s tru c tu re in whioh the f e r r io io n s a re joined lay hydroxy b rid g e s. Ho evidence was rep o rted f o r h ig h er polynuclear sp ecies fo r these f e r r ic complexes. M etal P ro tein s V allee (20) d iv id es the m etal p ro te in s in to two t c la s s e s ; the m etallo p ro teln s and the m etal p ro te in com plexes* The b a sis o f t h i s d iv isio n i s the stre n g th o f binding o f m etal ion to p rotein* A m etallo p ro tein binds th e m etal w ith such a v id ity th a t i t may be considered as an in te g ra l p a rt o f the p ro te in molecule* The m etal p ro te in complexes, on the o th e r hand, are in a tru e eq u ilib riu m w ith fre e m etal ions in s o lu tio n . Malms trom and Eosenberg (21) have w ritte n an ex cel le n t review on the m etal p ro te in oomplexes, whioh they r e f e r to as m etal a c tiv a te d p roteins* I t i s pointed out th a t a metal ion can influence the ao tlo n o f an enzyme in many ways, such as changing the io n io s tre n g th , s t a b i l i s a tio n o f p ro te in s tru c tu re and a lte r in g th e e le c tr o s ta tic n a tu re o f the protein* I t i s e s s e n tia l th a t the m etal 10 a c tiv a te d p ro tein s be ooneldered only ae those in vhloh the m etal ion e n te rs In to the o a ta ly tio a o t. The ro le o f m etal p ro te in s has been ex ten siv ely reviewed (20, 21, 18). I T ran sferrin H isto rio a l Since i t s discovery and is o la tio n an extensive amount o f work has been done on the p ro te in tr a n s f e r r in . I t i s now one of the b eet c h a ra c te rise d non-heme iro n pro te in s and i t s p ro p e rtie s have been reviewed many timee (22, 23, 2»f, 25, 26). Although i t has been known fo r a number o f y ears th a t blood contained iro n a s hemoglobin, i t was n o t u n t i l 1927 th a t Barkan (27) dem onstrated th a t iro n was eomplexed to another non-dlalyzable component o f the blood. L ater he shoved th a t the iro n was re le a se d and became re a d ily d ialy sab le a f te r a o ld lflo a tlo n of the serum (2 8 ). Holmberg and L au rell (29) in 19** 5 studied the iron-b in d in g cap acity o f serum by adding fe rro u s iro n in the presence o r the c h e la tin g agent a ,a - d ip y r id y l. No evidence o f the lro n - d ip y rid y l oomplex was n o ticeab le u n t i l about 315 ug o f iro n p er 100 ml serum had been added. The a d d itio n o f iro n below th e s a tu ra tio n p o in t caused the serum to change from a yellow to a yellow -red • The new ohromophore had an abso rp tio n mart m um o f about 500 mu. The v arious serum fra c tio n s whioh had been sepa ra te d by Cohn and co-w orkers were examined fo r iro n binding eap ao ity by Sohade and Caroline (3 0 ). The fra c tio n IV*3, b was shown to co n tain an iro n -b in d in g p ro te in , a s demon s tra te d by bloassay and speetrophotom etrio analysis* The iro n p ro te in oomplex had a salmon pink c o lo r, th e in te n s ity o f which was shown to be d ire c tly p ro p o rtio n al to the amount o f added iron* Surgsnor, e t a l . (31) f u rth e r p u ri fie d the iro n binding a c tiv ity * The p ro te in was designated "metal binding g lo b u lin " and appeared in F ractio n IV-7* Koechlln (32) in 1952 c r y s ta lliz e d from e th a n o l/ w ater an e s s e n tia lly iro n -fre e form o f the iro n binding p rotein* I t was determ ined th a t th e th re e tim es r e c r y s ta l liz e d p ro te in had a minimum m olecular w eight, based on the iro n c o n te n t, o f M-5,000* This inform ation coupled w ith a study in which Onoley, e t a l . (33) determ ined, by osmotic p ressu re and u ltra o e n trifu g a tlo n , th a t the m olecular weight o f the p ro te in o f F ractio n IV-7 i s about 90,000, led to the conclusion th a t the m etal binding g lo b u lin bound two iro n s p er p ro te in molecule* Koeohlln dem onstrated th a t iro n is bound in th e f e r r ic form* The iro n -p ro te in complex was f i r s t c r y s ta lliz e d by Laura 11 (26) by a d d itio n o f ethanol to a so lu tio n o f p ro te in a t pH 6* Sohade, e t a l * (3*0 e sta b lish e d th a t the iro n p ro te in oomplex re q u ire s b io a r- bonate iro n fo r f u l l c o lo r development* He suggested the 12 nans sld e ro p h ilin in view of the high a f f in ity of th e pro t e in fo r iron* The nano tr a n s f e rrin was suggested by Holmberg and L aurell (35) fo r th e p ro te in since i t had been shown th a t the noleoule o a rrie s predom inantly iro n under p h y sio lo g ical co n d itio n s. Physloohemioal p ro p e rtie s . Qnoley, Scatohard, and Brown (33) were the f i r s t to study th e phyaloal p ro p e rtie s o f the v arious p ro te in f n o ti o n s . They found th a t Cohn f n o t i o n IV-7 whioh i s predom inantly tra n s f e rrin (32) had a m olecular weight o f about 90,000 based on osmotio pressure stu d ies* Other measurements o f the m olecular weight o f tr a n s f e r r in have been made u sin g the various p h y sical tech niques available* These values support the values above o f Qnoley and co-workers: lig h t s c a tte rin g , 88,000 (36); sedim entation and d iffu sio n c o e ffic ie n t, 88,000 (37); 89>000 (38); 90,000 (39); Archibald technique, 93,000 0*0). In variance w ith these workers a n the fin d in g s of Charlwood 0*1), who n p o r t s a value f o r human tra n s f e rrin o f about 68,000* He suggests th a t th e o th er woxkers nay have had some dimer form ation in t h e i r p re p aratio n . Beskorovainy and B afelson 0*2) have c a rrie d out a o a n f u l study o f the hydrodynamic p ro p e rtie s o f the iro n - fre e human tra n s fe rrin * They re p o rted th a t the hydro- dynamio param eters a n b e st f i t by an oblate e llip s o id w ith an a x ia l ra tio o f 1:3* 13 A zarl and feeney (**3 > MO dem onstrated th a t th e iro n tr a n s f e r r in oomplex i s more s ta b le than the apo p ro te in to therm al d en atu ratio n and p ro te o ly tio a tta c k by chym otrypain. They suggested th a t th e m etal ion i s ab le to ohange the conform ation o f the p ro te in in such a way th a t i t s la b ile p o sitio n s a re hidden. S ch u ltz, e t a l . Of?) e sta b lish e d th a t tr a n s f e r r in i s a g ly co p ro tein co n tain in g hexose, hexosamine, and s ia li c a o id . Jamieson Of 6) ch a racteriz ed th e oarbohydrate por tio n s o f th e glyoopeptides obtained from a p ro te o ly tio d ig e s t o f tr a n s f e r r in . His fin d in g s in d icated th a t each mole o f tr a n s f e r r in o ontalns two branched heterosaooharide chains which are id e n tio a l in com position and contain two re sid u e s o f s ia lio a c id , fo u r re sid u e s o f H -acetyl glucos amine, two re sid u e s o f g alacto se and fo u r resid u es o f nannose. The carbohydrate chain appears to be linked to the p ro te in v ia asparagim yl lin k a g e s. The n atu re c f the binding s i t e . The n atu re o f the iro n binding s ite o f tr a n s f e r r in has been in v e stig a te d by a number o f la b o ra to rie s (3*f» *f7, *f8, ^9 , 50, 5 l) . The d e f in itiv e work by Aasa, e t a l . 0*9) in d icated th a t the p ro te in co n tain s two iro n binding s i t e s whioh are non in te ra c tin g and id e n tic a l by evexy param eter stu d ie d . Tata from u ltr a v io le t spectrosoopy, BSE sp e o tra , and pH t i t r a t i o n s in d ic a te d th a t the f e r r io ion i s bound by th ree Ik ty ro sy ls and two h ls tid y ls re a l due* and a bicarbonate ion* The la c k o f sp in -sp in in te ra c tio n in th e iro n -sa tu ra te d p ro te in in dloated th a t the two binding s it e s are separated o by a t le a s t 9 A * The oupric p ro te in oonplex was also studied and i t was deternined th a t the copper i s bound by two ty ro sy l and two h i s t id y l groups a t the sane two s p e c ific s i t e s . Vallee and U lnar (5 l) have stu d ied the o p tlo a l ro ta to ry d isp ersio n o f tra n s f e r r in . In the iro n -fre e form, the p ro te in e x h ib its a p la in negative ro ta to ry d isp ersio n between 300 and 675 nu • The a d d itio n o f f e r r ic ion to the p ro te in r e s u lts in the appearance of a negative Cotton e f f e c t, the nagnltude o f which i s p ro p o rtio n al to the anount of iro n added. The authors suggested th a t the n e ta l i s bound in a ste re o sp e c ific s i te which c o n s titu te s an as syne t r i e ehronophore. The fin d in g th a t oupric ion does n o t a l t e r the ro ta to ry d isp ersio n of th e p ro te in was in d ic a tiv e th a t the copper i s n o t bound in an a ssy n e tric nanner by the p ro te in . Sinoe the ehronophore o f the copper and the iro n are o f approxinately the sans nagnl tude, i t i s not a d iffe re n c e in s p e c tra l p ro p e rtie s which accounts fo r the d iffe re n c e o f the ro ta to ry d isp ersio n curves. The combination o f these observations w ith the in fo ra a tlo n av a ilab le on the ligands which bind these ions 15 in d ic a te s th a t the ab so lu te co n fig u ratio n o f the binding s i t e s s u s t bet Tyr Hie- H C O 3- His His- T rr Tyr • G % — ly r Tyr — His T yr. re p re se n ts the ty ro sy l resid u es and H is, the h is tid y l residues* This co n fig u ratio n i s reasonable since oupric ions o h a ra o te rls tio a lly bind in a square plan ar configura tio n w hile f e r r ic ion i s known to bind in an o ctahedral manner* I t can be seen th a t the copper i s bound by a symmetrical ligand f i e l d , whereas the iro n i s assym etrl- o a lly bound. Although tr a n s f e r r in in the physiological s ta te binds p rim arily f e r r ic io n ( 3* 0 , i t has also been shown th a t the p ro te in i s able to bind Cu2* , Zn2+, and Cr^* (52). These io n s are h o lle re d to be bound a t the sane spec if io m etal s ite * P erk in s, e t a l . (50) rep o rted th a t th e pro te in can also bind oalolum and c o b a lt, although n o t a t the same s i t e as the iron* Aasa, e t a l . (*f9) hare stu d ied the binding oonstant o f iro n tra n s f e rrin u sin g an eq u ilib riu m d ia ly s is tech nique w ith o ltr a te and BETA as oonpetlag c h e la te s. I t was 16 found th a t a much lo n g er tin e was neoessary f o r the fe-ESIA to come to eq u ilib riu m w ith the tr a n s f e r r in than was req u ired w ith the c i t r a t e iro n complex. In th e case o f the Ee-BDTA, sev e ral weeks were req u ired fo r e q u ilib ra tio n as noted by the f a e t th a t the re a c tio n in d iffe re n t d ire c tio n s y ield ed varying apparent binding c o n sta n ts. The eq u ilib riu m w ith l e - o i tr a t e was reached w ithin fiv e days. A K value fo r the binding o f both Bs^* atoms to tr a n s f e r r in was on the o rd er o f 10^°. This i s in d ic a tiv e th a t the binding o o n stan ts a t the two s ite s are independent o r vaxy by a sm all d iffe re n c e (^ 9 ). The lack o f a s i t e - s ite in te ra c tio n w ith tra n s f e r r in i s in opposition to what Wexner and Weber (53) found fo r oonalbumin. T heir evidence In d ic a te s th a t oonalbumin has a value which i s much g re a te r than the In s o lu tio n , th e average oonalbumin molecule would co n tain e ith e r two o r no iro n atoms. R ecently, however, Aisen (5*0 has presented evidence th a t in oonalbumin, as In tr a n s f e r r in , and K 2 valu es are q u ite s im ila r, and p ro te in can be Iso la te d con tain in g b u t a sin g le m etal io n . CHAPTER XI DISCUSSION 07 T H E PR O B L E M S A N D G E N E R A L EXPERIM ENTAL A PPR O A C H In the in tro d u c tio n , sev eral asp ects o f iro n chem i s t r y and metabolism were dieoueeed vhloh bear d i r e e t l j on the th e s is re se arc h . I t w ill be h elp fu l to re o a p itu la te some o f the more s a lie n t p o in ts. 1 . The ra te o f tr a n s la te s tin a l flu x and the u l t i mate s ite o f d ep o sitio n o f f e r r ic ion in the r a t 1b a fun ctio n o f the ohelate th a t i s used to complex the iro n (3 ). 2. The ra te a t which equilibrium i s reached w ith tr a n s f e r r in i s longer w ith Te-EDTA than w ith 7 e - c itr a te • The former does not reaoh equi lib riu m w ith in a month, while the l a t t e r i s a t equilibrium w ith in a week 0«-9)* 3 . A few examples of m n ltid en tate ch elate exchange re a c tio n s have been stu d ie d . The mechanism appears g en erally to involve the p a r tia l unwrapping o f the ch elate from the o e n tra l m etal ion (8 ) . D etailed inform ation 17 18 i s n o t a v a ila b le on the n atu re o f otaalate exchange re a c tio n ! involving p ro te in s , if. R ecently i t has been found th a t f e r r ic n i t r a t e (13) and f e r r ic fru c to se (3) fo ra high molecu l a r w eight polyaers in so lu tio n . P reviously i t had been thought th a t f e r r ic complexes e x iste d p rim arily as monomers o r dim ers. I t i s apparent th a t a number o f problems rem ain in the study o f f e r r ic c h e la te chem istry and m etabolism . What i s the n a tu re o f the d iscrim in atio n process in the a ssim i la tio n and u t i l i s a t i o n o f f e r r ie c h e la te s? D o the model fe r r io c h e la te s stu d ied e x is t as polymers in so lu tio n ? I f so , what i s the e f f e c t o f the polym erization on the re a c t i v i t y o f the f e r r ic c h e la te s? What mechanisms are opera tiv e in the tra n s f e r o f iro n from low m oleoular w eight c h e la tin g agents to tra n s fe rrin ? How do the mechanisms account fo r the ra te d iffe re n c e s? I t ie lik e ly th a t th e ra te o f tra n s f e r o f iro n from th e low m olecular weight o h elatin g agents to tra n s f e r r in p lay s an im portant p a rt in the u t iliz a t io n o f iro n in p h y sio lo g ical system s. The th e s is re se a rc h w ill have two p o in ts o f empha s i s . (1) To in v e s tig a te the f e rrio oomplexes o f ETA, EMA and o itr a te in o rd er to determine i f polymerio speoies are formed. (2 ) To study the k in e tic s and mechanisms involved in the tr a n s f e r o f iro n ( I I I ) from NTA, ED TA and o l tr a t e to tr a n s f e r r in . The fe r r io complexes w ill be stu d ied in a manner sim ila r to th a t developed by S piro , e t a l . (1 3 ). This w ill include the c o rre la tio n o f d ata from equilibrium d ia ly s is k in e tic s , spectrophotom etry and pH t i t r a t i o n . The k in e tic stu d ie s involving the ch elate exchange re a c tio n s w ill be follow ed spectrophotom etrieally a t **70 mu, the ab so rp tio n maximum o f the f e r r ic tr a n s f e r r in complex. CHAPTER III EXPERIM ENTAL M E T H O D S &*££& The b u ffe r used In the p re p aratio n o f a l l so lu tio n s ) ezoept as noted in the t e x t, was tris-hydroxym ethyl- auinome th an e, t r i e . I t was obtained as a reagent grads chem ical from Sigma Chemical C orporation and n o t fu rth e r p u rifie d before use* The t r i e was used a t a conoentration o f 0*005 M a t a pH o f 7*5* A fte r the pH was a d ju ste d , the b u ffe r so lu tio n was e q u ilib ra te d w ith a i r by rig o ro u s bubbling fo r a t le a s t two ho u rs, to assure a bicarbonate co n cen tratio n s u f f ic ie n t to s a tu ra te the protein* P rep aratio n o f the P ro tein Human a p o -tra n s fe rrin was obtained from Behringwerke (Marl burg-Lang, Germany). I t was found to be approxim ately 95£ iro n free* Before u se , i t was dialy sed a t le a s t 2*f hours a g a in st frequent ohanges o f t r i s b u ffe r. An a liq u o t o f the stock so lu tio n was t i t r a t e d w ith 10 ^ M Pe-NTA in o rd er to determ ine the iro n binding e q u ira le n te p er l i t e r * The tr a n s f e r o f the iro n from the N T A to the 20 21 p ro te in occurred r a p id ly • The endpoint o f the t i t r a t i o n was determ ined by follow ing the abeorbency a t *f70 mu, the ▼ ielble abeorptlon maximum o f the T e -tra n a fe rrin complex. The p ro te in was then d ilu te d w ith b u ffe r to the d eeired c o n c e n tra tio n . I t waa necessary th a t a l l glassw are be ac id rin sed to re more a l l tra o e s o f m etal contam ination* The p ro te in was n o t allowed to come in to co n tact w ith m e ta llic co n tain ers o r f i t t i n g s . P rep aratio n o f P srric B D TA A naly tical reagent grade B D TA was obtained from M alllnokrodt ae e ith e r the dlsodlum s a l t o r the a o ld . I t was n o t fu rth e r p u rifie d before use • Fe(NO^)^ was a lso a n a ly tic a l reagent grade from M alllnokrodt. The Fe-EDTA was prepared by th ree methods* The f i r s t two employed t r i s buffer* The th ird used no b u ffe r. 1* The dlsodlum s a l t o f ED TA was taken to pH 6*0 • Pe(HO^)^ and d ilu te H aQ H were added sim ultaneously w ith rap id s t i r r i n g . The pH was read continuously and m aintained between 5*0 and 6*0 by re g u la tin g the ad d itio n o f the base. A fter the re q u is ite amount o f iro n had been added, the pH was ad ju sted to 7*5 and the so lu tio n d ilu te d to rolume w ith t r i s buffer* This method oan be used to prepare Pe-BDTA so lu tio n s up to 0*005 H * At hig h er c o n c e n tra tio n s, a aox* concentrated baee ie req u ired fo r n e u tra l- i t a tio n . This o ften r e s u lts In the p re c ip ita tio n o f f e rrio hydroxide s o ls . 2* Equi-m olar q u a n titie s o f dlsodiun ED TA and Jto(V0 3> 3 were nixed. The pH was then ad ju sted from about 2*8 to 7.5 by the ad d itio n o f so lid ITaHCO^. The so lu tio n s were adjusted to volume w ith t r i s buffer* This nethod o f p rep aratio n avoids high lo o a l ooncentrations o f base and oan be used to prepare Ft-EDTA to concentra tio n s as high as 0*1 M. 3* Tor d ia ly s is experinents and c e rta in ra te stu d ie s y v a ria b le base eq u iv alen ts per mole o f iro n were added in the p rep aratio n o f Fe-EDTA * Te(NO^)^ and disodium ED TA were mixed and takoi to a volume such th a t the a d d itio n of base would b rin g the so lu tio n to the f in a l req u ired volume* The base, 0*01 M HaOH, was added to a ra p id ly s tir r i n g so lu tio n to avoid high lo c a l oo n cen tratio n s• P rep aratio n o f T erric C itra te Be agent grade o ltr io aold and trisodium c i t r a t e were obtained from A llie d Chemical C orporation and n o t fu rth e r p u rifie d before use* T e -c itra te was prepared by th ree procedures* 23 1* r e - c it r a te was prepared a t a 1*1 ra tio o f o itr a te to iro n by n ix in g o l t r i c acid and PeCNO^)^* She pH was ad ju sted by the ad d itio n o f d ilu te H aO H to a ra p id ly s ti r r i n g solution* A ll so lu tio n s were prepared in t r i s b u ffe r except a s noted in the t e x t. 2* In the p re p aratio n o f F e -o itra te a t v ario u s r a tio s o f o itr a te to iro n , fS(VO^)^ was added to a so lu tio n of c i t r l e a c id and the aaount o f HaClO^ o r NaNO^ neoessazy to M aintain oonstant io n ic s tre n g th through the serie s* The pH was adju sted w ith H aO H to 7*5, and the so lu tio n taken to volume w ith t r i s buffer* 3* The P e -o itra te so lu tio n s used in the d ia ly s is and t i t r a t i o n experim ents were aade up in d is t i l l e d water* Fe(HO^)^ was added to a so lu tio n o f tris o d iu n c itra te * The r e la tiv e q u a n titie s were ad ju sted according to the d esired o itr a te to iro n ra tio * Various amounts o f 0*01 M HaG B were added w ith rap id s tirrin g * The f in a l con c e n tra tio n o f P e -o itra te in these experim ents was l< f3 H. P rep aratio n o f P e rric H T A V T A was obtained in the a d d form from Geigy o r Matheson, Coleman and.B ell* I t was n o t p u rifie d before 2k u n • The Pe-NTA was prepared lay adding the r e q u is ite quan t i t i e s o f Pe(NO^)j and n e u tra lis e d NTA. The pH was ad ju sted to 7.5 w ith d ilu te NaOH and the so lu tio n was taken to Tolume in t r i e b u ffe r. P rep aratio n o f th e P errio T ra n sfe rrin Coupler To study th e removal o f iro n from tr a n s f e r r in , so lu tio n s o f th e iro n sa tu ra te d p ro te in were p repared. Pe-HTA was added in a liq u o ts to a p o -tra n s fe rrin to the p o in t o f s a tu ra tio n , as judged by the abeorbanoy a t **70 mu* In o rd er to remove the fre e N TA and any n o n -s p e c ifio a lly bound ir o n , the iro n p ro te in was d ialy sed ag a in st t r i s b u ffe r and then d ilu te d to the d e sire d oonoentration f o r study. Spectrophotom etrio Methods D iffe re n tia l spectrophotom etry stu d ie s employed a Cary Model 15 recording epeetrophotom eter. The instrum ent was equipped w ith tandem c e ll h o ld e rs. The sp e c tra could be obtained using two re a c ta n ts in the referenoe beam and the re a c tio n m ixture in the sample beam. I t was thus p o ssib le to make s p e c tra l stu d ie s o f th e in te ra c tio n of the f e r r io c h e la te s w ith tr a n s f e r r in . The k in e tic s o f the re a c tio n s o f the f e r r io che la te s and tr a n s f e r r in were follow ed by the absorbanoy change a t **70 * i , the ab so rp tio n m aximum o f P e -tra n s fe rrin . 25 The Instrum ents used v ers a Cary Model 15 and a Bookmen HJ w ith a G ilfo rd reco rd in g attachm ent • Both instrum ents were thermos t a t t e d . The tem perature was m aintained a t 2 5 -0 .1 °, w ith a Forma eo n stan t tem perature b ath . G enerally a 1 .0 a l a liq u o t o f the p ro te in was added to 0.1 a l o f the f e r r io c h e la te in a c u v e tte . The re a c tio n m ixture was thoroughly s tir r e d w ith a sm all s ti r r i n g ro d . The re a o ta n ts and the cu v ette were p re e q u ilib ra te d a t 25°• I t was p o ssib le by mixing the so lu tio n in a cu v ette alread y placed in the spectrophotom eter to o b tain the f i r s t reading w ith in te n seconds o f m ixing. The i n i t i a l ra te was e x tra p o lated back to time zero in o rd er to obtain the i n i t i a l absorbency v a lu e . This reading was co rrected fo r the absorbenoy o f the p ro te in and the f e r r io c h e la te . T r e a t y * ?+ K n m t i * T± The i n i t i a l r a te s o f the re a c tio n s were determ ined from th e slope disp layed on the re co rd er c h a rts . These v alu es proved to be reproducible w ith in 10#. The f i r s t o rder p lo ts were co nstruoted on a sem ilogarithm ic graph, on the lo g scale i s p lo tte d the fra c tio n of the t o t a l absorbency change rem aining a t a given time on the lin e a r s o a le . The d ia ly s is d a ta were tre a te d in a s im ila r manner. M a l y l . 8 tttd l.. The d ia ly s is o f a substance acro ss a membrane i s a f i r s t o rd er process* Tbs flu x depends on the eo n c en trstlo n g ra d ie n t and the e ise o f the aoleoulee in re la tio n to the pore else* To exanine the f e rrio o he l a te e fo r polym erisa tio n , eq u ilib riu m d ia ly s is experim ents were c a rrie d out* The methods o f Llndskog and Malmetrom (55) were followed* Two p la s tic chambers o f about th re e a l capacity were separated by a sh eet o f d ia ly s is tu b in g (T isking Corpora tio n )* In to both compartments were introduced equal v o l- unes o f the f e r r io c h e la te solution* The iro n on one side 59 was la b le d w ith tr a c e r amounts o f Be, while the o th e r sid e contained only the sta b le isotope* The apparatus was co n stan tly shaken and a t various tim es a 0.1 a l a liq u o t was removed from both sid es and the ra d io a c tiv ity determ ined u sin g a s c i n ti lla t io n counter (Jfuclear Chicago)* CHAPTER IV FERRIC CITRATE t ITS N A T U R E A N D T H E LIG A N D E X C H A N G E REACTION W ITH TRANSFERRIN Studies a t a l t l . C itra te i Iro n R atio T itra tio n S tudies A s e rie s of F e -c itra te so lu tio n s v ers prepared w ith f in a l co n cen tratio n s o f 10~^ M Fe(NO^)^, 10~^ M t r l - sodlun c i t r a t e , 0.1 H NaNO^ and v a ria b le amounts o f NaQH. The Tolumss vers ad ju sted so th a t the ad d itio n o f the base vould r e s u lt in the d esired f in a l volume• The base, 0*01 H HaOH, was ac c u ra te ly p ip e tte d in to a ra p id ly s t i r rin g so lu tio n of the trlsodlum c itr a te - ir o n complex* The fin a l pH vas determ ined on a Beckman Researoh Model pH m eter, vhlch had been standardised w ith 10“^ M HNOj in 0.1 M NaNO^* The re s u ltin g t i t r a t i o n curve i s presented In Fig* 1* The pH i s p lo tte d as a fu n ctio n o f base equlva le n ts added per mole o f iro n , OH/Pe, in ad d itio n to the th ree base eq u iv alen ts req u ired to n e u tra lis e the o l t r i e acid* The t i t r a t i o n curve e x h ib its two b u ffe r regions* The f i r s t i s a t a low pH and term inates a f te r the ad d itio n 27 Tig* 1*--'The t i t r a t i o n curve o f T e - c itr a te , super imposed on the.absorbanoy a t 270 mu. The f in a l concentra tio n s were 1 0 ^ M TeCHOa;?, 10“3 N trisodium o it r a te . 0*1 M NatfO* and v a ria b le amounts o f HaOH. Experim ental procedures are described in the te x t. 28 0.50 0.40 0.30 OH/Fe 30 o f 1 .5 OH/Pe. The second i s between pH 8 and 9 and t e r - n ln a te s a f te r the ad d itio n o f 3*0 OH/Pe • The a d d itio n o f fu rth e r base re s u lts in the p re c ip ita tio n o f fe rrio hydroxide s o ls . The r is ib le and u ltr a v io le t sp eo tra of eaoh o f the F e -c itra te so lu tio n s was obtained. Quartz c e lls o f 1 n path le n g th were employed. The ad d itio n o f the f i r s t base eq uivalent causes a oolor ohange from the o rig in a l lig h t yellow to a lime green . The ad d itio n of base in the seoond b u ffe r region produces a reddish brown hue. The sp eo tra obtained are presented in F ig. 2. I t w ill be noted th a t in the second b u ffe r region (1.6 to 3*0 OH/Pe) an is o b e s tie p o in t i s generated. This suggests th a t th ere are only two iro n chromophores in eq u ilib riu m . The absorption a t 270 m u fo r eaoh le v e l o f added base i s presented in F ig . 1, superimposed on the t i t r a t i o n curve. The abrupt change o f slope a t 1.5 base eq u iv alen ts supports the g lass elec tro d e r e s u lt th a t th is i s the end p o in t o f the f i r s t b u ffe r reg io n . The n o n -in teg ra l value o f the stoichiom etry i s o f in te r e s t and suggests e ith e r the sharing o f one hydroxyl group by two fe rrio ions o r the form ation o f more than one type o f f e r r ic complex. T ig. 2. The sp e c tra o f the T e -e itra te s e rie s described in T ig. 1 . A qu arts c e ll o f I n path le n g th was employed. The sp e c tra were obtained on a Cary 15 speotro photom eter. 31 ABSORBANCY 33 D ialy sis Experiments The P e -c itra te a e rie s described above was stu d ied by d ia ly s is techniques, as o u tlin e d in the experim ental se c tio n . D uplicate so lu tio n s were prepared using tra o e r 59 q u a n titie s of Pe. P ig . 3 p resen ts in senilogarlthm ic form the r e s u lts o f these s tu d ie s . P e -e ltra te so lu tio n co n tain in g zero o r 1 .0 OH/Pe e x h ib it only one ra p id ly d laly zin g component. At higher base le v e ls , the d ia ly s is p a tte rn s are b ip h aslo , in d ic a tin g the presence o f a t le a s t two types o f fe rrio complex. The fra c tio n o f the iro n th a t e x is ts as the slowly d laly zin g complex can be determ ined by the ex tra p o la tio n of th e lin e a r p o rtio n o f the curve to zero tim e. These v alu es are presented in Table 1. I t i s of i n t e r e s t th a t even a t the end o f the second b u ffe r region (3*0 OH/Pe) 2 0 % of the iro n remains in the ra p id ly d laly zin g form. An exam ination o f P ig . 3 shows th a t the ra te of d ia ly s is o f th e slow component does n o t appear to be a ffe c te d by a d d itio n of b ase. The observed f i r s t o rd er ra te co n stan t fo r the d ia ly s is of th e P e -o itra te w ith no _ o — 1 added base i s 3.1 x 10 J n in . • Por comparison the fo r the slow phases i s 0.*t x 10 mln. • This re p re se n ts an e ig h tfo ld d iffe ren ce in th e d ia ly s is ra te o o n stan t. . 1 P W Q t l g n g S J g a n M a tn ig m Three F e -c itra te so lu tio n s were prepared w ith the f in a l co ncentration varying over a 100-fold range. Eaoh 3»*“The tin e oourse o f e q u ilib ra tio n acro ss a d ia ly s is membrane f o r a s e rie s of J S - e itr a te so lu tio n s . The so lu tio n s are as described in Jig* 1* The curve num b ers r e f e r to the number o f base eq u iv alen ts added p er mole o f iro n . The d a ta are p lo tte d as the fra o tio n of ra d io a o tlv ity rem aining to e q u ilib ra te on the logarithm io soale a s a fu n ctio n o f time on th e lin e a r so a le . HOURS 36 TABLE 1 TH E TRACTION OF TH E IBON COMPRISING TH E S L O W L Y DIALYZING C O M P O N E N T OF TH E FERRIC CITRATE SERIES AS A FUNCTION O F ABLER B A SE EQUIVALENTS PER M O L E OF IRON (O ther co n d itio n s are noted in Fig* 1 and the t e s t ) OH/Fe Slow F ractio n Percentage 0.0 le s s than 10 1 .0 15 1.5 27 2.0 *3 2.5 59 3 .0 82 37 so lu tio n was 0*1 M in HaNO^ and ad ju sted to 2*5 OH/Ps* The d ia ly s is p a tte rn was stu d ied in the u su al Banner and the r e s u lts are presented in Fig* The 10~2 M F s -o ltra te i s seen to contain 65% the iro n as the slow ly dlalyxing compo n ent w hile the 10~^ M contains about 5 0 % • The r e s u lts o f the 10 M F e -o ltra te d ia ly s is show no evidence f o r a slow component* I t appears th a t the th resh o ld fo r form ation o f the slow ly d lalyxing fra c tio n i s between 10 end 10 M * I s o la ^ L o I weight The is o la tio n o f a polymeric fra c tio n was c a rrie d out by I t s * Paul Saltman and Tom Spiro and Mr* John Benner. Gel f i l t r a t i o n perm itted the sep aratio n of a polymeric fra c tio n of F s-o ltra te * M olecular weight determ inations o f the is o la te d F e -c itra te fra c tio n were made using osmotic pressure tech niques* These experim ents were c a rrie d out in our lab o ra to r ie s by Mr* John Benner* A Meohrolab Model 503 high speed membrane osmometer was used* The membranes employed were S 4 S B-20 o e llu lo e e a c e ta te 9 preconditioned in hepes b u ffe r a t pH 7*5* A 2 % so lu tio n o f th e iso la te d and d ried polymer p rep aratio n was made up in th is b u ffe r and su itab le * 5 d ilu tio n s were prepared* A value of 2*0 - 0*1 x 10 was determ ined fo r the m oleoular w eight. I t was found th a t th is value did n o t vary to a s ig n ific a n t ex ten t when F ig . The d ia ly s is p a tte rn s fo r a s e rie s o f F e -o itra te solutions* The f in a l ooncentrations are noted in the curve designations* Eaoh F e -c itra te so lu tio n was ad ju sted to 2*5 base eq u iv alen ts p er so le of iron* Other oonditions are as noted in the te x t. 38 (C-C#q)/Ctq 06 0 4 - 0.2 150 1 0 0 50 kO d iffe re n t le v e ls o f OH/Pe were used In preparing the Fe- c itr a te solutions* D lsousslon The experim ental r e s u lts can b e st be explained in terms o f an equilibrium between v ario u s F t- c itr a te com plexes* These may Include a monomer, a dimer and polymer* The evidenoe fo r those complexes and the proposed s tru c tu re s w ill be dlsoussed below* Varner and Weber (1 8 ), on the b asis o f th e ir t i t r a tio n d a ta suggested the exlstsnoe o f a P e -c itra te monomer, in which the iro n i s coordinated by two carboxyl groups and the alcohol group. The pH o f a 10~3 M so lu tio n of the trisodium c itr a te - ir o n oomplex i s about 3*0 before the a d d itio n o f any base* This i s probably due to the d is placement o f the alco h o lic proton. The evidence fo r the polymer i s quite c le a r out* The fin d in g o f a m olecular weight o f 2 x 10 i s in d ic a tiv e o f a polymer involving the linkage o f approxim ately 800 f e r r ic ions* The t i t r a t i o n d ata show th a t in a d d itio n to a proton displaoed from c i t r a t e , the polymer re q u ire s the n e u tra lis a tio n of two protons from w ate r. The s tru c tu re th a t b e st f i t s these d ata i s a polymer in which the f e r r ic ions a re linked by hydroxyl b rid g es, the o th er s ite s o f th s iro n being coordinated by the c i t r a t e lig an d s. The equilibrium between the polymer and the low k l m olecular m ig h t complexes i s re fle c te d in the d ia ly s is p a tte rn under the v ario u s co n d itio n s. The d ata in Table 1 show th a t as the le v e l o f added base i s in c re a se d , the fra c tio n p re se n t as the polymer i s enhanced. I t i s in t e r e s tin g th a t a t the endpoint o f the t i t r a t i o n about 20% of the iro n rem ains in a low m olecular weight form. The ex isten ce o f a dim eric P e -c itra te i s suggested by th e speotrophotom etrio t i t r a t i o n shown in P ig . 1 . I t w ill be seen th a t the absorbency a t 270 m u as a fu n c tio n of th e added base eq u iv alen ts e x h ib its an abrupt change o f slope a t 1 .5 OH/Pe. The f i r s t OH/Pe has been accounted f o r in th e n e u tra liz a tio n o f th e proton d isplaced from the c i t r a t e . Since the 0 .5 OH/Pe rem aining cannot be accounted fo r in term s of monomeric P e - o ltr a te , h ig h er complexes must be invoked. Tw o ex plan atio n s are p o s s ib le . (1) The Pe- c it r a te forms dim ers, in which two f e r r lo ions are bridged by one hydroxyl group from w ater. The a d d itio n o f fu rth e r base lead s to polym erization o f the f e r r ic ions and accounts fo r the change in sp e c tra l p ro p e rtie s . (2) That approxim ately 2 5 % o f the iro n i s being incorporated in to polymers w hile the bulk rem ains in monomeric complexes. This hypothesis i s supported by the d a ta from P ig . 3* I t w ill be seen th a t a f t e r th e a d d itio n o f 1 .5 OH/Pe approxi mately 7 0 % o f the iro n e x is ts as a ra p id ly d ialy zin g com plex and th e rem ainder i s o f a higher m olecular w eight. k 2 Of sp ec ia l i n t e r e s t to th e lig an d exohange re a o tlo n w ith tr a n s f e r r in i s th e concentration dependent polym erisa tio n of the P e - c itr a te . P ig . b shows th a t the th resh o ld fo r the form ation o f polymer i s between 10 and 10 M . This i s the co n cen tratio n range in vhieh the iro n tra n s fe r re a c tio n w ill be stu d ie d . I t should be borne in mind th a t t h is phenomenon may have an e f fe c t on the k in e tic s observed in th e re a o tio n w ith th e p ro te in . Por reference a s tru c tu r a l form ula f o r monomeric P e -c itra te i s given below. / ■k i/ /i P e rric C itra te a t V ariable C itra te xo Iron Baxioa P e -c itra te so lu tio n s were prepared w ith a f in a l co n cen tratio n o f 10 ^ M Pe(IT0^)^9 0.031 M trisodium o itra te , 0 .1 M VaHO^ and varying amounts o f 0.01 M C02 fre e HaOH. pH determ inations were made on a H eathkit recording pH ^3 m eter. The re su ltin g t i t r a t i o n curve i s shown in F ig . 5. I t should he noted th a t the pH i s 7*0 before the ad d itio n o f b ase, due to the b u ffe rin g e f f e c t o f th e ezoess c i t r a t e . The t i t r a t i o n curre e x h ib its a sin g le b u ffe r region which i s between zero and approxim ately 2.0 base eq u iv alen ts added p er mole o f iron* S peotral S tudies The v is ib le and u ltr a v io le t sp e c tra were obtained f o r each o f the F e -c itra te so lu tio n s described above. The r e s u lts are presented in F ig. 6 . The curve desig n atio n s r e f e r to the number o f added base e q u iv a le n ts. I t w ill be seen th a t an is o b e s tlc p o in t i s generated a t 315 mu. This suggests th a t th ere are only two chromophores in e q u ilib rium . A spectrophotom etric t i t r a t i o n a t 280 a|i i s super imposed on th e potent!om etrio t i t r a t i o n curve in F ig . 5* The abrupt change in slope a t 2.0 base eq u iv alen ts) sub s ta n tia te s th is as the endpoint o f the b u ffe r re g io n . D ialy sis Experiments A s e rie s o f so lu tio n s was prepared with f in a l con c e n tra tio n s o f 10 3 M Fs(NO^)^ 0.003 M COg free ffaOH, 0.1 M NaNO^ and v a ria b le amounts o f trisodium c i t r a t e . I t should be noted th a t th e re are 3.0 OH/Pe in th is s e rie s to allow fo r the p o ssib le polym erization. As u su a l a 59 d u p licate s e rie s was prepared u sin g tra o e r amounts o f Fe P ig . 5*—The superimposed potentlom etrlo t i t r a t i o n curve and epeetrophotom etrie t i t r a t i o n a t 280 mu fo r Pe- c it r a t e in the presence o f excess c itra te * The f in a l con c e n tra tio n s were 10*3 M Pe(KO^)^, 0*031 M trisodium c i tr a t e and 0*1 M VaNOa* Varying base eq u iv alen ts were added as 0*01 M NaCH, and are in d ic a te d as OH/Pe* Other cond itio n s are as noted in the text* 0. 35 0.30 0.20 OH/Fe -r vn J i P ig . 6 .—The r i s i b l e and u ltr a v io le t sp e c tra fo r the F e -o itra te so lu tio n s described in F ig . 5 and the te x t. The curve d esig n atio n s re f e r to the added OH/Fe. The d ata from t h i s fig u re were used in the co n stru ctio n o f the speotrophotom etrlc t i t r a t i o n shown in F ig . 5. (rtui)Y ABSORBANCY -r N j k& in o rd er to follow the d ia ly s is . The re s u lts o f the d ia ly s is experim ents are p re sented in F ig . 7. The curve d esig n atio n s r e f e r to the r a tio of the c itr a te to iro n in the f in a l s o lu tio n s. The d ia ly s is p a tte rn fo r r a tio s le s s than 21 i l are h ip h aslo . The fra c tio n present as the slowly d ialy zln g complex decreases as the c i t r a t e le v e l i s in c re a se d . At r a tio s o f 2 1 :1 o r g reater^ the e n tir e amount o f th e iro n e x is ts as a ra p id ly d ialy zln g complex. The fra c tio n of the Iro n th a t i s p re se n t as a ra p id ly d ialy zln g complex i s presented in Table 2 as a function o f the ra tio o f c itr a te to iro n . The k0 ba fo r the d ia ly s is o f the slow phase o f the 11:1 -3 - i complex i s 0.1 x 10 ■in* • The ko t 8 fo r the 21:1 r a tio f e r r ic c itr a te is l.M-5 x 1 0 ^ ®in. The sp ectra o f the F e -o itra te so lu tio n s described in the d ia ly s is experim ents above are presented in F ig . 8 . I t w ill be seen th a t th e re i s an is o b e s tic p o in t a t 315 mu, in d ic a tin g th a t th ere are probably only two species in eq u ilib riu m . There i s a monotonic v a ria tio n in the absor- bency a t 2 8 0 m u ending in a lim itin g spectrum a t r a tio s above the le v e l o f 2 0 : 1 . In co llab o ra tio n w ith Dr s . P h ilip Aisen and H askell Beisoh a t the IBM Watson la b o ra to rie s ) bulk so lv en t F ig . 7 .—The d ia ly s is p a tte rn fo r a s e rie s o f Fe c i t ra te so lu tio n s o f v a ria b le c itr a te to iro n r a tio s . Tbs curve d esignations ln d ie a te the r a ti o s . The so lu tio n s had a f in a l concentration o f 1 0 " 3 FeCJKMo, 3 x 1 0 " 3 C 0 2-fr e e NaCH, 0 .1 M IT aN O o and v a ria b le trie o d iu a c i t r a t e . The r e s u lts are p lo tte d as the lo g o f the fra c tio n of is o to p ic iron, re aain ln g to e q u ilib ra te as a function o f tia e on a lin e a r s c a le . ^9 (C-C«q)/Ctq HOURS VJl O 51 TA BLE 2 TH E RAPIDLY D 1A L Y Z X N G FRACTION OF A SERIES O F FERRIC CITRATE SOLUTIONS AS A FUNCTION OF TH E CITRATE T O IRON RATIO (The ta b le la constructed from the d ata o f Fig* 7) c itra te /F e Rapidly D ialyzlng F ractio n Percentage 1 . 0 2 0 6 . 0 * fO 1 1 . 0 57 1 6 . 0 85 2 1 .0 1 0 0 2 6 .0 1 0 0 31.0 1 0 0 P ig . 8 . —The sp ec tra fo r the s e rie s o f P * -o itra te so lu tio n s described In F ig, 7* The curve d esignations In d io ate the c itr a te to iro n r a tio s . 52 ABSORBANCY 5*t re la x a tio n etu d lea were made. A s e rie s o f F e -c itr a te solu tio n s were prepared by the ad d itio n o f Ve(NO^)^ to solu tio n s contain in g v a ria b le amounts o f e l t r i o a o id . The pH was ad ju sted to 7*5 w ith d ilu te base. I t was found th a t the bulk solvent re la x a tio n ra te i s a ffe c te d by the addi tio n o f exoess c i t r a t e . The re la x a tio n ra te in c re a se s as c it r a t e i s added up to a r a tio o f 20:1. At th is p o in t i t le v e ls o f f and i s r e la tiv e ly u n affected by the a d d itio n o f f u rth e r o l t r a t e . D iscussion The evidence obtained from the experim ental stu d ie s described suggests the form ation of a d ie itr a te iro n sp ec ie . I t i s the presence o f th is complex th a t accounts fo r the observed p ro p e rtie s o f F e - o itr a te . The evldenoe fo r th is oomplex and i t s e f fe c t on th e chemical behavior o f F e -c itra te w ill be d iscu ssed . The d ia ly s is stu d ie s d e a r l y in d ic a te th a t between the r a tio s 1 : 1 and 2 0 : 1 , c i t r a t e : iro n , th ere i s an equi lib riu m between the slowly d ialy zln g polymer and a low m olecular weight complex. In lig h t o f the f a c t th a t the low m olecular weight complex i s enhanced by the a d d itio n o f excess o l t r a t e , i t i s most lik e ly th a t th is oomplex has th e fozmula Fe ( c itr a te )n . For s te rio reasons i t i s d i f f i c u l t to b eliev e th a t n could be g re a te r than 2 . S tudies using spaoe f i l l i n g Courtlaud Models showed th a t s tr a in fre e 55 models o f f e r r ic d io l tr a t e , F e(C it) 2 could be c o n stru c te d . The f e r r lo ion i s bound by two carboxyls and the alc o h o lic oxygen o f each c i t r a t e . The sp e c tra l study presented In F ig . 5 supports the above proposal f o r the V e(C it) 2 oonplex. I t i s seen th a t th ere I s a aonotonio deorease In the absorbency a t 2 8 0 mu, which term in ates abruptly a f t e r the ad d itio n o f 2.0 OH/Fe. This i s the stoiohiom etzy th a t would be expected fo r n e u tra lis a tio n o f the proton d isplaced from each o f the o ltr a te alcohol groups by f e r r ic io n . The d ia ly s is p a tte rn s and th e s p e c tra l s tu d ie s nay be in te rp re te d in te rn s o f an equ ilib riu m between the poly meric specie and a F e (C it)2* F ig . 7 d e a r l y dem onstrates th is p o in t. The low m olecular weight fra c tio n in c re a se s as the c it r a t e to iro n r a tio is ra is e d . F in a lly a t the p o in t o f 2 1 * 1 the e n tire fra c tio n o f the iro n e x is ts as the low m olecular weight complex. The r a tio f o r to ta l incorpo ra tio n o f iro n in to th e low m oleoular w eight speoie c o rre s ponds c lo se ly w ith the r a tio o f the lim itin g sp e c tra shown in F ig . 8 and the d a ta obtained from proton re la x a tio n s tu d ie s • I t i s in te re s tin g th a t the r a te o f the d ia ly s is o f the slow fra c tio n i s re la tiv e ly u n affeo ted ty v a ria tio n c i t r a t e le v e ls . I t appears th a t the exoess o ltr a te does l i t t l e to enhance the breakdown o f th e polymer onoe i t has 56 formed* The ieo h ee tic p o in ts noted in the sp e c tra l stu d ie s suggest th a t only two chronophores a re in eq u ilib riu m , pre sumably the polymer and the fS(C lt> 2 « This hover*r does not ru le out the presence o f a sm all fra c tio n o f the iro n e x is tin g as oligiom ers, d in ars and monomers* Time Course o f the Be met ion The tra n s f e r o f iro n ( I I I ) from l t l , c itra te * iro n to tr a n s f e r r in was studied speotrophotom etrioally a t if70 mu, the r is i b l e abso rp tio n maximum o f the Yt-transflerxin complex* P e -c itra te stu d ied in th is se c tio n was prepared from c i t r i c sold and Pe(PO^)^* The pH was adjusted w ith d ilu te H aOH to 7*5* H I so lu tio n s including the p ro te in were made up in 0*005 M t r i s buffer* The tra n s f e r r in was a t a co n cen tratio n of 1 0 “* * binding eq u iv alen ts A i t e r . 1 0.1 ml a liq u o t o f 1 0 * 3 M P e -c itra te was added to 1*0 ml o f the p ro te in in a m ioro-curette* The re a c tio n m ixture was thoroughly s tir r e d and the absorbenoe recorded usin g a Beckman IX J spectrophotom eter equipped w ith a th e r mos ta tte d c e ll holder and a G ilford attachm ent. The re a c tio n follow s a blphaslo tin s course a s i s shown in Pig* 9* The f i r s t phase, which w ill be re fe rre d to as the I n i t i a l absorbenoy, i s oomplete w ithin the ten Hesul Fig* time course o f tr a n s f e r o f iro n (III) fro * o ltr a te to tr a n s f e r r in as determ ined spectrophoto- m e tric a lly . The f in a l concentration o f the F e -c itra te in m o larity and the binding eq u iv alen ts A l t e r o f the tra n s f e r r in i s 0*91 x 1 0 “* . The re a c tio n was c a rrie d out a t 25° in a therm ostatted c e ll h o ld er. The pH was m aintained a t 7*5 w ith 0.005 M t r i e b u ffe r. A ll o f the iro n (III) i s tra n s fe rre d to the p ro te in a t com pletion o f the re a c tio n . 57 Absorboncy 470 mp 0.2 0 . 1 0 20 15 1 0 Hours seconds neoeseary to o b ta in the f i r s t absorbency reading and accounts fo r about 20£ of th e to ta l reaction* I t was shown by d if f e r e n tia l spectrophotom etry th a t the i n i t i a l spectrum i s id e n tic a l w ith th a t o f the p roduct, fe - tr a n s f e r r in . I t appears th a t the i n i t i a l absorbenoy i s caused by a fra c tio n o f the iro n which re a c ts very rapidly* The second phase o f the re a o tio n follow s f i r s t o rd er k in e tic s as shown in fig * 10* The d ata are p lo tte d as the fra c tio n o f the tr a n s f e r r in e ite s u n fille d on a logarithm io scale a s a fu n ctio n o f tine* L in e a rity i s observed through a t l e a s t 85£ completion* The observed f i r s t o rd er ra te c o n stan t i s 2*5 x 10~^ min* 1 . I t i s apparent th a t a f te r the i n i t i a l absorbency, the rem ainder o f the iro n re a c ts w ith the p ro te in a t a uniform r a t e . Study o f the Bate Dependence Experiments were designed to in v e s tig a te the dependence o f the seoond phase o f the re a c tio n on th e con c e n tra tio n o f f e - o itr a te and tr a n s f e r r in . The i n i t i a l ra te s could be determ ined w ith an acouracy o f “ 1 0£ , on th e Cary Model 15 spectrophotom eter* A s e rie s o f l t l , c itr a te tir o n so lu tio n s was p re pared w ith f in a l co n cen tratio n s over a ten fo ld range* Each member o f the s e r ie s was combined w ith a tra n s f e r r in so lu tio n containing o f 0 * 9 1 x 1 0"* * binding e q u iv a le n ts / l i t e r * The r e s u lts are presented in fig * 11* The d ir e c t Tig* 1 0 .—S eallogarithm ic re p re se n ta tio n o f the tr a n s f e r o f Iro n (111) from o ltr a te to tr a n s f e r r in . Con c e n tra tio n s and co n d itio n s are as noted In f i g . 9 and the t e x t . The observed r a te co n stan t derived fro n t h i s p lo t I s 2 .5 x 10*3 m inutes"1 . The lin e a r ity i s an In d ic a tio n th a t th e seoond phase o f th e tr a n s f e r re a c tio n follow s f i r s t o rd er k in e tic s . 60 Hours » Fraction U n reacted U l o <* o u ro o Pig* 1 1 . —The i n i t i a l ra ta o f the aaeond phase o f the tr a n s f e r o f iro n (III) from c i t r a t e to tr a n s f e r r in , as a fu n ctio n o f the P e -c itra te concentration* The p ro te in co n cen tratio n was a a in tain ed a t 0 * 9 1 x 1 0 "* binding eq u iv alen ts A i t e r . The ra te was determ ined spec tro photo- m etrio a lly as described in the te x t. 62 4 .0 6 .0 8 .0 10.0 Ferric Citrate ( M x I04 ) Rate (A A 4 7 0 x se c .-1 x I0 5) o £9 Q * p ro p o rtio n a lity between the i n i t i a l v e lo o ity and the Pe- c i t r a t e concentration i s in d ic a tiv e of a ra te U n itin g step which i s f i r s t o rd e r w ith resp eo t to the iro n complex. In a sim ila r manner a s e rie s o f so lu tio n s co n tain ing v a ria b le concen trations o f tr a n s f e r r in were re a c te d w ith P e -c itra te a t a co ncentration of M-.55 x 10 M . The i n i t i a l v e lo c itie s of th e second phase a re presented in Table 3* I t w ill be seen th a t over a fiv e fo ld range o f tr a n s f e r r in co n cen tratio n th ere i s no s ig n ific a n t ohange in the rate* Thus the ra te expression i s sero o rder in p ro te in f i r s t order in P e -c itra te ; v*k ( F e - c itr a te ) . These re s u lts in d ic a te th a t the ra te U n itin g step must involve the fo ra a tio n o f a re a c tiv e P e -c itra te cou p le r before in te ra c tio n w ith the p ro te in occurs. Otherwise the ra te would be dependent on the p ro te in concentratio n as w ell as the P e - c itr a te . I t i s p o ssib le th a t the reao - tiv e f e r r ic complex is the sane as th a t responsible f o r th e i n i t i a l absorbency. Study o f Tni t j a i Absorbenov Sue to th e ra p id ity o f the i n i t i a l phase o f the re a o tio n o f P e -o itra te w ith tr a n s f e r r in , i t was n o t f e a s i b le to make ra te stu d ie s using the techniques d esc rib ed . I t was possib le however to estim ate aoourately the value o f the i n i t i a l absorbency under v arious conditions by e x tra p o la tin g the seoond phase baok to tin e se ro , and 65 TA B U S 3 TH E INITIAL H A TE 07 TH E SE C O N D PH A SE 07 TH E REACTION 07 TBBRIC CITRATE A T A 111 RATIO, W ITH TRANSFERRIN (The co n cen tratio n o f the 7 e - c itr a te vae *f. 5 5 x 1 0 ~*M in a l l oases) T ra n sferrin C oncentration Rate o f Reaction^ B in d in ^ E q u lralejjte A ^ 7 0 m c” 1 1 0 1 . 8 2 3.0 3 . 6M - 3.1 5 M ^ • 1 7 .2 8 2 . 6 9.09 3 .2 c o rre c tin g f o r the absorbency o f the p ro te in and F e -o itra te so lu tio n . The re la tio n o f the i n i t i a l absorbency to the F e -c ltra te co n c en tratio n , when the tra n s f e r r in concentra tio n i s held constant a t 0 .9 1 x 1 0 ”* * binding e q u iv a le n ts/ l i t e r , i s shown in F ig . 12. The data follow a hyperbolic fu n c tio n , which oould be in te rp re te d in te rn s o f a sa tu ra tio n s q u illb r la o r a s a re fle c tio n of a varying fra c tio n a l conposition o f a re a c tiv e F e -c itra te conplex, as the to ta l F e -c itra te co n cen tratio n i s in cre ased . ▲ re so lu tio n of th is question was achieved by a study o f the i n i t i a l absorbency a t varying concentrations o f tr a n s f e r r in . The i n i t i a l absorbenoy, measured a t a F e -c itra te co n cen tratio n of 0 . 9 1 x 1 0 "*^, as a fun ctio n o f tra n s f e rrin co n cen tratio n i s shown in F ig. 13* The p lo t i s d is tin c tly b ip h asio . The f i r s t rsg io n e x h ib its a d ire c t proportion a l i t y between the i n i t i a l absorbency and the tra n s f e rrin co n c en tratio n . ▲ sharp break ooours where the le v e l of the p ro te in concentration corresponds to about 20^ of th e F e -o itra te added. The fu rth e r a d d itio n of p ro tein does not s ig n ific a n tly enhance the i n i t i a l absorbency. These re s u lts c le a rly in d ic a te th a t a fra c tio n of the F e - c itr a te , approxim ately 20£, e x is ts in a form which i s a b ls to re a c t ra p id ly and oom pletsly with the p ro te in . The rem ainder o f the F e -c itra te i s unabls to oombins ra p id ly w ith th e p ro te in to produce the i n i t i a l absorbenoy. P ig . 12*—The i n i t i a l absorbency as a fu n c tio n of P e -c itra te co n cen tratio n . Tbs tr a n s f e r r in co n cen tratio n was held constant a t 0 .9 1 x 1 0~* binding eq u iv alen ts p er l i t e r . 67 Ferric Citrate ( M x I04 ) Absorbancy 470 mji o 8 * o CD b o o 89 * ig . 1 3 . —I n i t i a l absorbenoy a s a fu n ctio n of tr a n s f e r r in co n cen tratio n . The. P e -c itra te co n cen tratio n was held constant a t 0.91 x lCT^M. 69 Abtorboncy 470 mil OJOt 0. 01 0 . 0 0 0 . 0 0.4 0 6 1 . 0 0 8 Transferrin Binding Equivalents (M x I04) These re s u lts a llo v us to in te r p r e t the r e la tio n o f th e i n i t i a l absorbenoy and the ? e -o itr a te co n cen tratio n shown in T ig. 12* D ialy sis k in e tio s in F ig . b have shown th a t, as the to ta l F e -c itra te con cen tratio n in c re a se s, the fra c tio n th a t i s p re se n t as the polyner i s in oreased, and the low ao leo u lar w eight fra c tio n i s correspondingly low ered. The i n i t i a l absorbency appears to be a M anifes ta tio n o f the anount o f a low M olecular weight coMponent of fe rrio c itr a te p resen t a t the tin e o f n ix in g . The decreas ing fra c tio n a l conposition o f th is conponent through the F e -c itra te s e rie s g ives r is e to the hyperbolic r e la tio n of the i n i t i a l absorbenoy and the F e -c itra te co n cen tratio n . The molar a b so rp tiv ity determined from the propor tio n a l region of F ig . 13 i s approxinately 5o£ of th a t expected fo r the product F e -tra n s fe rrin . This suggests the form ation o f an interm ediate tern a ry complex o f the form C it-F e-T rf, w ith a lower molar ab so rp tiv ity * I t has n ot been possible to o b tain d ire c t experim ental evidence fo r an interm ediate complex such as w ill be shown fo r the re a c tio n o f Fe-EDTA and tra n s f e rrin . I t was shown by d ia ly s is techniques th a t the ad d i tio n o f base to a 1 * 1 , c itra te * iro n oomplex led to an enhancement of the polymeric fra c tio n . A p a r a lle l study 72 was Bade on the re a c tio n of tr a n s f e r r in and F e - c itr a te , which had been prepared a t v a ria b le OH/Fe levels* 1 1 : 1 , tris o d iu n c i t r a t e : iro n oonplex was prepared in d i s t i l l e d w ater. The volumes were ad ju sted so th a t the ad d itio n o f the d esire d amount o f base would r e s u lt in the c o rre c t f in a l volume. The base was added slowly to a ra p id ly s ti r r i n g solution* The f in a l co n cen tratio n o f the F e -c itra te was 10 M. A 0.1 ml a liq u o t o f th e F e -o ltra te was added to 1 * 0 ml o f tra n s f e r r in co n tain in g 1 0 binding eq u iv alen ts A lte r * The pH o f the re a c tio n m ixture was m aintained a t 7 * 5 - 0 * 2 by the b u ffe rin g ac tio n o f the pro te in and 0.005M t r i e b u ffe r. Fig* lb p re se n ts the r e s u lts o f the i n i t i a l ra te o f the second phase o f the re a c tio n as the base r a tio i s increased* I t w ill be seen th a t th e re i s a la rg e decrease in th e ra te between zero and 1*5 OH/Fe* This i s follow ed by a lin e a r decrease to 3*0 OH/Fe* The i n i t i a l absorbenoy i s presented in Fig* 15 fo r the ease F e -c itra te se rie s* The lin e a r deorease in the I n i t i a l absorbenoy as a fu n ctio n of the added base equiva le n ts suggests th a t th e re a c tiv e fra o tio n of the F e -c itra te i s being converted in to the un reao tlv e polymeric form* I t i s o f in te r e s t th a t a t 3*0 OH/Fe an i n i t i a l absorbenoy i s s t i l l observed, even though i t re p re se n ts only about b% o f the t o t a l . This i s in agreement w ith the fig * l*f. —The i n i t i a l ra ta o f the tra n s f e r o f iro n ( 1 1 1 ) fro n c i t r a t e to tr a n s f e r r in a s a fu n ctio n o f the base eq u iv alen ts p e r ao le o f ir o n , added to the f e - c i t r a t e p r io r to re a c tio n . The m olar co n cen tratio n o f the f e - c i t r a t e and th e tr a n s f e r r in binding eq u iv alen ts l i t e r was Q.91 x 10“* . The pH was m aintained a t 7.5 t 0*1 w ith 0.005- t r i s b u ffe r. 73 0.5 1.0 1 .5 OH/Fe 2.0 2.5 3.0 Tig* l 5 .~ T h e I n i t i a l absorbency In the re a c tio n o f F e -c itra te and tr a n s f e r r in as a fu n ctio n of the base eq u iv alen ts p e r so le o f iro n used in the p rep aratio n o f the F e -o itra te . D e ta ils of the spectrophotonetrio tech niques a re presented in the experim ental se c tio n . 75 OH/Fe Absorbancy 470 mp o o I n O ai to O r o ui 91 77 d ia ly s is experim ents which in d io a te the presence o f a low m olecular w eight fra c tio n even a t th is base r a ti o . BeT~ 23 The E ffe c t o f Bxoeaa C itra te in the The e ffe o t o f excess c i t r a t e on the chemical n atu re of F e -c ltra te complexes has been d iscu ssed . I t was pro posed th a t an eq u ilib riu m e x is ts between a F e -c itra te monomer, Fe-CIT, a polym eric sp ec ie , (FeC lt)n and the d i c itr a te iro n oomplex, F e(C it)2 . The fra c tio n e x is tin g as th e l a t t e r i s enhanced by the presence o f exoess o f o itr a te a t the tin e o f n e u tra lis a tio n . A 1 :1 , c itr a te :I r o n complex i s predom inantly polym eric; a 20:1, c itr a te : iro n p re p aratio n i s predom inantly F e(C it>2 end a t the i n t e r m ediate r a tio s both sp ecies are in eq u ilib riu m . The re ac t i v i t y o f the F e(0it>2 was studied w ith re sp e c t to both the i n i t i a l absorbenoy and the ra te o f the re a c tio n . A s e rie s o f F e -c itra te so lu tio n s were prepared with iro n r a t i o s . The so lu tio n s were prepared by tb s a d d itio n of Fe(HO^)^ to a so lu tio n o f c i t r i c aold in 0.005 M t r i s b u ffe r. The pH was a d ju sted to 7 .5 by the slow ad d itio n of base to a ra p id ly s t i r r i n g so lu tio n . The io n ic stren g lh of th e so lu tio n s were ad ju sted w ith H&CIO^ so th a t the io n ic s tre n g th o f the re a c tio n would be 0.028M. As p re vio u sly describ ed , 0.1 ml o f the F e -c itra te was added to an iro n co n cen tratio n o f 10~3 M , and v a ria b le o itr a te to 78 1,0 ml o f tra n s f e rrin containing 10*^ binding e q u iv a le n ts/ l i t e r . A Cary Model 15 was employed fo r the spectrophoto- m etric determ in atio n s. The f i r s t fiv e m inutes o f the re a c tio n i s presented in P ig . 1 6 . The curve designations r e f e r to the c it r a t e to iro n r a tio s o f the P e -c itra te prepara tio n . The l i l y o itr a te iir o n complex has been discussed e a r l ie r and i s shown here fo r comparison. The re a c tio n p a tte rn i s blphasio. There i s a ra p id i n i t i a l absorbency follow ed by a slow r a te c h a ra c te ris tic o f the polymer breakdown. The re a c tio n p a tte rn o f th e 20:1 c itr a te tir o n com plex i s a lso b ip h a slc . There i s an i n i t i a l absorbenoy, amounting to about 10£ o f the to ta l re a c tio n and a second phase w ith a g re a tly enhanced r a te . The bulk o f the re a c tio n i s a re fle c tio n o f the P eC C lt^ re a c tiv ity and is completed w ith 15 m inutes, q u ite ra p id compared to the 20 hours required fo r the l t l , c itr a te iir o n complex. The 50:1, o itr a te tir o n complex shows a sim ila r re a c tio n p a tte rn b u t is somewhat slower and re q u ire s 30 minutes fo r com pletion. P ln a lly the 10:1 o itr a te : iro n oomplex, whloh con ta in s (1) a re a c tiv e low m olecular weight oomplex, (2) th e P e(C it)2 and (3) the polymeric (PeC lt)n , e x h ib its a three phase re a c tio n p a tte rn . The f i r s t phase i s an i n i t i a l F ig . 16.—The tin e course o f th e re a c tio n o f F e -c itr a te and tr a n s f e r r in , a t v ario u s o itr a te to iro n r a t i o s . The co n cen tratio n o f the iro n in m olarity and the tr a n s f e r r in binding eq u iv alen ts A l t e r was 0.91 x 10"H M . The c i t r a t e to iro n r a tio s are in d ic a te d by the curve d e sig n stio n s. These re a c tio n s were c a rrie d out a t a con s ta n t io n ic stre n g th o f 0.028 M , u sin g N aC lO fc. as the com pensating e le o tro ly te . 79 T T 0. 0 1 . 0 2. 0 Minutes T 3.0 4.0 5.0 00 o absorbency which i s complete w ith in ten seconds* The second interm ed iate phase, c h a ra c te r is tic o f the P eC C lt^* ends ab ru p tly a f te r 2*5 minutes and i s follow ed by a slow th ir d phase c h a ra c te r is tic of polymer breakdown* The f in a l phase accounts fo r approxim ately 30£ o f the re a c tio n and was found to follow f i r s t order k in e tic s* The observed -3 -1 ra te co n stan t was 1*7 x 10 min* , in close agreement w ith the 2*5 x 10~^ min* 1 determined fo r the polym eric 11 1, c it r a t e : i r o n complex* The I n i t i a l Absorbenoy a t Various Oitrate to Iron Batios The I n i t i a l absorbenoy has been shown to be a mani f e s ta tio n of a h ig h ly re a c tiv e P e -c itr a te complex p re se n t a t the time o f the a d d itio n of th e protein* The i n i t i a l absorbency fo r a s e r ie s o f so lu tio n s o f in creasin g c i t r a t e to iro n r a tio s i s shown in P ig. 17* The conditions are the same as described f o r Pig* 16* As the r a tio in c re a se s from 1<1 to 5*1 th ere i s an enhancement o f the i n i t i a l absorbency however a s the r a tio ie fu rth e r increased th ere i s a mono to n ic decrease in the in i tie d absorbenoy* I t is proposed th a t the re a c tiv e specie i s a monomerio P e -c itra te oomplex, designated as Pe-Cit* The r e s u lts o lte d above etre c o n siste n t w ith such a speole in eq u ilib riu m w ith both (P e-C it)n and PeC C lt^* Apparently a t a 5:1 r a tio th is specie i s maximised, however the Fig* 17.—The i n i t i a l absorbenoy in the re a c tio n s o f tr a n s f e r r in w ith F e -o itra te a t ra rla b le c itr a te to iro n ra tio s * Conditions and co ncentrations are as noted in F ig . 16* 82 0 .0 4 0.03 O N- > N o c o -Q o CO < 0.02 0.01 0.0 L 0 _L 10 J__________I __________I __________ 20 3 0 4 0 50 Citrate /F e a> u* Qk a d d itio n o f fu rth e r o itr a te favors the fo ra a tio n of F eC C lt^f which does n o t co n trib u te to the I n i t i a l absor benoy • The Bate as a fu n ctio n o f the C itra te to Iron B atio The ra te o f re a c tio n follow ing the I n i t i a l abeor- bency was examined a s a fu n ctio n of the c itr a te tiro n r a ti o . The r e s u lts are presented in F ig . 18. I t w ill be seen th a t between l t l and 20:1 th ere i s am arked enhancement o f the r e a c tiv ity . This i s the region in which th ere i s an eq uilibrium between the (F e-C it)n and the F e(C it)2 « Thus the i n i t i a l r a te s in th is region are p rim arily a measure o f the F e(C it) 2 fra c tio n , since depolym erisation i s r e la tiv e ly slow. The optimum enhancement i s a t a r a tio o f 20 c itr a te s p er iro n . At th is p o in t a l l of the iro n appears as the F e(C it)2* Two hypothesis might be suggested to acoount f o r the in h ib itio n of th e ra te a t o itr a te to iro n r a tio s g re a te r than 20:1. (1) The f e ( C it >2 re a c ts through the form ation of the m onooitrate iro n oomplex and the a d d itio n o f the excess o itr a te s h if ts the equilibrium away from th e re activ e F e-C it specie and toward the u n reaotive F e(C it)2 * (2) At the higher o itr a te to iro n r a tio s the rev erse re a c tio n , th a t i s the removal of iro n from tra n s f e r r in beoomes a meaningful fa c to r. S tudies o f the ra te P ig . 18 •—The i n i t i a l ra te o f tra n s f e r o f iro n fro * F e -c itra te to tr a n s f e r r in as a fu n ctio n o f c itr a te to iro n r a ti o s . The co n cen tratio n of the iro n in m o larity and the binding e q u lr a le n ts A ite r o f th e tra n e fe rr ln is 0.91 z 10 M . C onstant io n ic stre n g th o f 0 .0 2 8 was s a in - tain ed u sin g HsCIOl as a compensating e le c tr o ly te . Other co n d itio n s and methods as noted In t e x t . 85 Rate (A A a th x s e c . 86 2.5 2.0 K o X 0.5 0.0 10 2 0 30 40 50 60 70 Citrate/Fe 87 o f removal of iro n from tra n s f e rrin in d io ate th a t the rev erse re a c tio n would n o t have a s ig n ific a n t e f fe e t on the r a te a t these concentrations* To determ ine i f the e f fe c t o f c itr a te might be on the p ro te in , two co n tro l experiments were c a rrie d out* F e -o itra te was prepared a t a 1>1 r a ti o . J u s t p rio r to re a c tio n w ith the p ro te in , a tw enty-five fo ld excess o f c i t r a t e was added to one a liq u o t o f F e -c itra te and to an o th er, an amount o f NaClO^ c a lc u la te d to give a f in a l ionio stre n g th o f 0*01^ M * I t was found th a t the kQl)8 in the presenoe o f added c itr a te was l*0*t x 10 ^ min* ^ and -1 in the presence o f the n e u tra l s a lt was 0*89 x 10 min* • I t i s apparent th a t the added c itr a te does l i t t l e to enhance the breakdown o f the polymer and has l i t t l e i f any e f f e c t on the r e a c tiv ity o f the p ro te in . Klne^io Study o f thy R eaction o f rerrlo Jloltrate with Transferrin ▲ s e rie s o f so lu tio n s o f F e -o itra te o f varying co n cen tratio n were prepared* The c it r a te to iro n r a tio in a l l oases was 25*1. The ionio stre n g th in the re a c tio n was m aintained a t 0*28 M by the ad d itio n o f HaClO^,* The i n i t i a l ra te o f the re a c tio n w ith tra n s f e r r in as a fu n ctio n of the F e(C it>2 concentration i s shown in Fig* 19* At the higher co n cen tratio n s, the tw enty-five fo ld exoess o f o itr a te causes the rev erse re a c tio n to become a s ig n ific a n t P ig . 1 9 .—The ra te o f tra n s f e r o f iro n from P e (C it) 2 to tra n s f e rrin as a fun ctio n of the concentration o f F e -o itra te a t a molar r a tio o f 25*1 o itr a te tiro n . The J ro te in contained 0.91 z 10“* bindingw equivalente A l t e r , onio stre n g th was m aintained a t 0.28 uy the a d d itio n of MaClO^. 88 Rate ( A A 4 7 0 x sec*1 x I03) 0 2 4 6 8 10 1:25 Ferric citrate (MxlO4 ) a> > © 90 f a c to r in the ra te * At the lo v e r co n cen tratio n s th e r a te ia d ir e c tly dependent on th e P e (C it)2 co n c en tratio n . The ra te dependence on th e tr a n s f e r r in concentra tio n in th e presence o f a o onstant co n cen tratio n o f 25*19 c i t r a t e t i r o n was stu d ie d . The f in a l iro n co n cen tratio n in «4f the re a c tio n m ixture was 0.91 x 10 M. I t w ill he seen in Table k th a t the ra te does n o t vary to a s ig n ific a n t ex ten t as the p ro te in c o n c en tratio n i s v a rie d over a fo u rfo ld range o f binding c a p a c ity . This in d ic a te s th a t th e r a te lim itin g step o f the re a c tio n i s sero o rder w ith re sp e c t to the p ro te in . A pparently in the case o f the P « (C it)2 r a tio as w ell as (P e-C it)n th e ra te lim itin g step involves the conversion of a n o n -re activ e f e r r ic c i t r a t e complex to a re a c tiv e one, p rio r to in te ra c tio n w ith tr a n s f e r r in . In view o f preceding d isc u ssio n s, the most lik e ly p o s s ib ility in th is case would be the form ation o f Pe-C it from Pe(C lt> 2 * I t should be emphasised th a t the ra te o f conver sion o f P e(C it)2 to the a c tiv e oomplex i s approxim ately e ig h t tim es f a s te r than th a t observed f o r the conversion o f the polymer. The E ffe c t o f B icarbonate Ion As discussed in the in tro d u c tio n , the e f fe o t o f nonodentate ions on m u ltid en tate lig a n d exchange re a c tio n e can be considerable (2 1 ). Since bicarbonate ion i s known to be d ire c tly involved in the tr a n s f e r r in binding s i t e 91 TA B LE k T H E REACTION O F FERRIC CITRATE A T A RATIO O F 25 CITRATES PER IRON W ITH V A RY IN G C O N C EN TR A TIO N OF TRANSFEREE (The f in a l fe rrio io n concentration was 0.91 x 10”^ M ) T ra n sfe rrin Conoantration I n i t i a l Rata h Binding Eqi l e n t s A i t e r k . 1 O x 1 0 Binding Equiva- A A ^ ^ q x a a e x 1 0 ° 0.22 5.2 0.»f5 5.0 0.68 k.2 0.91 5.3 92 and i s known to complex f e rrio io n , a study o f the e f f e c t o f HaHCO^ on the k in e tic param eters was made. A . s e rie s o f P e -c itra te so lu tio n s were prepared w ith a f in a l iro n con cen tratio n of 10”^ M and v a ria b le c i t r a t e to iro n ra tio * A 0.1 a l a liq u o t o f the P e -c itra te so lu tio n was added to 1.0 a l o f a tr a n s f e rrin so lu tio n containing — 10 binding eq u iv alen ts A i t e r . The p ro te in so lu tio n con tain ed 0.1 M HaHCO^ and 0*005 M t r i e b u ffe r. The pH was aa in ta in e d a t 7*5* The re a c tio n was followed a t M -7 0 m on a Cary Model 15 spectrophotom eter. I t was found th a t the presence o f the HaH CO ^ enhanced the ra te o f re a c tio n o f the P e(C it)2 complex to an e x te n t th a t i t was f u lly reacted w ith in the te n seconds req u ired to ob tain the f i r s t absorbenoy reading* The ra te o f the polymer re a c tio n was not s ig n ific a n tly a ffe c te d . A comparison o f the k in e tic p a tte rn observed w ith and w ithout the added VaHCO^ i s presented in Table 5* I t w ill be seen th a t th ere i s a d o s e p a r a lle l between the i n i t i a l absorbency in the presence o f H aHCO^ and the com bined i n i t i a l absorbency and interm ediate P e(C it>2 phase in the absence o f the VaHCO^, in d ic a tin g an enhancement o f the P e (C lt) 2 re a c tiv ity * There are e s s e n tia lly two means by whioh the H C O ^ can enhance the r e a c tiv ity of the P e (C it)2 * (1) Through the form ation o f a re a c tiv e oomplex such as P e-C lt (HC0^)n y 93 TA B L E 5 C O M PA R ISO N O F T H E INITIAL A B SO R B E N C Y IN TH E REACTION OF FERRIC CITRATE W ITH TRANSFERRIN IN T H E PRESENCE OF BIC A R B O N A TE ION W ITH C O M B IN ED INITIAL A B SO R B E N C Y A N D T H E INTERM EDIATE PHA SE IN TH E A B SEN C E O F B IC A R B O N A TE A T VARIOUS CITRATE T O IRON RATIOS (The iro n ( I I I ) m olar co n cen tratio n and binding eq u iv alen t A l t e r o f tra n s f e rrin are 0*91 x 10~*7 C itra te /Ira n Combined F ractio n Reacted w ithin Ten Seconds and F ractio n in Interm ediate F e(C it)5 Phase, in Absence “o f N aH C O o (Percentage) F raotlon Reacted w ithin Ten Seconds in Presence of NaHC03 (P ercentage) l s l 20 23 5*1 63 65 10*1 80 78 20*1 100 100 o r (2) by enhancing the ra te a t which the F e(C it) 2 la con v erted in to a re a c tiv e sp e c ie . I t i s n o t fe a sib le to te s t these p o s s ib ilitie s u sin g the techniques a t hand. D iscussions The evidence presented concerning the tr a n s f e r of iro n fro n F e -o itra te complexes to tr a n s f e r r in can be u n i fie d by proposing the follow ing re a c tio n soheae. (F e-C it )n f » _______ > C it-F e-T rf < ■ — * Fe-T rf + C it Both the polymerio fra c tio n , (F e-C it)n , and the d io ltr a te iro n , F e (C it)2 are envisioned as re a o tin g w ith the tra n s f e r r in through the form ation o f a re ao tlv e species, the m onooitrate iro n , F e-C it. This oomplex would be pre sumed to be in equ ilib riu m w ith the polymer and F eC C it^ and fu rth e r would be expeoted to be q u ite re a c tiv e , slnoe th ree o f the co o rd in atio n s ite s o f the iro n are exposed to n u cleo p h ille a tta c k . I t i s also proposed th a t the i n i t i a l absorbency noted in the re a c tio n of F e -o itra te w ith tra n s f e r r in i s due to the re ao tlo n o f the F e-C it th a t i s in eq u ilib riu m a t the time th a t the p ro te in i s added. TJhder co n d itio n s where the F e -c itra te e x is ts p r i m arily as a polymer, th a t is a t a 1*1 r a tio of iro n to o itr a te and a t pH 7 .5 , the re a o tio n i s f i r s t o rd er w ith F e (C it)0 95 re sp e c t to the F e -c itra te and zero o rd er w ith re sp e c t to the protein* This c le a rly in d ic a te s th a t the ra te U n itin g step i s the form ation o f a re a c tiv e oonplex from the poly mer, follow ed by a ra p id re a c tio n w ith tr a n s f e r r in . The tra n s fe r o f iro n from F e(G it)2 to tr a n s f e r r in i s zero order w ith re sp e c t to the protein* The ra te i s dependent on the t o ta l iro n co n cen tratio n , in d ic a tiv e th a t the ra te lim itin g step again involves the conversion to a re a c tiv e specie* The enhancement of the re a c tio n o f F e(C it)2 w ith tra n s f e r r in by high concen tratio n s o f bloarbonate may occur by the displacem ent o f one o f the c itr a te s to form a reao - tiv e oomplex o f the form Cit-Fe-CHCO^ )n . This might re a o t d ire c tly w ith the p ro te in . At ra tio s between 1 and 2 0 tl, c itra te x iro n , the re a c tio n p a tte rn s were c h a ra c te ris tic o f a mixture o f the (F e-C it)n , F e(C it)2 and F e-C it. Eaoh form o f the Fe- o itr a te appeared to re a c t a t i t s own c h a ra c te ris tic rate* Thus the rap id I n i t i a l absorbency of the Fe-C it binding to the p ro te in was follow ed by the interm ediate k in e tic s of the breakdown o f F e (C it)2 to F e-C it, and u ltim a te ly the very slow depolym erization o f (Fe-Cit>n to Fe-C it • CHAPTER 7 FERRIC NTAi ITS N A T U R E A N D TH E LIGAND E X C H A N G E REACTION W ITH TRANSFERRIN The Nature o f FerricN T A t R esu lts and D iscussions Gustafson and M artell (1 9 ), on the baala o f p otentiom etrio t i t r a t i o n d ata found th a t the Fe-NTA forms a monohydroxy sp ec ie . Evidence i s presented th a t th is complex i s involved in the form ation o f dim eric Fe-NTA sp e c ie s. Physical-chem ical stu d ies were n o t reported whioh would dem onstrate the p o ssib le ex isten ce o f Fe-NTA polym ers. In view of the fin d in g o f F e -c itra te polym ers, i t was considered e s s e n tia l to ca rry out sim ila r e x p e ri ments w ith Fe-NTA. Equilibrium d ia ly s is experim ents were o a rrie d out to determine i f th ere was a polymeric fra c tio n . Fe-NTA 59 was prepared w ith and w ithout added tra o e r Fe. The solu tio n s were prepared a t various le v e ls o f added base up to a r a tio of 5 QH/Fe-NTA. I t should be noted th a t th is in clu d es th ree base eq u iv alen ts req u ired to n e u tra lis e the protons o f NTA. I t was shown th a t the d ia ly s is p a tte rn was monophasic over the e n tire range, in d ic a tin g the lack 96 o f a polymeric fra ctio n * At the sane tin e in our la b o ra to rie s Dr. Thomas Spiro studied the p ro p e rtie s o f Fe-NTA on a BioGel P-30 column. The column a o ts as a m olecular sie v e . Complexes of m olecular weight g re a te r than 30,000 are elu te d w ith the void volume. Sm aller molecules are held up to an ex te n t which i s in r e la tio n to t h e i r s is e . I t was found th a t the Fe-NTA complex a t a 1:1 r a tio moved down the c o l umn in a manner c h a ra c te ris tic o f a low m olecular weight complex. The above s tu d ie s , coupled w ith the fin d in g s o f Gustafson and M a rte ll, in d icate th a t the Fe-NTA e x is ts in an eq u ilib riu m between a monomeric and dim eric form, but does n o t fo ra larg e polymerlo complexes. The d iffe ren ce in th e a b ility o f F e -c itra te and Fe-NTA to form polymers may w ell l i e in the th re e fo ld co ordination of the form er and fo u rfo ld coordination of the l a t t e r . ...... from N TA to T ra n sferrin A s e rie s o f Fe-NTA so lu tio n s was prepared a t 10 ^ M and a t ch e la ts to m stal mole r a tio s o f 1 , 10 and 30:1* A 0.1 ml o f Fe-NTA a liq u o t was added to 1*0 a l o f tra n s f e r r in so lu tio n containing 10 binding eq u iv alen ts A l t e r . The tr a n s f e r o f the iro n was found to be completed w ith in the te n seconds required fo r s tir r in g and obtaining the f i r s t 98 read in g . D iffe re n tia l epeetra were obtained ae d escrib ed in the experim ental s e c tio n . The sp eo tra obtained were id e n tlo a l w ith those o f the f e rrio tr a n s f e r r in complex. The molar a b so rp tiv ity a t *t70 m from the t i t r a t i o n o f tr a n s f e r r in w ith Fe-NTA was found to be 0.26 to 0.30 x 10 > a value c o n siste n t w ith th a t found in the l i te r a t u r e (21) and th a t obtained w ith o th e r f e r r ic c h e la te s . This in d i ca te s th a t a l l o f the Fe-NTA i s im mediately re a c tiv e and i s bound a t the normal tra n s f e rrin binding s i t e . Experiments were c a rrie d out to in v e stig a te the e f fe c t o f the OH/Fe r a tio used in preparing the Fe-NTA, on the r e a c tiv ity o f Fe-NTA w ith tr a n s f e r r in . Fe-NTA was p re - -3 pared to a fin a l con cen tratio n o f 10 M and w ith 3*0, lf.0 , 5*0 o r 6.0 base eq u iv alen ts added p er mole o f iro n . A 0.1 ml a liq u o t o f the Fe-NTA was added to 1.0 ml o f tra n s f e r r in containing 10 binding e q u iv a le n ta A ite r. I t was found th a t in each case 100# o f the iro n had been tra n sfe rre d to the p ro te in w ithin the time required f o r the f i r s t absorbency re ad in g . D iscussion The ra p id ity o f the tra n s f e r o f iro n ( I I I ) from N T A to tr a n s f e rrin preoludee a study o f the mechanism o f the re a o tio n from a k in e tic view point using the sp eo tro - photom etrie methods d escribed. Based on a h a lf l i f e o f 30 seconds, whioh i s about h a lf the tin e required fo r the f a s te s t read in g , the minimal f i r s t o rd er ra te co n stan t would be lif.O x min”1 . This i s 10^ tim es the ra te observed fo r the re a c tio n o f Pe-EDTA and tr a n s f e r r in . I t would he expected th a t the Pe-NTA monomer would be q u ite re a c tiv e since th ere are two coord in atio n s i te s on the f e r r ic ion which are not occupied by the c h e la te m olecule, and henoe su scep tib le to n u cleo p h illc a tta c k by the p ro te in . This i s q u ite sim ila r to the case o f the re a c tiv e complex observed w ith P e - c itr a te . I f the Pe-NTA does form dim eric complexes they are e ith e r very re a c tiv e o r in a rap id equ ilib riu m w ith the monomeric form. Por re fe re n c e , a s tru c tu ra l form ula o f Pe-NTA i s included below. CHAPTER 71 FERRIC EDTA: ITS N A T U R E A N D TH E LIGAN D E X C H A N G E REACTION W ITH TRANSFERRIN R esults and D iscussions: The N ature o f T s rr£ e % !l!l------- G ustafson and M artell (19) havs in v e stig a te d the h y d ro ly sis o f Fe-EDTA and re la te d f e r r ic ch e la te complexes. P otentiom etric t i t r a t i o n s showed the re le a se o f one proton authors provide evidence fo r the form ation of a dimerio complex and suggest th e stru ctu re in which f e r r ic ions a re linked by two hydroxyl b rid g es. A p o in t, im portant to th e stu d ies o f the re a c tiv ity o f Fe-EDTA, i s the concentration dependence o f the dim eriza- tio n p ro cess. I t is ty p ic a l o f suoh eq u ilib riu m th a t poly m erisatio n i s favored by increases o f t o t a l co n cen tratio n . Although the work o f Gustafson and M artell sug gested the absence of high m olecular weight polymers o f Fe-EDTA, no p h y slcal-ch en lcal stu d ies had been rep o rted to -1 - 2 from Fe-EDTA leading to a Fe(OH)EDTA complex. The O H / \ O H 100 101 v e rify th is p o in t. The stu d ie s rep o rted below e s ta b lis h th a t Fe-EDTA does n o t form high m olecular weight polym ers. E quilibrium d ia ly s is experim ents were c a rrie d out a s desoribed in the experim ental methods c h a p te r. Fe-EDTA was prepared a t mole r a tio s o f 1*1 and 5*1, EDTAtFe. FeCNO^)^ was added dropwise to a s o lu tio n o f EDTA in 0.005 M t r i s b u ffe r. The pH was m aintained a t 5*5 to 6 .5 by the sim ultaneous ad d itio n o f d ilu te HaOH. A fter the re q u is ite amount of iro n had been added the pH was taken to 7 .5 . The f in a l iro n co n cen tratio n was 10 3 M . An id e n tic a l s e t 59 o f so lu tio n s was prepared u sin g tr a c e r amounts o f Fe. The r e s u lts o f the d ia ly s is experim ents fo r th e 1*1 p re p a ra tio n are presented in F ig . 20. I t w ill be seen th a t the d ia ly s is p a tte rn i s monopbaslc over the e n tire range, in d ic a tin g th a t th ere i s no slowly e q u ilib ra tin g polymerio sp e c ie . The d ia ly s is ra te co n stan t wae determined as 2 jf x 10 3 min. 1 fo r both r a tio s stu d ie d . This value corresponds c lo se ly to th a t found f o r the f e r r io d lo ltr a te _0 .1 complex, 3*1 x 10 J m in. , suggesting th a t the Fe-EDTA i s approxim ately the same else as the f e r r io d i c it r a te . The physical c h a ra c te r is tic s o f Fe-EDTA were studied by chromatography on a Bio-Gel P-30 column. The experim ents, done in co lla b o ra tio n w ith Dr. Thomas S p iro , pexm ltted the d e te c tio n of any polymer fra o tio n g re a te r than 30,000 m olecular w eight. Hone was d e te c te d , i t was Fig* 20*— The d ia ly s is p a tte rn o f a 1*1 prepara tio n o f Fe-EDTA. The f in a l co n cen tratio n o f Fe-EDTA was 10"5 H. The pH was m aintained a t 7.5 w ith 0.005 M t r i e b u ffe r. The ca lc u la te d f i r s t o rd er ra te oonstant f o r d ia ly s is was 2.*t x 1 0“3 m in."1 . 102 Fraction to Equilibrate 8 l(b found th a t the Fe-EDTA moved e n tire ly as a low m olecular w eight complex. P relim inary in v e stig a tio n o f the c e n trifu g a l p ro p e rtie s o f Fe-EDTA was made w ith the a n a ly tic a l u l t r a c e n trifu g e by Dr s . A llerto n and Saltman and Mr. Benner. There was no evidence f o r the form ation of high m olecular weight polymers o f the type found fo r F e -c itra te . The above work, ooupled w ith the stu d ie s o f Gustafson and M artell (19) show conclusively th a t high m olecular weight polymers o f Fe-EDTA are n o t formed, but th a t h y d ro ly sis o f Fe-EDTA lead s to the form ation o f dim eric complexes. S p ectral fin d in g s are in agreement w ith th ese fin d in g s. The absorbency a t **70 over a tw entyfold range o f Fe-EDTA co ncentration i s p lo tte d in F ig . 21. The n o n -lin e a rity in d ic a te s th a t B eer's law i s not follow ed, due in a l l p ro b a b ility to the monomer-diner e q u ilib riu m . A pparently a t **70 mu, the dimer has the higher m olar a b s o rp tiv ity , leading to the p o sitiv e devia tio n in the B eer's law p lo t. The k in e tic s o f the tra n s f e r o f iro n ( I I I ) from ED TA to tr a n s f e rrin must be considered in te rn s o f the chemical nature o f the Fe-EDTA oomplex and the oonoentra- tio n dependent d im erizatio n p ro cess. For reference a s tru c tu ra l form ula fo r Fe-EDTA i s included on the follow ing page 107. P ig, 21,—A B eer's law p lo t fo r Pe-EDTA prepared a t a mole 1*1 r a t i o . The n o n -lin e a rity in d ic a te s an eq u ilib riu m between a t leaB t two Fe-EDTA complexes. The s e rie s was prepared in 0.005 M t r i s b u ffe r and m aintained a t pH 7 ,5 . 105 Fe-EDTA ( M x 10s) Abtorboncy 4 7 0 mp 901 107 / ° ° \ O CH, ,C O — CH J J f ^ V I /V 7 i co— ch; CH, I CH, CH, R e s u l ts and D is c u s s io n s * The T ra n w ia r o f I r o n ( I I I ) fro m EDTA t o T r a n s f e r r i n Time Courae of the R eaction The time oourae of the tr a n s f e r o f iro n ( I I I ) from ED TA to tr a n s f e rrin was studied* Fe-EDTA was prepared a t a 111 r a t i o , as described in the preceding section* The f in a l concentration was 10~^ M * A 0.1 ml a liq u o t o f the Fe-EDTA so lu tio n was added to 1*0 ml o f tra n s f e rrin con- -4 ta in in g 10 binding eq u iv alen ts A l t e r . The m ixture was s tir r e d and the re a c tio n followed sp ectrophotom etrieally a t *f70 mu. In Fig* 22 i s presented the k in e tic s the f i r s t fiv e days, during which time the re a c tio n has reached approxim ately 65£ completion* Longer stu d ie s were p re vented by d enaturation o f the p ro te in a t 25°• I t w ill be seen th a t the re ao tio n follow s a b iphasic p a tte n * The f i r s t phase, c o n sistin g o f about 5 % o f the to ta l ab so r- benoe, ocours w ithin the ten seconds req u ired fo r F ig . 22.—The tin e course o f the tr a n s f e r o f iro n ( I I I ) from EDTA to tr a n s f e r r in . The co n o an tratio n o f the Fe-EDTA in m olarity and the binding e q u iv a le n ts /lite r o f the tr a n s f e r r in was 0.91 x 10“^ . The pH was m aintained a t 7 .5 w ith 0.005 M t r i e b u ffe r. The f ra c tio n of th e re a c tio n completed i s based on the absorbency a t **70 bm> 108 i r i 1 r i 3 Days S 110 o b tain in g the f i r s t read in g . The second phase o f the re a c tio n i s com paratively q u ite slow. The d ata are re p lo tte d in f i r s t order form in Fig* 23. The lin e a r ity observed in d ic a te s th a t the seoond phase o f the re a c tio n follow s f i r s t o rder k in e tic s . The -3 -1 ra te co n stan t is 0.12 i 10 min. • I n i t i a l Absorbency S tudies The ex ten t o f th e f i r s t phase of the re a c tio n , which w ill be re fe rre d to as the i n i t i a l absorbency, was stu d ied as a funotion o f Fe-EDTA and p ro te in concentrations. I t was noted th a t a t th e time of mixing the 9S-E0TA and the p ro te in , which are them selves c o lo rle s s , the so lu tio n became a d is tin c t v io le t c o lo r. T his co lo r ohanged g rad u ally over a number of hours to the salmon pink c o lo r c h a ra c te r is tic o f the F e -tra n s fe rrin complex. D iffe re n tia l sp e c tra were obtained a t v arious tim es in the re a c tio n o f Fe-EDTA and tr a n s f e r r in . A Cary Model 15 spectrophotom eter equipped w ith tandem c e ll hold e r s was employed. In th e reference compartment the Fe-EDTA and tra n s f e rrin so lu tio n s were kept in separate c u v e tte s . In the sample compartment the re a c ta n ts were mixed in the same cu v ette and th e seoond cuvette was f i l l e d w ith t r i s b u ffe r. The Fe-EDTA m o larity and th e tra n s f e rrin binding eq u iv alen ts A l t e r were 10 in both compartments. The v is ib le sp ec tra obtained a t v ario u s tim es F ig . 23•—A f i r s t o rd er p re se n ta tio n o f the tin e course o f the tr a n s f e r o f iro n ( I I I ) from ED TA to tra n s f e r r i n . The co n d itio n s are as noted in F ig . 2 *f and the t e x t . The observed f i r s t o rder r a te co n stan t was calcu la te d to be 0*12 x 10“^ n i n . '1 . The p lo t was co n stru cted as th e f ra c tio n o f the tr a n s f e r r in s i t e s u n f ille d on the lo g arith m ic scale a s a fu n ctio n o f t ia e on the lin e a r s c a le . Ill Days Fraction Unreacted 112 113 during the re a c tio n are presented in F ig . 2 M -. The speotrum runs im m ediately a f t e r the a d d itio n of the re a c ta n ts has a v is ib le maximum a t 515 to 520 n*i. A fter *t.5 hours, the peak has s h ifte d to *+90 mu and a f t e r 18 hours the spectrum c h a ra c te r is tic of th e F e -tra n s fe rrin complex, w ith a M -7 0 m u maximum i s observed. The to ta l blue s h if t i s on the o rd er o f 50 mu. These sp e c tra c le a rly in d ic a te the ex isten ce o f an in term ed iate f e r r ic complex in th e tra n s f e r o f iro n (111) from ED TA to tr a n s f e r r in . The n atu re o f the complex cannot be determined by sp e c tra l means, however i t seems lik e ly th a t the interm ediate i s o f the form EDTA-Fe-TRF. The dependence o f the i n i t i a l absorbency on the p ro te in and Fe-EDTA co n cen tratio n was in v e stig a te d . Fe-EDTA was prepared w ith the f in a l concen tratio n s varying over a tw en ty -fiv efo ld range. The method o f p re p aratio n was the same as o u tlin e d above. In th is s e t o f experim ents a 0.85 ml a liq u o t o f each Fe-EDTA so lu tio n was added to 0.25 ml o f tra n s f e r r in containing b x 10 binding e q u iv alen ts A l t e r tr a n s f e r r in . In th is way the d ilu tio n fa c to r o f the Fe-EDTA was minimized. The i n i t i a l absor bency o f the re a c tio n m ixture obtained a t 520 mu, and c o rre c te d fo r the absorbency o f the p ro te in and Fe-EDTA i s a hyperbolic fu n ctio n o f the Fe-EDTA co n cen tratio n , as i s shown in F ig . 25* Two hypotheses might account fo r the hyperbolio Pig* 2*tv—V isib le sp ec tra a t v ario u s tim es in the tr a n s f e r o f iro n ( I I I ) from ED TA to tr a n s f e r r in . The molar co n cen tratio n o f the Fe-EDTA and th e binding e q u iv a le n ts / l i t e r o f the tra n s f e rrin were 10~3. The ourve d esig n atio n s r e f e r to the time elapsed since the beginning o f the re ac tio n . Absorboncy 0 .7 OJB 0.5H 4.5 0 .3 h 0. 2 0 . 1 0.0< 420 540 620 Wavelength (mjj) ti M l Pig* 2 5 .—The i n i t i a l absorbency a t 520 in the re a c tio n o f Pe-EMA w ith tra n s fe rrin as a function o f the co n cen tratio n of PerEDTA. The tr a n s f e r r in was held con s ta n t a t 0*91 x 10“^ binding eq u iv alen ts A lte r* The spectrophotom etrie techniques involved are discussed in the experim ental methods se c tio n . 116 Fe-EDTA ( M x I04 ) Absorboncy 520 mji U 118 re la tio n s h ip ! (1) the fu n ctio n re p re se n ts a s a tu ra tio n eq u ilib riu m o f the ty p e , Fe-EDTA + t r f EDTA-Fe-trf, (2) the hyperbolic curve i s a re fle c tio n of the concentra tio n dependent eq u ilib riu m 2 Fe-EDTA^ (Fe-EDTA^, the form er being the re a c tiv e sp ec ie . As the co n cen tratio n of the Fe-EDTA i s ra is e d , the fra c tio n e x is tin g as the monomer becomes le s s , thus a hyperbolic re la tio n s h ip . The question was p a r tia lly resolved by examining the i n i t i a l absorbency as a function o f the p ro te in con c e n tra tio n over a tw entyfold range, while the Fe-EDTA con- -L c e n tra tio n was m aintained constant a t 1.82 x 10 M. The r e s u lts are presented in F ig . 26. As in the case o f the v a ria b le Fe-EDTA, the i n i t i a l absorbency i s a hyperbolic fu n ctio n o f the p ro te in binding o ap a city . This r e s u lt supports the concept o f an equilibrium between a te rn a ry in term ediate complex, unbound p ro te in , and Fe-EDTA. A double re c ip ro c a l p lo t o f the d a ta from F ig . 26 i s presented in F ig. 27. From th is p lo t a molar a b so rp tiv i t y o f the interm ediate a t 520 m u was determined as 1 .6 x 10^ and the apparent binding constant was ca lc u la te d to be 1.3 x 10~^ M . I t should be noted th a t the v a lid ity o f these values i s dependent on the amount o f Fe-EDTA a v a il able f o r interm ediate form ation. Two hypotheses were presented above to aooount fo r the hyperbolic re la tio n s h ip o f the i n i t i a l absorbency and F ig . 2 6 .--The i n i t i a l absorbency a t 520 m u in the re a c tio n o f tr a n s f e rrin and Fe-EDTA a s a fu n ctio n o f the binding eq uivalents of the p ro tein .^.T h e Fe-EDTA concentre tio n was held constant a t 1.82 z 10 M . The p ro te in was v aried over a tv en ty fo ld range. 119 Absorbancy 520 my 0.14 0.02 0.10 0 .0 8 0 .06 0 .0 4 0.02 0.0 0 2 4 6 8 10 Transferrin Binding Equivalents (M x I04 ) 120 f i g . 27.—A double re c ip ro c a l p lo t o f the d a ta p resented in f i g . 2 6 . from t h i s p lo t the n o la r ab so rp tiv i t y a t 520 m fo r the in term ed iate was determ ined a t 1 .6 x 109 . This Talue i s based on th e assumption th a t a l l o f the fe-EDTA i s a v a ila b le f o r te rn a ry ooaplez form ation. An a g g jre n t eq u ilib riu m co n stan t was determ ined as 1.3 x 121 (Absorbancy 520 m y )' 70 6 0 4 0 3 0 20 (Transferrin Binding Equivalents (M x lO9))**1 122 123 Fe-EDTA oonoentration • These vara a s a tu ra tio n eq u ilib riu m o f tha type fa-EMA + T rf ■ B U T A -Fe-Trf and the oonoentra- tio n dependant d in e rle a tlo n o f the Fe-BDTA. Both o f theae fa o to re have been shown to be v alid * The o v e ra ll hyper b o lic fu n ctio n then i s probably a r e s u lt o f both o f th en being o p e ra tiv e . Hate Dependence S tudies The ra te o f the second phase o f the re a c tio n was stu d ied a s a fu n c tio n o f p ro te in and Fe-BDTA co n cen tratio n s, A s e rie s o f Fe-BDTA so lu tio n s was prepared as desoribed previously* The f in a l co n cen tratio n s were v a rie d over a tw en ty -fiv efo ld ran g e. A 0*85 u l a liq u o t o f eaeh Fe-BDTA e o lu tio n was added to 0*25 a l o f tr a n s f e r r in co n tain in g I, 10 binding e q u lv a le n ts A ite r* The re a c tio n wae follow ed e p e o tro p h o to n e trio a lly a t M -7 0 eu f o r fiv e hours* The d ata were p lo tte d aooordlng to f i r s t o rd er k ln e tle s as the lo g o f the fra o tlo n o f the tr a n s f e r r in s i t e s u n f ille d as a fu n ctio n o f tin e* The observed f i r s t o rd er ra te o o n sten t was determ ined f o r eaoh oonoentration o f Ve-BDTA end i s p resen ted in Fig* 28* The lin e a r re la tio n s h ip in d ic a te s th a t the r a te U n itin g ste p o f the re a c tio n i s f i r s t o rd er w ith re ep eo t to the Fe-EDTA oonoentration* I t i s o f i n t e r e s t to co n sid er th ese r e s u lts in li g h t o f th e Fe-BDTA nononer-diner equilibrium * As has been d iscu ssed , a t h ig h e r iro n c o n c en tratio n s th e f ra c tio n Tig* 28.—The observed f i r s t o rd e r ra te 0 one ta n t obtained fo r a s e rie s o f Te-EDTA so lu tio n s as a fu n c tio n o f the concentrations* The r a te co n stan ts were obtained from p lo ts o f th e form shown in Tig* 23* The binding e q u lv a le n ts A ite r o f th e tra n s f e r r in in. th e f in a l re a o tio n m ixture was held o o n stan t a t 0*91 x 10“* . In th ese expe rim ents* the Te-EDTA was added in a volume o f 0*85 ml to a p ro te in so lu tio n o f 0*25 ml* The purpose o f t h i s was to minimise the Fe-BDTA d ilu tio n facto r* 25 20- 0 5 10 1 5 2 0 2 5 Fe-EDTA (MxlO4) 126 o f dim er in c re a s e s . Thus the lin e a r ity observed in f i g . 28 o re r a tw en ty -fiv efo ld range o f Fe-BDTA oonoentration m a t be in te rp re te d in term s o f (1) an eq u iv alen t r e a c tiv ity o f • , the d in e r and the monomer, o r (2) a r a te U n itin g ste p that! i s independent o f th e nononer-diner eq u ilib riu m . In a s im ila r s e t o f experim ents, th e fe-EDTA was all m aintained co n stan t a t 1 . 8 2 x 10 M w hile th e tr a n s f e r r in binding cap acity was v a rie d o re r a tw entyfold ran g e. The i n i t i a l r a te o f the seoond phase o f th e re a c tio n was d e te r mined o re r th e f i r s t 300 m inutes a f t e r m ixing. The r e s u lts a re p resen ted in f i g . 29* A hyperbolic fu n c tio n , charac t e r i s t i c o f a s a tu ra tio n eq u ilib riu m i s e x h ib ite d . A double re c ip ro c a l p lo t o f th ie d a ta i s presented in fig * 30. from th is p lo t, an apparent eq u ilib riu m oonatant — N " was determ ined as 2.8 x 10 N. The diacrepanoy noted in the apparent eq u ilib riu m co n sta n t from f i g . 27 (13*0 x 10~** M ) and th a t obtained ab o re, suggests a d iffe re n c e In the fe-EDTA oom positlon a t the tim e o f mixing and a t th e steady s ta te during the re a c tio n . E o m ally i t would be expected th a t th e eq u ilib riu m o o n stan t determ ined fo r complex form ation and fo r the r a te o f th e re a o tio n would be th e same. These r e s u lts nay be considered in term s o f th e follow ing h y p o th esis. In o rd er fo r th e fa-EDIA to re a o t w ith tr a n s f e r r in , the o h elate mast f i r s t p a r tia lly u nfold from the f e r r i c io n . This r e s u lts RLg* 29.—The i n i t i a l ra ta o f the tr a n s f e r o f iro n ( I I I ) from EMA to tr a n s f e r r in . The r a te was d e te r mined from the absorbeney obangs a t *f70 m u o re r the f i r s t 300 m inutes, i The Fe-EDTA o o noentration was held co n stan t a t 1 . 8 2 x 10“* M . 127 R ot* (AA 4 7 0 k mln.-i x 10*) 2 4 6 8 Transferrin Binding Equivalents (M x I04 ) F ig . 30*—A double re c ip ro c a l p lo t o f th e d a ta shown in F ig . 29* The apparent eq u ilib riu m co n stan t fo r the s a tu ra tio n fu n c tio n involved was determ ined as 2,&x 10 M. The maximal r a ts was determ ined a s l.*f x 10“* absorbency u n its p e r m inute. 129 (Rot* (AA4 7 0 x min.- ' x I04) ) “ ' 0 4 8 12 16 2 0 (Tronsferrin Binding Equivalents (MxlO8))’ 1 O f o 131 I n th e f o r m a tio n o f a l a b i l e f e r r i c c o m p le x , Fe-EDTA t h a t l a a u a c e p t i b l e t o n u o l e o p h l l i o a t t a c k by th e p r o t e i n * A t t h e t i n e o f a d d i t i o n o f t h e p r o t e i n a p o r t i o n o f th e Fe-EDTA i e i n e q u i l i b r i u m w ith th e o t h e r fo rm e o f Fe-BDTA, a n d r e a o t a r a p i d l y w i t h t h e p r o t e i n t o g lY e t h e i n i t i a l a b a o rb e n o y * The l o v e r e q u i l i b r i u m c o n s t a n t d e te rm in e d f o r t h e r a t e e x p e r l n e n t a r e f l e c t s t h e lo w e re d am ount o f le-BDTA i n th e s te a d y e t a t e i n o o m p a rie o n t o th e am ount p r e s e n t a t t h e tim e o f a d d i t i o n o f t h e p r o t e i n * A r e l a t e d s tu d y v a a made i n w h ic h th e r a t e o f t h e r e a c t i o n v a a o o n p a re d t o t h e I n i t i a l a b a o rb e n o y f o r a a e r i e s o f Fe-EDTA c o n c e n t r a t i o n s a t a c o n s t a n t t r a n s f e r r i n - i . oonoentration of 0*91 x 10 binding eq u iv alen ts A lte r * The o o m p a rie o n i s show n i n F ig * 31* * h e *m te 1* M e n t o l n o r e a s e o u t o f p r o p o r t i o n to t h e i n i t i a l a b a o rb e n o y , e m p h a s is in g th e d i f f e r e n c e i n th e c o n d i t i o n s a t th e tim e o f m ix in g an d a t t h e s t e a d y s t a t e * V iew ed i n te rm s o f th e monomer d im e r e q u i l i b r i u m , t h e s e r e s u l t s s u g g e s t t h a t t h e d im e r f o r m a tio n c a u s e s a g r e a t e r i n h i b i t i o n o f th e i n i t i a l a b a o rb e n o y th a n i t d o e s o f th e r a t e o f se e o n d p h a se o f th e r e a c t i o n * Ih » C a t a l y t i c E f f e c t o f B io a rb o n a tc Fe-EDTA w as p r e p a r e d w ith a f i n a l o o n o e n tr a ti o n o f l ( f 3 M fro m FsCVO^)^ a n d d is o d iu m SDTA* The pH was a d j u s t e d to 7*5 by t h e a d d i t i o n o f s o l i d VaHCO^* The f i n a l Fig* 3 1 .—Tli* i n i t i a l r a ta o f the tr a n s f e r o f iro n (111) fro n BETA to tr a n s f e r r in a s a fu n c tio n o f the i n i t i a l absorbenoy a t ^70 m . In th is , a e rie s the tra n s f e r r i n was held co n stan t a t 0*91 x 10 bindi ng eq u iv alen ts A l t e r . The Fe-EDTA oonoentration was v a rie d . 132 Rot«(AA470 x min.-i x I04 ) 0 .0 0 0.01 0 . 0 2 005 0.06 Absortoancy 470 mjj M u> u> I3h c o n c en tratio n o f b icarbonate was about 0.05 M . A ll so lu tio n s were made up in 0.005 M t r i s b u ffe r. A 0.1 ml a l l - _k quot o f the Fe-EDTA was added to 1 .0 ml o f 10 N tr a n s f e r r i n . The re a c tio n was follow ed sp ectro p h o to m etrlcally a t if70 mu. The d a ta f i t f i r s t o rd er k in e tic s w ith an - O - T observed f i r s t o rd er r a te co n stan t o f 0*85 x 10 min, , approxim ately e ig h t tim es the ra te observed in the absence o f the added b icarb o n ate. In experim ents s im ila r to those described b e fo re , d iffe re n c e sp ec tra were obtained a t v ario u s tim es during th e re a c tio n o f the NaHCO^ n e u tra liz e d Fe-EDTA w ith tra n s f e r r i n . The 520 m |i peak and the v io le t c o lo r noted p re v io u sly was m issing. The only spectrum obtained was th a t o f the f e r r ic - tr a n s f e r r in complex. In another experim ent, Fe-EDTA which had been neu tr a liz e d w ith N aO H was added to tr a n s f e r r in . As u su al the 520 mi peak was g en erated . To th is s o lu tio n was added enough NaHCO^ to b rin g the co n cen tratio n to 0.1 M . I t was found th a t whereas the normal time f o r the peak to s h i f t to *f70 mu i s 20 h o u rs, the presence o f NaHCO^ ac celera te d the process so th a t only 1 .5 hours were re q u ire d . As was discussed e a r l i e r , the 520 m u peak i s ap p aren tly due to an interm ed iate te rn a ry oomplex. The n atu re o f the oomplex i s unknown, however i s p o stu la te d to be o f th e fo ra EDTA-Fe-Trf • I t i s apparent th a t the 135 bicarbonate ion i s a c tin g to e ith e r prevent the form ation o f th e in term ed iate, a l t e r i t s sp e c tra l p ro p e rtie s o r cause i t to break down much more ra p id ly . The c a ta ly tic e f f e c t o f the bioarbonate suggests the l a s t i s a reasonable p o ssi b i l i t y . Proposal Of The r e s u lts and d iscu ssio n s presented in the pre ceding sec tio n w ill be summarized by the proposal o f a re a c tio n scheme fo r the tra n s f e r o f iro n ( I I I ) from EDTA to tr a n s f e r r in . (Fe-EDTA)g « 2 Fe-EDTA (Fe-EDTA)2 - 2 Fe-EDTA* Fe-EDTA « Fe-EDTA* Fe-EDTA* ♦ T rf « EDTA-Fe-Trf EDTA-Fe-Trf « Fe-T rf ♦ ED TA In th is scheme the dimer (Fe-EDTA)2 and the # monomer, Fe-EDTA, give r is e to a re a c tiv e complex Fe-EDTA in which the cordinate s ite o f iro n are p a r tia lly exposed. The Fe-EDTA* rap id ly e q u ilib ra te s w ith the p ro te in to form an interm ediate complex in which the iro n i s held by both the amino acid ligands o f the p ro te in and the ED TA c h e la te . The EDTA i s then displaced from the interm ediate w ith the concom itant form ation o f the product, f e r r ic tr a n s f e r r in . The involvement o f p a r tia lly unfolded Fe-EDTA 136 complexes in lig an d exohange re a c tio n s has heen proposed toy Margerum, e t a l . (10) and Basolo and Pearson (6 ). EDTA, a hexadentate c h e la tin g ag e n t, i s able to com pletely surround the f e r r i c ion so th a t i t i s w ell sh ield ed from nucleo - p h ilic a tta c k . The p a r tia l unwrapping o f the ohelate exposes the iro n , making i t much more re a c tiv e . At the time o f a d d itio n of th e p ro te in a c e rta in fra o tio n o f the iro n i s able to re a c t ra p id ly w ith the pro** te in to form the in term ed iate, d isp lay in g the 520 mu peak. I t is suggested th a t th is i s due to a fra c tio n of the iro n * e x is tin g in a p a r tia lly exposed complex, the Fe-BDTA • Both the monomer and the dimer are shown in an * eq u ilib riu m w ith the Fe-EDTA • I f the ra te o f the second phase o f the re a c tio n were a ffe c te d by the d im e risa tio n , a hyperbolic r e la tio n o f ra te and the Fe-EDTA co n cen tratio n would be expected. Since the ra te o f the second phase o f the re a c tio n i s apparently u n affected by the p o sitio n of the monomer-dimer eq u ilib riu m , the re a c tiv e specie must be provided by both complexes and a t approxim ately the same r a te • In comparison w ith fe rrio c i t r a t e , the e x te n t to which the Fe-EDTA forms a condensation complex has l i t t l e e f f e c t on the ra te o f the re a c tio n . Both f e r r ic c h e la te s re q u ire the generatio n o f a re a c tiv e specie which then re a o ts w ith the p ro te in . However, in the oase o f the Fe-EDTA, the ch elate binde the iro n tig h tly enough th a t a tru e equilibrium ie e e t up between the interm ediate and the r e a c ta n ts . Thus i t i s not p ossible to determine the reac tiv e fra c tio n as was the case w ith f e r r ic c i t r a t e . CHAPTER 711 TH E R E M O V A L OF IRON ( I I I ) F R O M TRANSFERRIN B7 CH ELA TIN G A G E N TS The k in e tic s o f removal o f iro n ( I I I ) from tra n s f e r r in by c i t r a t e , E D T A . and N T A was in v e s tig a te d • Fe- tr a n s f e rrin was prepared as described in the experim ental section* The stock so lu tio n was d ilu te d so th a t the absorbency a t if 70 m u was 0*10* This corresponds to a con- c e n tra tlo n o f 0.^ x 10 binding e q u lv a le n tsA ite r* To 1*0 ml o f the tra n s f e rrin was added 0*1 ml o f a 1.0 M ch elate solution* The mixture was s tir r e d and the re a c tio n was followed spectrophotom etrically a t *f70 mu. The re s u lts are presented in Fig* 32* The th re e c h e la te s , c itr a te EDTA, and N T A re a c t a t d iffe re n t r a te s and reach d iffe re n t e q u ilib ria which are dependent on th e ir binding a f f in itie s * The d ata are re p lo tte d in Fig* 33 as the fra c tio n of th e to ta l absorbency change as a fu n ctio n o f time* I t can be seen th a t the c itr a te and N T A reaoh equilibrium a t approxim ately the same time* EDTA, on the o th er hand, req u ires a longer time fo r eq u ilib ratio n * The v a r ia b ility in the r a te s a t which the ch e la tin g agents 138 F ig. 3 2 .—The tin e course o f the removal o f iro n from tra n s f e rrin by various ch elatin g agents. The f in a l concentration o f iro n in the re a c tio n mixture was 0.36 x 10**^ M . The ch e la te concentration in each case was 0.091 N. 139 Abtorbancy 470 mp < H | I Fig* 3 3 .—The f ra c tio n of eq u ilib riu m a tta in e d as a fu n c tio n o f tim e. The d a ta were taken from F ig . 3^. I k l f • - Fraction Equilibrated o p p f — f ? 2*C i b y remove th e iro n suggests th a t the r a te lim itin g ste p in th e re a c tio n meohanlsm may be the form ation o f an interm ed iate oomplex, C hel-P e-T rf, and the subsequent removal o f the iro n from the p ro te in . The removal o f th e iro n ( I I I ) from tr a n s f e r r in by ED TA was found to follow pseudo - f i r s t o rd er k in e tic s a t these co n cen tratio n le v e ls . The dependence o f the ra te o f removal o f iro n from tr a n s f e r r in was stu d ied as a fu n ctio n o f ED TA and Pe- tr a n s f e r r in co n c en tratio n . A s e rie s o f re actio n s were c a rrie d o u t w ith a co n stan t P e -tra n s fe rrin co ncentration o f 0.36 x 1 0 ^ binding eq u iv alen ts A l t e r and v aria b le ED TA c o n c en tratio n . The lo n io stre n g th was m aintained co n stan t a t 0.27M w ith JTaClG^. The r e s u lts , in P ig . 3*S show a lin e a r re la tio n s h ip between the ra te o f the re a c tio n and the EDTA co n c en tratio n . This in d ic a te s th a t the r a te lim itin g step o f the re a o tio n i s f i r s t o rd e r w ith resp eo t to th e EDTA co n c en tratio n . In a sim ila r s e t o f experim ents the ED TA concentra tio n was m aintained a t 0.091 M and the P e -tra n s fe rrln con c e n tra tio n was v a rie d . The i n i t i a l r a te o f re a o tio n as a fu n ctio n o f the P e -tra n s fe rrin i s p resen ted in P ig . 35. I t w ill be seen th a t the re la tio n i s l i n e a r , in d ic a tin g the re a o tio n i s f i r s t o rd er w ith re sp e c t to the P e -tra n s fe rrln . The sim plest sohene whioh i s c o n siste n t w ith these d ata i s shown below. F ig. 3 ^ .—Hate o f removal o f Iro n from iro n tra n s f e r r in by ED TA as a fu n ctio n o f th e EDTA co n c en tratio n . The iro n -tra n s fe rr in con cen tratio n in these re a c tio n s was 0.36 x 10"* binding e q u iv a le n ts A ite r • The co n cen tratio n o f the EDTA w as-varied as shown. The pH was m aintained a t 7 .5 w ith 0.005 M t r i s b u ffe r. The io n lo stre n g th was 0.28 H throughout th e s e r ie s . NeClO^ was used as the com p ensating e le c tr o ly te . 0 . 0 0 . 0 1 0 . 0 2 EDTA I I 0 .0 5 H • r s n Tig* 3 5 .—Tii« i n i t i a l r a ta o f th e renoval o f iro n from tr a n s f e r r in as a fu n ctio n o f iro n tr a n s f e r r in oonoen- t r a t io n . The EDTA co n c en tratio n in th ese re ao tlo n e was 0.091 M. The iro n p ro te in co n c en tratio n as M atured sp e o tro p h o to a e tric a lly was v a rie d over the range shown. The pH was B aintained a t 7*5 w ith 0.005 M T rie b u ffe r. 0 z 3 4 FeTransferrin (N x I05) lMJ BDTA-le-Trf Ve-EDIA ♦ T rf I t appears th a t ED TA and F e -tra n s fe rrin fo ra an in te ra e d i- a te te rn a ry ooaplez in which the iro n i a held by both the EDTA and th e p ro te in . The in te ra e d ia te can rev erse to fo ra the F e -tra n s fe rrin o r th e p ro te in can re le a s e the iron; to fo ra th e p ro d u cts, Fe-EDTA and iro n fre e tr a n s f e r r in . The ra te o f th e removal i s then a fu n ctio n o f the c h e la te 's a b i li ty to fo ra the in te ra e d ia te complex and to fin d the iro n tig h tly enough to e f fe o t the removal fro a the tra n s f e r r in m olecule. fb -T rf + EDTA EDTA-Fe-Trf CHAPTER Till GBHERAL DISCUSSIOH The prim ary observ atio n s which gave im petus and I n te r e s t to the th e s is researoh were th e d if f e r e n tia l r a te s a t whloh the v ario u s c h e la te s re le a se d th e ir Iro n to tra n s fe rrin * In Pig* 36 i t w ill be seen t h a t the o h e la te s e x h ib it a wide spectrum o f r e a c t iv it y . The Pe-HTA re a c ts w ithin s ix seconds | th e P e -c itra te re q u ire s some tw enty hours and the Pe-EDTA, a number o f days* The r a te o f re a o tio n w ith the Pe-HTA i s a t le a s t 10^ tin e s th a t o f the Pe-EDTA and p o ssib ly much more* In o rd er to understand th is phenomenon we must f i r s t oon sid er th e ro le o f tr a n s f e r r in in accepting iro n from a c h e la tin g agent* With a l l o f th e o h elates te s te d , th ere i s a fra o tlo n o f th e iro n w ith whloh the p ro te in i s able to re a o t q u ite re ad ily * In th e oase o f Pe-HTA, a l l o f the iro n i s re ao tiv e * Experim ents w ith Pe-EDTA and P e -o itra te show th a t approxim ately 5 to 2 0 % o f the t o t a l iro n r e a c ts immediately* The o b serv atio n th a t the tr a n s f e r r in i s an extrem ely e f f io ie n t n u cleo p h ile in a tta e k in g la b ile iro n oomplexes forms a b a sis f o r our understanding F ig . 3 6 . —The tin e o o q tn o f the tr a n s f e r o f iro n ( I I I ) f r o s EDTA| ETA and o ltr a te to th e p ro te in tr a n s f e r r i n . The re a c tio n i s follow ed sp e c tro p h o to se trio a lly a t b70 m i, the ab so rp tio n a a rlau n o f the F e -tra n s fe rrin ooap lez. The s o la r oonoentratlon o f f e r r lo o h sla te and the bin d in g eq u iv alen ts A l t e r o f tr a n s f e r r in were 0.91 z 10“* in a l l o ase s. Absorbancy 4 7 0 mp 0.25 NTA 0 2 0 — Citrate 0.15 EDTA 2 0 Hours the d if f e r e n tia l r a te s o f re a o tio n fo r the v arious f e r r i c complexes tested * The re a c tiv ity o f the f e r r ic complexes w ith tr a n s f e r r in i s a m an ifestatio n o f the ra te a t whloh the f e r r i c complexes them selves form la b ile iro n species* This was shown q u ite c le a rly in the case o f the F e -c itra te , where both the polymer and the d ic i t r a t e iro n complex were converted to a re a c tiv e m onooltrate iro n complex before in te ra c tio n w ith th e p rotein* In the case o f the Fe-NTA, i t appears th a t a l l o f the iro n i s q u ite la b ile to a tta c k by the tr a n s f e r r in m oleoule. The chenioal n atu re o f the Fe-NTA may be q u a lita tiv e ly s im ila r to th a t o f the Fe- c i t r a t e monomeric complex* The Fe-EDTA a lso e x h ib its a ra p id ly re a c tin g fra c tio n a t the time th a t the p ro te in i s added to th e iro n complex* In th is case however th ere i s the a d d itio n a l fa c to r o f the form ation o f a reasonably sta b le te rn a ry complex* The ED TA competes e f f ic ie n tly enough f o r the iro n th a t a tru e eq u ilib riu m i s estab lish ed ; Fe-EDTA + T rf ; » EDTA-Fe-Trf * I t was proposed th a t the fra c tio n o f the Pe-EDTA th a t re a c ts immediately w ith the p ro te in c o n s is ts o f complexes in which the ED TA che la te i s p a r tia lly unfolded from the f e r r ic ion* I t i s lik e ly th a t the e f f io le n t nucleophilio c h a ra c te r o f the p ro te in i s due in p a rt to the two h is tid y l groups known to p a r tic ip a te in the iro n binding s ite * The im idazole residue has o fte n been im plicated as an e f fic ie n t 153 nucleophile in bioorganie M mechanisms (5 6 ). I t i s now p o ssib le to advance reasonable explana tio n s fo r b io lo g ic a l observations concerning the metabolism o f f e r r ic c h e la te s discussed in the intro d u ctio n * The ra te o f tr a n s in te s tin a l flu x o f the f e r r ic c h e la te s i s c lo se ly re la te d to th e form ation o f polym eric sp e c ie s. The Fe-EDTA and the Fe-NTA which do not form polymers are able to ra p id ly cro ss the g u t w all, while the F e -c itra te and the F e-fru cto se which a re predom inantly polym eric are slow er to be absorbed. The most lik e ly explanation o f the d iffe re n c e observed in the organ and tis s u e d ep o sitio n o f the iro n complexed to the EDTA and N TA l i e s in the r e a c tiv ity o f the f e r r ic complex w ith sp e c ific c e ll tra n sp o rt systems as w ell as th e tr a n s f e r r in binding s i t e s . The Fe-NTA which e n te rs the blood stream would be expected to im mediately e q u ilib ra te w ith tr a n s f e r r in which would then tra n s p o rt the iro n to re tic u lo c y te s o r depot tis s u e s in a normal p h y sio lo g ical manner. The Fe-EDTA ra p id ly passes through the in te s tin a l w a ll, i s unable to re le a se the iro n to the o e lls o r tr a n s f e r r in and in ste a d , passes through the kidney tubules and i s found in the bladder o r u rin e . In co n clu sio n , we have stu d ied a re p re se n ta tiv e sample of f e r r ic o h e la te s from sev e ral p o in ts o f view to a s c e rta in th e ir chemlo&l and physical n a tu re . These p ro p e rtie s have been re la te d to the lig an d exchange re a c tio n w ith h u m a n tr a n s f e r r in and the mechanism by which the re a c tio n takes p lao e. In tu rn , the b io lo g ic a l u t i l i s a b l l - i t y o f the iro n o h elates oan be d ire c tly c o rre la te d w ith the chem ical and p hysical p ro p e rtie s o f the iro n as mediated by the n atu re o f the c h e la tin g ag en t. CHAPTER IX SUMMARY T ra n sfe rrin , a g lo b u lin , a c ts to tra n sp o rt and d ep o sit iro n in ph y sio lo g ical system s. The d is s e rta tio n research is concerned w ith the exchange o f iro n between tra n s f e r r in and the c h e la tin g agents c i t r a t e , eth y len ed i- am in etetraao etic a c id , EDTA, and n i t r i lo tr ia c e tl o a c id , N T A. I t was necessary to study the chemioal n atu re o f the f e r r ic complexes o f th ese ch elatin g agents in o rd er to e lu c id a te the mechanism by which the iro n i s tra n sfe rre d to the p ro te in . At a r a tio of l t l , c i tr a te iir o n , polymers of Fe* c i t r a t e , (Fe-C it )n , are formed. D ialy sis techniques demonstrated th a t the high m olecular weight polymer was in equilibrium w ith low m olecular w eight complexes. At a 20*1, c itra te * iro n ra tio n the iro n e x is ts predom inantly as a low m olecular weight complex, te n ta tiv e ly id e n tifie d as f e rrio d ic i t r a t e , F e-C it2 * At interm ediate r a tio s an equilibrium between (F e-C it)n , F e -C it, and F e-C it2 e x is ts . Both the (F e-C it)n and F e-C it2 re a c t w ith tra n s f e r r in v ia the form ation of a highly re a c tiv e fe rrio 155 156 complex, probably a monomeric F e-C it, The re a c tio n of the (F e-C it)n has a h a lf l i f e of approxim ately 10 hours, the F e-C it2 » 10 m inutes, and the F e-C it, le s e than 5 seconds. Fe-EDTA and Fe-NTA do n o t form polymeric complexes. The l a t t e r re a c ts com pletely w ith tra n s f e r r in w ith in 5 seconds, while the form er req u ires more than a week. D irect sp e c tra l evidence fo r the form ation o f an in t e r mediate EDTA-Fe-transferrIn complex is p resented. I t would appear th a t both Fe-NTA and F e -c itra te also form such an Interm ediate since i t i e u n lik e ly th a t Fe^+ i s p resen t in an appreciable co n cen tratio n . The removal of iro n from tr a n s f e rrin by c i t r a t e , N TA and ED TA was stu d ie d . The c itr a te and N TA reaoh eq u ilib riu m a f te r approxim ately two hours; the ED TA how e v e r, re q u ire s twelve hours. Various asp ects of f e r r ic ion metabolism, in clu d ing i t s tra n sp o rt and u t ili z a t io n , are discussed in lig h t o f the fin d in g s re p o rte d . LI TERATURE CI TED LITERATURE CITED 1. B e u tle r, E .. F airbanks, V. F ., and Fahey, J . L .. in "C lin ic a l D isorders of Iro n M etabolism," Grune & S tr a tto n , New York, (1963). 2. E ichhom , G. L ., in "Iro n M etabolism." CIBA Symposium, S pringer-V erlag, B erlin , (19#+), pp. 9. 3 . S a lt man, P ., J . Chem. E d ., k g , 682 (1965). *+ , K atz, J . H .. and Ja n d l, J . H ., in "Iron M etabolism," CIBA Symposium, S pringer-V erlag, B e rlin , (196*0, pp. 103. 5. P ollycove, M., in "Iro n M etabolism," CIBA S y m p o s i u m , S pringer-V erlag, B erlin , (196*+), pp. l*+8. 6. Graddon, D. P .. "An In tro d u ctio n to C oordination C hem istry," Pergamon P re ss, New York, (1961). 7. Dwyer, F. P ., and M ellor, D. P ., "C helating Agents and Metal C h elates," Academic P re ss, New York, (1961), p p . 18. 8. B asolo, P ., and P earson. R. G., "Mechanisms o f Inorganic R eactions," Jo h n Wiley and Sons, I n c ., New York, (1958), pp. 91. 9 . Marge rum, D. W., James, D. L ., and Rosen, H. M., J . Am. Chem. S o c., 82, * + * + 6 3 (196?). 10. Margerum, D. W., Zabin. B. A ., and James, D. L ., In o rg . Chem., 2 , 250 (1966). 11. Hedstrom, B. 0. A ., Arkiv fo r Kemi, 6 , 1 (1953)* 12. Malay, L. N ., and Selwood, P. W., J . Am. Chem. S oo., 2&, 6207 (195*+). 13* S p iro , T. G ., A lle rto n , S. E ., Renner, J . , T e r ris , A ., B ile , R ., and Saltman, P ., J . Am. Chem. S o c., 8fi, £721 (1966). 158 159 l*+« Aasa, R ,, Malmstrom, B ., Saltman, P ., and Vanngard, T., Biochim. e t Biophys. A cta, §£, ^30 (19&+). 15* B obtelaky, M.t and Jordan, J . , J* Am. Chem. S oc., 69, 2286 a * ? ) . 16. Lanford, 0 . E ., and Qulnan, J . R ., J . Am . Chem. S oc., 2Q, 2900 ( 1 9 W . 17. Vo8burg, W . C ., and Cooper, G. R ., J . Am . Chem. S oc., 61, W (19^1). 18. Warner, R. C ., and Weber, I . , J . Am. Chem. S o c., 75, 5086 (1953). 19# G ustafson, R. L ., and M a rte ll, A. E ., J . Phys. Chem., 62, 576 (1963). 20. V allee , B. L ., Adv. P ro tein Chem., ^0, 317 (1955). 21. Malms trom , B. G ., and Rosenberg, A ., Adv. in Enzymol, s i , 131 (1955). 22. Putnam, P. W . in "The P ro te in s," N eurath, H ., E d ., Vol. I l l , Academio P re ss, New York, New York, pp. 211. 23. L a u re ll, C. B. in "The Plasma P ro te in s," Vol. 1, Putnam, P . W«, E d ., Academio P ress, New York (I960), pp. 3*9. 2*+. Malms trom , B. G ., and N eilands, J . B ., "Annual Review o f B iochem istry," 31* 331 (19o*t). 25. Gurd, P. R. N ., and Wilcox, P. E ., "Advan. P ro t. Chem. / l i , 311 (1956). 26. L a u re ll, C. B ., Pharmacol, R evs., 371 (1952). 27. Barkan, G., Z tsch . fu r p h y sio l. Chem., 171. 19^ (19271 28. Barkan, G., Z tsch. fu r p h y sio l. Chem., 216. 1 (1933). 29. Holmberg, G. C ., and L a u re ll. C. B ., Acta P h y sio l. Soand., 1£, 307 (19^5). 30. So hade, A. L ., and C aroline, L ., Science, 10M -. 3^0 (19^ 6). 31. Surgenor, P .. S trong, L ., T aylor, H ., Gordon, R. J r . , "•* *• S oc., 21* 1 2 2 3 160 32. K oechlin, B ,, J . Am• Chem. S o c., £+, 26*+9 (1952). 33. One le y , J . L ., Sc a t chard, G ., and Brovn, A ., J . Phys. C olloid Chem., & , 18*+ (19*+7). 3*+. Schade, A. L ., R einhart, R. W., and Levy, H ., Arch. Bloohem., .gg, 170 (19*+9). 35. Holmberg, C. G ., and L a u re ll, C. B., Acta Chem. Scand., 1 , 9 * + * + (19*+8). 36. Schonenberger, M. Z ., N a tu rf., 10, * + 7 * + (1955). 37. L a u re ll, C. B ., and Ingelman, B ., Acta Chem. Scand., 1 , 770 (19*+7). 38. S ch u ltze, H. E ., Heide, K ., and M uller, H ., Behrlngtrerk, M itteilungen, 32$ 25 (1957). 39* D reyfus, J . C ., and Schaplro, G ., B u ll. Soc. Chem. B io l., 3 2 , 5^1 (1955). *+0. Bezkorovainy. A ., R afelson, J r . , M . £ . . and S ik h lte . V ., Arch. Blochem. ana B lophye., 103, 371 (1 9 6 3 ). *+1. Charlvood, P. A ., Bloohem. J . , 88, 39*+ (1963). if2. Bezkorovainy. A ., and R afelson, M . E ., Arch. Blochem. and B iophys., 107. 302 (196*0. *f3. A zari, P. R ., and Feeney, R. E ., J . B io l. Chem., 232. 293 (1958). Mt. A zari, P. R ., and Feeney, R. E ., Arch. Bloohim. B iophys., 2g, M + (1961). 1+5. S ch u ltz, H. £ ., Schm idtberger, R ., and Haupt, H ., Blochem. Q e itsc h ., 329. *+ 90 (1958). *f6. Jam ieson, G. A ., J . B io l. Chem., g*+0, 2 91* + (1965). *+7. B uttkus, H ., C lark , J . R .t and Feeney, R. E ., Blochem., *+ , 998 (1965)• *+8. W indle, J . J . , Wierama, A. K ., C lark. J . R.« and Feeney, R. E ., Bloohem., £ , 3 * + l (1963). *+9. A ssa, R ., Malms trom , B. G ., Saltm an, P ., Vanngard, T ., Biochim. e t Biophys. A cta, 203 (1963). 1 6 1 50. P ark in s, D. J . , and Jones, H. D. C ., Biochim a t Biophys A cta, l<j)£, 122 (196$). 51. V a lle s, B. L«, and Ulmer, 3). D ., Blochem. and Biophys. Res. Comm., £ , 331 (1962). 52. Hopkins, J r . , L. L ., and Sohwars, K ., Biochim. e t Biophys. A cta, (196^5. 53* Warner, R. C ., and Weber, 1 ., J . Am. Chem. S o c., 75, 5 0 * (1953). 5*+. A isen, P ., J . B io l. Chem., 1666 (1965). 55. Lindakog, S ., and Malmstrom, B. 0 ., J . B io l. Chem., i22» H28 (1962). 56. Ingraham, L. L ., in "Biochemical Mechanisms," John Wiley and Sons, I n c ., New York (1962), pp. M-l. This dissertation has been microfilmed exactly as received 67-6489 BATES, George Winston, 1939- THE FERRIC COMPLEXES OF CITRATE, EDTA AND NT A: THEIR NATURE AND THE LIGAND EXCHANGE REACTION WITH HUMAN TRANSFERRIN. University of Southern California, Ph.D., 1967 Chemistry, biological University Microfilms, Inc., Ann Arbor, Michigan

Asset Metadata

Creator Bates, George Winston, 1939- (author)

Core Title The ferric complexes of citrate, ETDA and NTA: their nature and the ligand exchange reaction with human transferrin

Contributor Digitized by ProQuest (provenance)

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1967-01

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Advisor Saltman, Paul (committee chair), Allerton, Samuel E. (committee member), Fife, Thomas H. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-120407

Unique identifier UC11360786

Identifier 6706489.pdf (filename),usctheses-c18-120407 (legacy record id)

Legacy Identifier 6706489.pdf

Dmrecord 120407

Document Type Dissertation

Rights Bates, George Winston

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA