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Disulfide exchange derivatives of human hair keratins

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Disulfide exchange derivatives of human hair keratins

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Content DISULFIDE EXCHANGE DERIVATIVES OF HUMAN HAIR KERATINS by Richard Joseph Schlesinger A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry) June 1969 UNIVERSITY O F SOU TH ERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA S 0 0 0 7 This dissertation, written by .............EicJtiar.d...jQS.eplx..5.Qbleging.er..... under the direction of Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Gradu ate School, in partial fulfillment of require ments for the degree of D O C T O R O F P H I L O S O P H Y Ju^j..jL969 DISSERTATION COMMITTEE ACKNOWLEDGEMENTS The report of this work would be incomplete without a sincere expression of appreciation for the help given by Dr. Earl McNall, my adviser. I also wish to thank Dr. Lucien A. Bavetta and Dr. Norman Kharasch for their cooperation and assistance as members of my dissertation committee and Dr. John Mehl for his encouragement and support as my adviser during the early part of my graduate studies. Grateful appreciation goes to Cary Instrument Co. for the use of their latest instruments and to Dr. Thomas Fife for the use of his Programma 101 desk top computer. TABLE OF CONTENTS Page LIST OF TABLES ...................................... v LIST OF ILLUSTRATIONS .............................. vi INTRODUCTION Definition of Keratins Soft and Hard Keratins Alpha and Beta Keratins Keratin Fiber Histology Cuticle Cortex Preparation and Isolation of Soluble Derivatives Reductive Reactions Oxidative Reactions Fractionation of Keratin Derivatives Characterization of Derivatives Electrophoresis Molecular Weight Amino Acid Analyses • - Infrared Spectrophotometry Conformation STATEMENT OF PROBLEM............................... 22 EXPERIMENTAL APPROACH .............................. 24 MATERIALS AND METHODS ........................ 26 Hair Samples Reagents Preparation of Hair Samples Solubilization of Keratin by Disulfide Exchange Determination of Thiol Determination of pH Determination of Solubilized Protein Kinetic Studies Experiments Varying Hair Concentration Peptides Released by 6 M Urea Reexchange Experiments iii Page Disc Electrophoresis Fractionation of Derivatives Chromatography Characterization of Fractions Molecular Weight Stokes Radius Determination Ultraviolet Absorptivity and Spectro photometry Moisture Content Infrared Spectrophotometry Nitrogen Analyses Amino Acid Analyses Optical Rotatory Dispersion Circular Dichroism RESULTS .............................................. 51 Kinetics of The Keratin DTDGA Exchange Reaction Fractionation of Derivatives Initial Fractionation Molecular Sieve Chromatography Additional Experiments on the Disulfide Exchange Reaction Effect of Hair Concentration Six Molar Urea Extracts Reexchange and Urea Fraction Exchange Influence of Temperature Characterization of Derivatives Disc Electrophoresis Effect of Varying Pore Size Molecular Weight by Molecular Sieve Chromatography Stokes Radius from Molecular Sieve Chromatography Ultraviolet Spectrophotometry and Absorptivity Infrared Spectrophotometry Nitrogen, Sulfur and Amino Acid Analyses Optical Rotatory Dispersion Circular Dichroism, DISCUSSION 122 SUMMARY 139 REFERENCES CITED 143 iv LIST OF TABLES Table Page I. Amino Acid Composition of Hair, Wool And Keratin Derivatives ....................... 15 II. Infrared Assignments for Wool, Carboxymethyl Keratins And Hair Keratin DTDGA Exchange Derivatives .-............................... 18 III. First And Second Order Rate Constants For The Hair Keratin DTDGA Disulfide Exchange Reaction .................................... 52 IV. Initial And Final Conditions Of The 1% Keratin DTDGA Exchange Reaction ....... 63 V. Properties And Yields Of Fractions In Various Steps In The Isolation Of Keratin Disulfide Exchange Derivatives ...................... 87 VI. Summary Of The Number Of Bands And Migration Distances In The Keratin DTDGA Reexchange Experiments In Disc Electrophoresis ...... 89 VII. Molecular Weights Of Keratin DTDGA Fractions From Molecular Sieve Chromatography ...... 95 VIII. Stokes Radius Determination Of Peaks Of Keratin DTDGA Exchange Derivatives From Molecular Sieve Chromatography .............. 96 IX. Summary Of Chemical And Physical Properties Of Principal Peaks Of Keratin DTDGA Exchange Fractions From Sephadex Chromatography ... 101 X. Far UV Cotton Effect Characteristics From Optical Rotatory Dispersion Data of Principal Peaks Of Keratin DTDGA Exchange Derivatives ................................ 113 XI. Optical Rotatory Dispersion Constants For Keratin-DTDGA Exchange Derivatives ....... 114 XII, Circular Dichroism Of Principal Peaks Of Keratin-DTDGA Exchange .................... 120 v LIST OF ILLUSTRATIONS Figure Page 1. Molecular weights estimated from Sephadex G-100 chromatography ........................ 37 2. Molecular weights estimated from Sephadex G-200 chromatography ........................ 39 3. Molecular weights estimated from Bio Gel P-100 chromatography ........................ 41 4. Molecular weights estimated from Bio Gel P-300 chromatography ........................ 43 5. Kinetics of keratin disulfide exchange with equivalent ratios of TA:DTDGA .............. 53 6 . Kinetics of keratin disulfide exchange with 1:10 ratios of TA:DTDGA .................... 55 7. Kinetics of keratin disulfide exchange with 1:50 ratios of TA: DTDGA .............. 57 8 . Kinetics of keratin disulfide exchange with 1:100 ratios of TA:DTDGA ................... 59 9. Kinetics of keratin disulfide exchange with 1:1000 ratios of TA:DTDGA .............. 61 10. Decay of thiol concentration for the keratin DTDGA exchange reactions ................... 65 11. Keratin solubilization as a function of reagent concentration ................... 67 12. Fractionation scheme in the isolation of keratin DTDGA exchange derivatives ........ 69 13. Disc electrophoresis densitometer tracings of fractions from reexchange experiments with DTDGA .............................77____ 73 14. Chromatography of acid insoluble exchange derivatives from 1% hair on Sephadex G-100 . 75 15. Chromatography of acid insoluble exchange derivatives from 1% hair on Sephadex G-200 . 77 vi Page 79 L 81 83 85 90 99 keratin DTDGA fraction ..................... 102 23. Infrared spectrum of 120,000 MW acid insol uble keratin DTDGA fraction ................ 105 24. Far ultraviolet optical rotatory dispersion of the 6,000 MW acid soluble keratin DTDGA fraction in various solvents ............... 108 25. Far ultraviolet optical rotatory dispersion of the 120,000 MW acid insoluble keratin DTDGA fraction in various solvents ........ Ill 26. Ultraviolet circular dichroism of the 6,000 MW acid soluble keratin fraction in var ious solvents ............................... 116 27. Ultraviolet circular dichroism of the 120,000 MW acid insoluble keratin DTDGA fraction in various solvents ............................ 118 Figure 16. Chromatography of acid soluble exchange de rivatives from 1% hair on Sephadex G-100 .. 17. Chromatography of acid insoluble exchange derivatives from 5% hair on Bio Gel P-100 . 18. Chromatography of acid insoluble exchange derivatives from 5% hair on Bio Gel P-300 . 19. Chromatography of acid soluble fraction from 5% hair on Bio Gel P-100 ............. 20. Chromatography of urea extract of 1% hair on Bio Gel P-100 ............................ 21. Ultraviolet absorption spectra of disulfide exchange derivatives and cystine .......... 22. Infrared spectrum of 6,000 MW acid soluble vii INTRODUCTION Definition of Keratins Keratins are fibrous proteins, crosslinked by cystine and occur principally in the horny layers of the epidermis and related appendages such as horns, hooves, scale, hair and feathers (40). Block and Bolling defined keratins on the basis of enzymatic susceptibility and sol ubility properties as follows: "Keratins are proteins, which in their native state are resistant to digestion by pepsin and trypsin and are insoluble in dilute acids, alkali, water and organic solvents” (28). Several clas sifications of keratins are defined. Soft And Hard Keratins These categories can be distinguished on the basis of physical properties, histology and chemical com position (39). The outer layer of the epidermis is an example of soft keratins. Horn, nail and claw are typical examples of hard keratins. Hair, wool, feathers and nail are principally composed of hard keratins, but are asso ciated with soft keratins in the central canal and the eponychium (35). 1 Alpha And Beta Keratins There are two additional major chemical classifi cations based on the fact that keratins give alpha or beta x-ray diffraction patterns (18). Keratin fibers usually exhibit extensibility i.e. the ability to undergo revers ible stretching on treatment with heat or alkali. In the contracted or alpha state, the molecule may be half the length of the extended or beta form. The configuration may also be induced by crystallization from appropriate solvents e.g. m-cresol or dichloracetic acid yields the alpha form and formic acid the beta (56). Fiber diagrams of both the alpha and beta forms have equatorial reflec tions which correspond to crystal spacings at 4.7 A° and 10.0 A°, the exact value of the latter is dependent upon the density of the side chains. Both equatorial reflec tions are perpendicular to the fiber axis. Stretching the alpha form to produce the beta form does not alter the equatorial reflections, but the meridional (vertical) re flections at 5.1 A° and 1.5 A° of the alpha form are re placed by 3.3 A° and 1.1 A° reflections in the beta state (18). Proteins in the epidermal hard parts of reptiles and birds have beta x-ray characteristics, but mammalian keratins are mostly of the alpha type. The medulla, when present at the center of some animal hairs, shows the beta pattern, so that both the alpha and the beta forms are sometimes present, in the same keratinized structure. Hair cuticle presents a special situation. In addition to the alpha characteristics given by the cortex, a very insoluble and highly crosslinked amorphous keratin is present. This is sometimes referred to as a gamma com ponent (39). Keratin Fiber Histology Cuticle Hair or wool fibers are surrounded by a flattened cuticle membrane made up of scale cells. The cuticle consists of two distinct layers; a keratinous outer layer termed the exocuticle and a non-keratinous inner layer derived from cytoplasmic debris, termed the endocuticle. In coarse fibers the cuticle may be many scale cells thick. Where the cells overlap they are separated by an intercellular cement deposited between the cell membranes (40). Cortex The central or main portion of hair fibers from all species is made up of consolidated spindle shaped cell residues which constitute the macrofibrils. Electron microscopy has been used to elucidate structural details of the cortex. The interior is a keratinized mass of fibrillar aggregates, the macrofibrils, which run parallel to the length of the cell and are made up of microfibrils, about 60-70 A° in diameter and about 100 A° apart, center to center. The microfibrils are embedded in more or less regular arrays in a non-fibrous protein matrix and are arranged in whorls in the macrofibrils of the ortho seg ment. These whorls are not usually found in the macro fibrils of the para segment which has regions with nearly regular hexagonal packing (38). Electron microscopy and x-ray diffraction evi dence suggest the presence of fibrillar subunits in wool and hair, termed protofibrils, that are made up of three protein chains in the form of modified alpha helices (51). Rogers’ electron microscope evidence shows a structure of nine protofibrils surrounding a central pair (2 0). Preparation And Isolation of Soluble Derivatives The earliest methods used to solubilize hard keratins employed concentrated acids and bases. Many of the more recent studies still employ pH’s lower than 2.0 or higher than 11.0 (25). These extremes in pH cause random bond cleavage (42). Aqueous extractions of wool or hair yielded remarkably little solubilized protein. Matoltsky obtained small quantities of water soluble peptides from hair subjected to grinding, which migrated as two separate bands in disc electrophoresis (41). 5 The most useful methods of extracting keratins involved those reagents which selectively cleave disulfide bonds. These methods produced substantial yields of soluble materials. Keratins have been solubilized by many oxidizing and reducing reagents as well as by electrolytic reduction (13). Reductive Reactions Solubility of wool, feather, hoof and hair kera tins by sulfide under various conditions of concentration and temperature, involves the formation of reduced pro tein and polysulfides as follows: K-S-S-K + 2Na2S + 2H20 2KSH + Na2S2 + 2NaOH 1/ In the case of wool 70% was dissolved in 0.1 M sodium sulfide in three hours at 30°C. Nearly all of this was precipitated by acid. The dissolving of keratins by sulfide is complicated by the existence of at least two categories of disulfide bonds which are cleaved at different rates. These groups were revealed by the de crease in acid precipitable material and the change in the character of the precipitate when preparations were made with longer times of treatment or higher concentra tions of sulfide (33). -/k-S-S-K refers to keratin in its native state in the hair. Subsequent use of K refers to keratin moiety of peptide chain. 6 Alkaline cyanides react with cystine residues in proteins to form a reduced protein-half cystine and a protein-thiocyanate. Cyanide dissolved smaller amounts of keratin than thioglycollate or sulfides, unless high alkalinity was used (29). In addition to cleaving cys tine, cyanide has also been effective for converting combined cystine to combined lanthionine (3, Bdi-thio di-alanine) making the residue resistant to solubilization with other reducing agents (13). Soluble keratin derivatives, prepared by alkaline thioglycollate reduction followed by alkylation, were pioneered by Goddard and Michaelis (29). Some of the reduced keratin derivatives were studied in the thiol state, but alkylation yielded stable derivatives. Reduced wool was converted to the S-ethylamino, S-benzyl, S-carboxyphenylmercuric, S-carbamidomethyl or S-carboxy- methyl derivatives. The S-carboxymethyl keratins were usually obtained by reaction of reduced keratins with iodoacetate. Since the pK of the sulfhydryl group was greater than 9.1, these reactions were generally performed at an alkaline pH, usually between 9.0 and 11.0 (30). The reduced carboxymethyl keratin derivatives proved to be the most satisfactory derivatives of reduced keratins studied until now (13). Acrylonitrile derivatives were prepared from thioglycollated reduced wool keratins. Bartulovich and 7 Ward obtained reproducible preparations of 30,000 M W from cortical cell residues. These corresponded to the low sulfur fractions obtained by other methods. In their procedure the low molecular weight, high sulfur fraction was lost by dialysis (6). Reduction in neutral solutions was applied to the solubilization of keratin from a number of sources. Jones and Mecham obtained solubilization in the amount of 28% for wool keratin and 40% for human hair keratin, which can be compared to over 80% for the solubilization of these two keratins in alkaline thioglycollate (34). When' wool proteins were reduced in neutral solutions, then alkylated and extracted at pH 11.0, the soluble proteins extracted in this way were equivalent to about 75% by weight of the original wool (37). Electrolytic reduction of wool keratins at pH 7.0 and 0.075 M mercaptoethanol was performed by Leach. The mercury cathode was maintained at -1.3 volts, using an automatic potentiostat. The progress of reduction was followed by amperometric titration. Since no derivatives were prepared, it was necessary to retain the keratins in a reducing environment, otherwise oxidation to insoluble disulfides occurred (37). The borohydride reduction of the disulfide bonds of wool, bovine and human plasma albumin was studied by Gillespie (21). When this reagent was applied at pH values of 7 to 10, about 30% of the proteins became dialysable, which indicated considerable peptide bond hydrolysis. Reductive fission of disulfide bonds of wool keratins was first employed by Jones and Mecham (34). The amounts of proteins extracted from various keratins with 0.3 M NaHSO^, 10 M urea solutions during 18 hours at 40°C were 80% for chicken feathers, 50% for wool and only 5% for ovokeratin (33). The fission of disulfide bonds of keratin by means of sulfite takes place by an asymetric mechanism and can be represented by the reaction K-S-S-K + SO" t Ks” + K-S-S0~ More recent work has been concerned with techniques to cause complete fission of the cystine bonds as a means of solubilizing keratins, since earlier work indicated that some disulfide bonds varied in their susceptibility to cleavage by sulfite (13). Oxidative Reactions Complete reaction of all of the disulfide bonds by sulfite in wool was accomplished by two methods. The first method involved the use of mixtures of sulfite and a mercurial to immediately convert the thiol formed to a mercaptide. The second method, which purported to react ; with all the disulfide bonds present, was oxidative in nature. Several mechanisms have been proposed for this reaction. Generally it is thought to occur in two stages; first the fission of the disulfide bond by sulfite to produce a thiol and S-sulfocysteine, followed by oxida tion of the thiol to disulfide, which again reacts with sulfite. The overall reaction is the conversion of disulfide to S-sulfocysteine (48). The oxidants used were cupric ions, iodobenzoate, tetrathionate or atmos pheric oxygen. Symmetrical fission of the disulfide bonds was achieved when the action of sulfite was supple mented by one of these oxidants. Under these conditions each disulfide bond yielded two S-sulfocysteine groups. The S-sulpho derivatives of the wool proteins, formed by the use of either cupric ions or tetrathionate, were also extracted with slightly alkaline solutions (61). Oxidation of wool keratins with aqueous, chlorine free, chlorine dioxide was described by Das and Speakman. This reagent dissolved 40% in 120 hours and an additional 47% upon subsequent aqueous treatment. The residue contained 1.3% sulfur compared to 5.3% for the dissolved portion (15). Chlorine dioxide does not attack the peptide bonds of such peptides as leucylglycine and most others, but is known from its action on silk, which does not contain disulfide bonds, to labilize the peptide bonds adjoining tyrosine or proline residues. Wool treated with chlorine dioxide undergoes a series of color changes that are believed to be related to the oxidation of tyrosine, as well as other changes in amino acid 10 composition. The cystine is largely oxidized to cysteic acid. Alexander and Earland prepared oxidized keratin derivatives using peracetic acid. They found that after 24 hours at room temperature, in a 1.6% solution of peracetic acid, little or none of the wool dissolved. Subsequent treatment of the oxidized wool with 0.1 N ammonium hydroxide, however, dissolved 90-92% of the original wool (3). Peracetic and performic acids, as well as chlorine dioxide, bromine and chlorine, can convert cystine to cysteic acid and intermediate oxida tion products, which on acid hydrolysis decompose to give cysteine and cysteic acid. Amino acid analyses on partially oxidized keratins are complicated by these reactions and therefore are somewhat unreliable. The exact oxidation pathway is a function of the nature of the oxidizing agent and the conditions under which it is used (4). Fractionation of Keratin Derivatives All of the keratin derivatives have been fractionated into two classes on the basis of the contents of sulfur containing amino acids. Successive extractions of the reduced carboxymethyl keratin deriv atives with potassium thioglycollate at pH 10.5 removed most of the high sulfur and high glycine proteins from the fiber. Subsequent extraction at pH 11.3 yielded a 11 solution containing mostly low sulfur proteins. The Gillespie preparation of low sulfur proteins was separated into high and low sulfur fractions by precipitation at pH 6.0 with zinc acetate or acidification to pH 4.4 in the presence of 0.3-0.5 M KC1 (23). The principal low and high sulfur containing oxidized keratin derivatives were separated by Alexander and Earland (3). The low sulfur, oxidized derivatives were precipitable by acidifying to below pH 3.0. This fraction had the alpha x-ray configuration and contained 1.6% sulfur. It was electrophoretically homogenous with a M W 'of 70,000 and accounted for 50 to 60% of the original wool. The high sulfur fraction was recovered only in the beta x-ray form and was electrophoretically polydisperse with a M W of 4,500. It contained 6% sulfur and accounted for 35% of the original wool (4). Characterization of Derivatives Electrophoresis Woods prepared the S-sulpho keratin and the S-carboxymethyl derivatives from wool and compared the starch gel electrophoretic properties of the acid insol uble derivatives. Both were found to have four principal components and possible additional minor components (61). Using starch gel electrophoresis, Thompson and 0*Donnell also obtained five slow moving bands in addition to traces of faster moving components from their wool carboxymethyl, acid insoluble, keratin preparation. These authors interpret their results to indicate two principal diff erent proteins. They obtained three smaller, slow moving bands and traces of faster moving components. They considered the slower moving bands to be aggregates of the two principal proteins and the faster components were thought to be traces of contaminating high glycine and high sulfur proteins (57). As many as eight frac tions have been associated with reduced acid soluble car boxymethyl keratin derivatives from electrophoresis and chromatography on DEAE cellulose (25). Molecular Weight Aggregated molecular weights.ranging from 200,000 to 1 ,000,000 were obtained by sedimentation and light scattering for the low sulfur reduced carboxymethyl keratin derivatives from wool (24,32). When 8 M urea, 5 M guanidine or 14 M formamide were used to disaggregate the low sulfur reduced carboxymethyl derivatives, a M W of 45,000 was obtained from the elution volumes on Sephadex G-200 (58). Similar values were obtained from the corresponding fraction of oxidized derivatives (45). DeDeurwarder and Harrap studied low sulfur reduced carboxymethyl keratin derivatives in 14 M formamide and obtained a molecular weight of a stable polymer of 13 70,000 from sedimentation studies. This polymer dis aggregated on dilution and these authors assign a molecular weight of 35,000 to the subunit (17). The problem of aggregation observed with the low sulfur proteins has not been found to be troublesome in the case of the high sulfur proteins. The acetylated keratin derivatives in these fractions are soluble and do not aggregate over a much wider pH range than those in the low sulfur groups. Ultracentrifuge, sedimentation measurements and data from light scattering experiments give molecular weights of 27,000 and 22,000 for the first two reduced carboxymethyl keratin fractions respectively (25, 26). Amino Acid Analyses Analyses of skin, hair, fibers, feathers, horn, tusk and quill have been covered in several recent reviews (1,. 13, 39, 40, 59). A comparison of the amino acid composition of Merino wool, human hair and some derivatives is given in Table I. Similarities exist between Merino wool and hair, but noticeable differences occur in more than six amino acids. The amount of 1/2 cystine residues in human hair is notably greater than that found in Merino wool (Table I). This is consistent with the fact that human hair is a harder keratin than Merino wool. The difference in the basic 14 amino acid, arginine is approximately three times greater in human hair than in Merino wool. This trend is reversed for the other amino acids, histidine and lysine, which are both approximately twice as great as the values found in human hair. Thus the total basicity is about the same. The acidic amino acids in general are slightly higher in human hair, making its total acidic character approximately one and one half times greater than that of Merino wool. In general there is order of magnitude agreement between the number of acidic and basic residues per thousand MV<W for the insoluble reduced carboxymethyl keratins and the corresponding fractions of the oxidized keratin derivatives (24, 27, 42, 52). Infrared Spectrophotometry A number of spectra of keratin fibers were reported by Ambrose and Elliott, whose investigations of synthetic polypeptide and proteins led them.to suggest certain frequency criteria for alpha and beta structures in proteins and polypeptides (5). A spectrum of beta keratin from steam stretched horse hair demonstrated the presence of polypeptide chains in the antiparalell arrangement (11). Strasheim and Bujis obtained infrared spectra of wool by grinding in a special mill and using the KBr disc technic (55). A comparison of the spectra of virgin wool fiber and wool powder revealed that the Table I. Amino Acid Composition of Hair, Wool And Keratin Derivatives,/Imoles P.er Gram. The major DTDGA exchange fractions were hydrolyzed in 6 N HC1 at 110 C for 20 hours. The analyses were performed using a Model TSM Amino Acid Analyzer. Merino1 Wool HumanZ Hair Oxidized Wool? Acid Acid Insol. Sol. Frac. Frac. Reduced Wool^ Acid Acid Insol. Sol. Frac. Frac. DTDGA Exchanged Hair Acid Acid Insol. Sol. Frac. Frac. (120,000 (6,000 MW) MW) Alanine 470 345 551 249 588 253 390 160 Arginine 149 476 490 455 676 506 720 472 Aspartic acid 366 415 702 163 798 251 420 380 1/2 Cystine 720 1422 424 1394 445 1599 470 1420 Glutamic acid 584 885 1246 565 1249 724 1390 510 Glycine 1024 512 676 549 692 678 770 240 Histidine 120 62 47 51 65 68 60 104 Isoleucine 198 212 284 208 330 305 170 76 Leucine 450 464 831 246 864 331 440 292 Lysine 314 178 265 50 359 53 190 286 Methionine 26 72 131 62 147 9 70 290 Phenylalanine 134 143 224 112 251 162 120 50 Proline 401 753 307 951 307 1225 570 480 Serine 696 851 764 934 722 1279 670 367 Threonine 389 542 394 718 417 1003 280 340 Tyrosine 172 126 278 135 313 183 110 78 Valine 382 490 452 399 486 482 350 360 Table I— Continued ^Analysis of completely digested Merino Wool taken from O’Donnell, I. J. and Thompson* E. 0. P., Austr. J. Biol. Sci., 15,740 (1962). o Analysis of completely digested human hair taken from Simmonds, D.H., Textile Res. J., 28, 314 (1958). 3Analysis of peracetic acid oxidized derivatives taken from Gillespie, J.M. and Simmonds, D. H., Bioch. Biophys. Acta, 39, 538 (1960). 4Analysis of reduced carboxymethyl keratin derivatives taken from Gillespie, J. M., O'Donnell, J. J., Thompson, E. 0. P. and Woods, E. F., J. Text. Inst., 51, T703 (1960). '1 H1 a > 17 crystallinity was reduced. This was verified by x-ray diffraction data (55). Mohair and human hair, prepared by the same methods, had spectra practically identical to that of wool, except the C-0 stretching vibration at 1690 cm~l was less intense in wool than in hair. Assign ments of new bands present in the infrared spectrum of wool, made by these authors, is summarized in Table II together with the amide I and II assignments, taken from Bellamy (7). The infrared spectrum of both the oxidized and reduced keratin derivatives have been studied and the spectrum of the soluble wool protein reduced acid insoluble carboxymethyl keratin derivatives exhibits the diagnostic alpha helix peak at 1545 cm-1 and the spectra of the wet film (93% RH)1 is similar to the dry film (0% RH) except for a general shift to higher wave numbers. The presence of the underlying water bands (1750-1500 cm_l) (3800-2800 cm-1) was present in both the wet and dry films. The spectrum of the film exposed to an atmosphere of 98-100% formic acid yapor also had a sim ilar shape, somewhat modified by the presence of a strong underlying band due to the acid. This latter spectra is broader on the low frequency side than the curves for the wet and dry films and probably reflects the presence of ■^-Relative Humidity Table II. Infrared Assignments For Wool, Carboxymethyl Keratins And Hair Keratin DTDGA Exchange Derivatives. ( Keratin or Fraction Peak Band Frequency or Range (cm-1) Assignment 6,000 MW acid soluble pep 3690-2800 Water absorption tide isolated from the DTDGA 1750-1480 Water absorption exchange reaction with human 1380 hair, KBr pellet. 1230 Carbonyl stretching modes 1065 600-400 Disulfide stretching 120,000 MW acid insoluble 3680-2800 Water absorption peptide isolated from the 1750-1480 Water absorption DTDGA exchange reaction with 2920 CH2 stretching mode human hair, KBr pellet. 1410-1370 C=0 stretching mode 1150-1025 C-0 stretching mode 500-400 Disulfide stretching Wool, KBr pellet1 2870 Symmetric CH3 and CH2 stretching 2800 2 x CH3 deformation frequency 1570 Antisymmetric stretching 1399 Symmetric C«««0 stretching 1377 Symmetric CH3 deformation, frequency 1342 CH deformation frequency Amide I2 1690 C»*«0 stretching 2940 CH stretching 3460 NH stretching Amide II2 1550 NH stretching oo Table II— Continued Keratin or Fraction Peak Band Frequency Assignment or Range (cm-1) Wool reduced carboxymethylS keratin, high molecular 1545 Helix peak, weight, low sulfur KBr pellet. ^Wool assignments taken from Strasheim, A. and Bujis, K., Spectrochim. Acta., 16, 1010 (1960). o Amide I and II assignments taken from Bellamy, L.J., "The Infrared Spectra of Complex Molecules", John Wiley & Sons, Inc., New York (1956). ^Reduced carboxymethyl keratin derivative assignments taken from Bendit, E. G., Nature, 211, 1257 (1966). 20 additional non-crystalline material, which in dry keratin has been shown to absorb at 1520-1525 cm-! and absorbs at 1 £ j i 5-10 cm-1 higher in keratin exposed to saturated water n vapor (9). Conformation Harrapp studied the Optical Roratory Dispersion of the low sulfur proteins and calculated the percentage alpha helix content from the Moffitt-Yang parameter, bQ . When the fractions were dissolved in 2-chlorethanol, the alpha helix content increased from 48% to about 60%. This helix content was consistent with the authors assumption that these proteins originated in the microfibril. After heating to 70° or after dissolving sufficient urea in the aqueous solution of the reduced acid soluble carboxymethyl derivatives to bring its concentration to 8 M, the alpha helix content was reduced to zero percent. This change with urea'was found to be reversible upon dialysis. A similar effect was observed when increased formamide concentrations, instead of urea, were studied with this system. There was decrease in helical content with increased concentrations up to 50% formamide. This decrease was also found to be reversible upon dialysis or dilution. The high sulfur acid soluble carboxymethyl keratin fraction behaved as random coils in aqueous solu tion. Very little alpha-helix structure was formed even 21 when the S-carboxymethyl keratin derivatives were dissolved in the helix forming solvent, 2-chloroethanol. The absence of helical content is consistent with the assumption that these fractions compose the matrix keratin of the wool fiber (26, 31). STATEMENT OF PROBLEM The human hair keratins and their counterparts in other species have been studied mainly in the intact fiber by means of x-ray diffraction, infra-red spectro photometry and electron microscopy. Thus there is con siderable knowledge of the gross structural characteris tics of hair fibers, but information regarding the chemical relationship of peptides, though voluminous, is confusing. Soluble peptides from hair and wool have been obtained by harsh chemical treatment, which resulted in the cleavage of many types of bonds and the destruction of many amino acid residues. "Keratins are high molecular weight polymers of hydrogen bonded peptides containing interchain cystine crosslinks. In order to solubilize keratins it has been necessary to disrupt both the hydrogen bonds and the disulfide crosslinks. These reactions up to now have been carried out at either high or low pH values, which also resulted in the cleavage of acid or alkali labile peptide bonds and affected the integrity of the residues that are sensitive to pH extremes. t» In order to stabilize reduced keratins and to prevent random reoxidation, it has been necessary to 22 block the reactivity of the sulfhydryl group. This was accomplished by alkylating the reduced forms and usually the carboxymethyl or carboxamidomethy1 derivatives were prepared. Since the pK of the sulfhydryl lies above pH 9, the cumbersome alkylation steps have been carried out at pH's higher than 9. To varying clegrees other atoms such as the imidazole and epsilon amino nitrogens also became alklylated. The additional factor of non specificity further increased the number of disadvantages of these methods. Important species differences have been noted among the crude derivatives studied. Marked variation in the quantities of different keratins dissolved in solvent media, showed clearly that even samples from similar keratin sources and different species were not identical, but differed significantly from one another, not only in amino acid composition, but also varied in configuration, alpha helix content, molecular weight and other properties. It is the purpose of this study to obtain a method of solubilizing water insoluble keratins, which will result directly in stable and specific derivatives of thevdisulfide crosslinks in the keratin molecule. It is a further purpose to investigate the reactions in volved and the derivatives obtainable by such procedures that are developed. EXPERIMENTAL APPROACH The principal reducing agents effective in solubilizing keratins include mercaptoethanol, mercapto- acetic acid and other thiols. These reagents are capable of disulfide exchange, particularly if their bis dithio dimers are used and the equilibria are suitably adjusted. In the present study a soluble, stable deriv ative, related to one of the above thiols, was prepared directly from water insoluble keratins in the presence of a hydrotropic reagent and at a neutral pH, by shifting the equilibria to favor a mixed disulfide derivative. The dithiocarboxymethyl keratin derivatives were prepared directly by using dithiodiglycollic acid as the bis dithio dimer and thioglycollic acid as the monomer thiol The conditions of this exchange reaction were systematically varied and such parameters as the rates of phase change (or peptide solubilization), sulfhydryl decay and pH changes were measured. As a measure of overall efficiency, the ratio of total sample that under went phase change to the net residue remaining when the reaction was terminated, was determined for all condi tions studied. The acrylamide gel electrophoretic properties of the derivatives were followed through all 24 25 phases of the study. Two major fractidns were separated by pH fractionation and molecular sieve chromatography. I Molecular weights and Stokes Radii of the principal frac tions were estimated from the elution volumes on molecular sieve chromatography. Amino acid analyses, sulfur, nitrogen and moisture content were also determined. The spectroscopic properties of the derivatives were studied in the ultraviolet, visible and infrared regions of the spectrum. The extinction coefficients at 280 nm1 were determined for the two principal fractions. Alpha helix contents from optical rotatory dispersion measurements were calculated using the coefficients of the modified two term Drude equation (MTTDE) and the Moffit Yang bQ parameter. A study of the far UV optical rotatory dis persion and the far UV circular dichroism was made for each of the principal fractions. *A nanometer is equivalent to a millimicron. MATERIALS.AND METHODS Hair Samples Several samples of brown hair were obtained from a 22 year old Caucasian male, who did not use hair preparations of any type. Reagents All chemicals and reagents were reagent quality, with the exception of 2-chloroethanol obtained from Matheson,Coleman and Bell, which was chromatoquality. Dithiodiglycollic acid was obtained from Evans Chemetics. Hydrochloric acid, sodium acetate and glacial acetic acid were obtained from J. T. Baker Chemical Co. Petroleum Ether, 30-60° C (Ligroin) was obtained from Mallinckrodt Chemical Works. Thioglycollic acid 97%, urea crystals and potassium ferricyanide were obtained from Matheson, Coleman and Bell. Acrylamide, N,N-methylenebis acryl- amide, N,N,N'-tetramethylenediamine, Riboflavin, ammonia free glysine and bromphenol blue were obtained from Eastman Kodak Company. Ammonium persulfate, silver nitrate and sodium nitroprusside were all Merck Blue Label. Amidoschwarz was obtained from National Aniline Division of Union Carbide. 5,5' dithiobis (2-nitro- benzoic acid) was obtained from Nutritional Biochemical . 26 Corp. Bio Gel P-100 and P-300 were obtained from Bio- Rad Laboratories. Sephadex G-100 and G-200 were obtained from Pharmacia Laboratories. Absolute ethanol was obtained from U. S. Industrial Chemicals Co. Preparation of Hair Samples The hair samples were defatted by repeated washings in petroleum ether, air dried at room tempera ture, cut into small lengths with scissors and further subdivided by means of an Oster electric, small, animal clipper. The petroleum ether rinses were then repeated to remove exogenous oil that may have resulted from contact with the clipper or handling during processing. Solubilization of Keratin by Disulfide Exchange The disulfide exchange derivatives:were pre pared in accordance with the following equation: KSSK + TSST T+~ 2KSST1 The keratin thioglycollate derivatives were prepared with the following fixed conditions: 6 M urea, 2 M Tris-HCl buffer, pH 7.50, 1% hair w/v, incubated at 50° C. A duplicate set of reaction vessels lacking hair served as reagent blanks. Four levels of thioglycollate (TA) The symbols refer to the following: KSSK— native keratin; TSST— dithiodiglycollic acid; KSST— keratin DTDGA exchange derivative; TS-— thiogly- collic acid. j 1, 10, 50 and 100 millimolar, were mixed with each of the following dithiodiglycollic acid (DTDGA) concentrations; 50, 100, 200, 500 and 1000 mM. These served as the principle variables. The reaction was performed in dis posable, plastic syringes. The neoprene syringe plungers were separated from the reaction mixture by multiple thicknesses of Saran Wrap. Samples were taken at 2, 4 and 8 hours and at 1', 3, 5, 7 and 14 days. In each case 0.5 to 0.6 ml of the supernatant was collected in 0.1 ml autoanalyzer cups through a U shaped, number 21 gauge, hypodermic needle by depressing the plungers of the syringes upward until the desired amount of sample was displaced. Determination of Thiol Thiol analyses were performed immediately after removal of the sample by the method of Ellman (19). This analysis was performed essentially as described by the author. The dilution and sample volume were selected to give absorbancy reading in a satisfactory range. The extiiaction coefficient reported by Ellman for 5,5’-dithio bis 2-nitrobenzoic acid (DTNB) was verified. The pro cedure used for the analysis was as follows: To a mixture of 11.0 ml of distilled water and 1.0 ml of 0.1 M tris buffer, pH 7.5 was added successively 10 micro liters of sample, blank, or standard, 20 microliters of 29 DTNB solution (39.6 mgs DTNB per 10 ml of 0.1 M phos phate buffer, pH 7.0). The solution was mixed, allowed to stand 3 to 5 minutes and read against the blank at 412 nm in the Beckman Model DB spectrophotometer. The molar, concentration was determined from the following formula: C0 = A D A(0.08845) C C0 = Molar concentration C = Extinction coefficient at 412 nm (13,600) D = Dilution factor (1203) A = Absorbancy at 412 nm The formula was tested with standard solutions of known concentration and was found to give the predicted thiol levels. Determination of pH The pH measurements were made either with a Radiometer pH meter, using an ultramicroelectrode requir ing 10 microliters of sample, or with a Beckman research model pH meter, utilizing a combination glass microelec trode requiring a volume of 0.1 to 0.3 ml of sample. A calomel reference electrode was used in both instances. While it was possible to read the pH to one thousandth of a pH unit, the buffer available limited the accuracy of the standardization to one hundreth of a pH unit. 30 Determination of Solubilized Protein The balance of the samples were transferred to 3/8 inch Visking dialysis tubing and dialyzed exhaustively against distilled water at 5° C to remove the excess reactants from the solubilized keratin derivatives. Dialysis was continued until no thiol was detectable by aqueous silver nitrate in the external dialysis solutions. For protein measurement each sealed dialysis membrane was removed from the distilled water, wiped dry and the peptide solution expressed into a 1.0 ml grad uated cylinder to check for any volume change, which in most cases was insignificant. Where a volume change was found to occur, an appropriate correction factor was applied to the absorbancy measurements at 280 mu. The absorbancy of the dialyzed supernatant was read in a Beckman model DB spectrophotometer in matched Beckman silica cuvets, adapted to accommodate 0.2 ml of sample. Kinetic Studies The raw data for absorbancy vs time were con verted by means of an Olivetti Programma 101, desk top computer, to first and second order rate data. Concen trations of soluble peptide were obtained using an extinction coefficient of 1 2 .8 , determined as described below on unfractionated disulfide exchange derivatives at 280 nia. The first order constants k^s^ . and the 31 correlation coefficient were computed from the slope of the line of the common logarithm of the concentration of peptide vs time, in hours. Second order rate constants, k2nd and corresponding correlation coefficients were computed similarly from the slope of the reciprocal concentration vs time in hours. The computer was pro grammed to compute the correlation coefficient: (Xj - X) (Y2 - Y) fx± - X)2(Yi - y)2]1/2 where the X coordinate was the solubility in grams per deciliter, the y coordinate time in hours and the bar values were the respective means. Experiments Varying Hair Concentration Samples of 1.0, 2.0 and 5.0 grams percent of finely cut human hair were each suspended in-the reaction medium consisting of 0.5 M DTDGA, 40 mM TA, 6 M tris-HCl buffer, pH 7.50. Anaerobic conditions were maintained by vacuum deaeration, flushing with nitrogen and subse quent prevention of atmospheric oxygen from entering vessels. The reactions were carried out in polypropylene disposable syringes, which facilitated the anaerobic sampling. The reactions were conducted at 50° C for two weeks, after which time the final pH and SH levels were verified as in previous experiments. At the termination of the reaction period, the solid phase residues were 32 separated from the supernatant and weighed. The solu bilized peptides were then subjected to acid fractiona tion and disc electrophoresis. Peptides Released by 6 M Urea Experiments were set up to determine if any peptides were released when no reductant or disulfide exchange reactions were employed and if so, to ascertain the relation of these peptides to those obtained in the disulfide exchange experiments. In these experiments all conditions were similar to the exchange reactions except the sulfhydryl and disulfide reagents were omitted. The experiments were terminated after two weeks of incubation at 50° C. The peptides released were measured and subjected to disc electrophoresis and Bio Gel chroma tography . Reexchange Experiments The peptides released by the disulfide exchange reactions from the 1% and 5% hair experiments, after pH fractionation and lyophilization were again allowed to undergo disulfide exchange under identical conditions of the original exchange reaction, except for the absence of any solid phase. The lyophilized urea fraction was treated in an identical manner. The disulfide exchange reagents were in sufficient excess over the peptides in solution to shift the equilibrium reaction toward complete 33 exchange. Disc Electrophoresis Acrylamide gel, disc electrophoresis, according to Ornstein and Davis (16, 44, 46) were performed on the keratin derivative produced. The following acrylamide concentrations; 5%, 7.5%, 10% and 15% were used to more exhaustively study the molecular sieving property by • varying the pore size of the lower gel. Separation in 7.5% gel were found to adequately employ the sieving property. Sixty microliters of sample containing four micrograms per microliter were applied to the top of the spacer gel from a Hamilton microsyringe. The electro phoresis was performed using a current of 5 milliamps per gel tube and was allowed to continue until a tracer dye (bromphenol blue), placed in the upper reservoir chamber, had migrated within five millimeters from the end of each gel tube. The acrylamide gel cylinders were removed from the tubes and transferred to a small test tube containing 1% amidoschwarz in 7.5% acetic acid and 35% ethanol and stained for periods from 15 minutes to 1 hour. The gels were then washed in several changes of 7.5% acetic acid, which was followed by electrophoretic destaining, using 7.5% acetic acid in the upper and lower buffer reservoirs and employing a current of 10 to 12.5 milliamps per gel tube for 30 minutes or less. The 34 destained discs were scanned in a Joyce-Loebl Chromoscan densitometer. Standardization of the integrator and the scanner were performed periodically. The dry weights of the washed hair residues were determined on an Ainsworth Type 12 analytical balance at the end of the last sampling interval. Fractionation of Derivatives A method combining acid fractionation followed by reduction of ionic strength was utilized for the ini tial preparation of derivatives. The procedure for pH and ionic strength reduction follows: The chilled (5° C) supernatant solubilized keratins were ajusted to pH 4.3 with 1M acetic acid. No visible phase change occurs at this pH. The pH 4.3 supernatant was dialyzed at 5° C, exhaustively against distilled water. A precipitate of the high molecular weight keratins formed in the dialysis bag as the ionic strength and urea concentration were reduced by dialysis. The supernatant was separated from the precipitated keratins by centrifugation for ten minutes in a refrig erated (5° C) International Centrifuge, Model PR 2, at 2000 RPM (c.a. 700 times £) . The precipitate which contained mainly acid insoluble 120,000 MW peptides and the supernatant which contained the acid soluble lower molecular weight peptides 35 were lyophilized for subsequent chromatographic purifica tion and analysis. Chromatography Preparative columns were poured to a height of 45 centimeters in ’’ Double Tough” Pyrex pipe columns, 2 1/2 centimeters i.d. The top of the column was connected to a one liter polyethylene reservoir chamber, which contained the developing buffer. The bottom of the column was connected to a one liter polyethylene reser voir chamber, which contained the developing buffer. The bottom of the column was connected to the flo cell cuvet of a Model UA 2 Isco Ultraviolet Analyzer. The outlet from the Isco monitor was directed to a Microchemical Specialties fraction collector. Samples of uniform volume were collected. The volume and absorbancy of the fractions were verified manually at the end of each run. The void volume (VQ) was determined for all of the molecular sieve columns by using Dextran Blue 2000. This determination was repeated before and after each run. The acid soluble and acid insoluble fractions were initially chromatographed on Sephadex G-100 or Bio Gel P-100. Fractions under each peak were pooled after verifying absorbancies and fraction volumes, then were lyophilized for further study. The peaks excluded from Sephadex G-100 and Bio Gel P-100 were rechromato- 36 graphed on Sephadex G-200 and Bio Gel P-300. These fractions, corresponding to the peaks, were also pooled after verifying absorbancies and fraction volumes, then were lyophilized for subsequent analysis. Characterization of Fractions Molecular Weight The proteins used and data pertaining to the molecular weight standardization are summarized in the figure legends (Figures 1 to 4). The standard molecular weight curves for each of the gels were prepared by plotting the distribution coefficient against ithe log arithm of the molecular weight. Kd - Ve/VQ where Ve is the effluent volume at the peak and V0 is the void volume of the column. Linear relationships were obtained in all instances. The purified fractions were chromatographed on 150 cm water jacketed, analytical columns. The distribution coefficient was determined for each peak and found to remain constant for a given gel column. The molecular weights were then determined by reference to the appropriate standard curve. Stokes Radius Determination The Stokes Radius of a macromolecule is pred icated on the assumption that it can take spherical 37 Fdg. 1— Molecular weights estimated from Sephadex G-100 chromatography. The column dimensions were 1.0 x 150 cm. The buffer system was 0.1 M NaCl in 0.1 M tris HC1, pH 7.50. The flow rate was maintained at 5 ml per hour with a Lambda pump. 1.0 ml fractions were collected. The void volume (VQ) and intrinsic volume (Vi) determined as described in the text, were 35.0 ml and 11.0 ml respectively. Peaks 1 and 2 of pH 4.3 soluble fraction from 1% hair experiments are represented. 38 I 4.0 V N^Peak No.2 (M.W. 5 ,800) S.Bovine Insulin 3.0 \ ,P e a k No. KM.W. 13,400) Kd ^-.Soybean Trypsin 2.0 Inhibitor Bovine Serum Albumin 1.0 \ 0 i i j — i— \ 3.0 3.5 4.0 4.5 5.0 Log Molecular Weight » 39 Fig. 2— Molecular weights estimated from Sephadex G-200 chromatography. The column dimensions were 1.0 x 150 cm. The buffer system was 0.1 M NaCl in 0.1 M tris HC1, pH 7.50. The flow rate was maintained at 5 ml per hour with a Lambda pump. 1.0 ml fractions were collected. The void volume (Vq ) and intrinsic volume (Vi)determined as described in the text, were 14.5 and 47.2 ml respectively. Peaks 1 and 2 of the acid insoluble fraction from 1% hair experiments represented. Peak number 3 is not represented. 40 I ! | 4.0 | 3.0 K d : 2.0 i ‘ 1.0 4.0 4.2 4.4 4.6 4.8 5.0 Log Molecular Weight L Peak No. 2 (M.W. 13,200) Soybean Trypsin Inhibitor Bovine Serum Albumin Peak No. 1 (M .W . 120,300)" Bovine Gamma Globulin 41 Fig. 3— Molecular Weights estimated from Bio Gel P-100 chromatography. The column dimensions were 2.5 cm x 45iO.Gm. The buffer system was 0.1 M NaCl in 0.1 M tris HC1, pH 7.50. The flow rate was maintained at 5.0 ml per hour with a Lambda pump. 1.0 ml fractions were collected. The void volume(VQ) and intrinsic volume (V^) determined as described in the text, were 60.0 ml and 153.0 ml respectively. Both peaks from pH 4.3 soluble fractions from 5% hair experiments are represented. 2.5 \ x^Peak No.2 (M.W. 5 ,4 0 0 ) X^Bovine Insulin 2.0 \ s P e a k No. 1 N. (M.W. 13,600) Kd ^S oybean Trypsin 1.5 Inhibitor Bovine Serum A lb u m in \i 1.0 \ . 0.5 i i i i 3.0 3.5 4.0 4.5 5.0 Log M olecular Weight \ 43 Fig. 4— Molecular weights estimated from Bio Gel P-300 chromatography. The column dimensions were 2.5 x 45.0 cm. The buffer system was 0.1 M NaCl in 0.1 M tris HC1, pH 7.50. The flow rate was main tained at 5 ml per hour. 1.0 ml fractions were collected. The void volume (Vo) and intrinsic volume (Vi), determined as described in the text, were 48.0 and 147 ml respective ly. The three peaks from the pH 4.3 insoluble fraction are represented. I 3.0 - Kd 2.0 - Peak No. 2 (M.W. 14,200) .Soybean Trypsin Inhibitor Bovine Serum Albumin Peak No.l (M .W . 124,000)’ Bovine Gamma Globulin 1.5 - 1.0 4.0 4.2 4.4 4.6 4.8 Log. Molecular Weight 5.0 \ 45 shape. A Sephadex G-200 column and the protein standards with known E^Ow’ were employed to determine the pore radius of the Sephadex G-200. The retardation R is obtained from the relationship: Ve - VQ r s ---------- 2. x 1 0 0 Vi where Ve is the effluent volume at the peak, Vo is the c void volume and is the intrinsic volume of the column determined according to the method of Ackers (2). R is related to the a/r ratio i.e. the ratio of the Stokes radius of the particle (a) to the effective pore radius (r) by the Renkin equation (47). w -l-/a? 1 - 2.104/a. - 2.09,a? - 0.957ap 5 ' ( ? ) ( ? ) ( ? ) ( ? ) The experimentally determined R values from the analyt ical Sephadex G-200 chromatography were converted to a/r ratios from a graphic solution of the Renkin equation by Ackers (2). The known or calculated a2o,wva^ues of the standards were determined from the relationship: a2 0 ,w " 213.6 ( D 2 0 , w x 1q7) These values were used to calculate a mean effective pore size from which the Stokes Radii of the purified keratin fraction were determined. Ultraviolet Absorptivity And Spectrophotometry Purified major components of the acid soluble and acid insoluble fractions were dialyzed exhaustively 46 against distilled water. The absorbancy of the diluted samples were measured against water at 277-289 nm in a Beckman Model DU spectrophotometer. Measured volumes (10 ml each) of protein solution and dialysate were placed in dried, tared weighing bottles and dried to constant weight under reduced pressure over P2O5 at 60° C. A Mettler, semimicro balance was used for weighing. o Ultraviolet and visible spectra of the aqueous solutions of the purified keratin derivatives were measured in a Cary Model 15 spectrophotometer in the range of 750 to 200 nm. Estimates of moisture content of the lyophilized fractions were determined from the difference between the originally weighed sample and the constant dry weight determined above. Moisture Content The dry weights of the fractions were deter mined by repeated differential gravimetry after drying over P2O5 under nitrogen at 50° C for a period of several days until a constant weight was reached. Infrared Spectrophotometry The samples dried out for the moisture analyses were used to prepare potassium bromide pellets. The spectra were measured over a range of 2.5 to 40 11. 47 Nitrogen Analyses Nitrogen contents of the purified fractions were determined by a modified semi micro Kjeldahl technic employing Nesslerization and spectrophotometry in the final steps (36). Amino Acid Analyses Samples for amino acid analyses were hydrolyzed in 6 N HC1 at 110° C under nitrogen. Reported composi tions are based on the values obtained after 20 hour hydrolysis. No corrections were made for the destruction of labile amino acids or for the partial release of amino acids resistant.to hydrolysis. Half cystine was determined mainly as cysteic acid. The analyses were performed using a Technicon Model TSM Amino Acid Analyzer according to the procedures of Spackman, Moore and Stein (54). The results were expressed in micromoles per gram. Optical Rotatory Dispersion The optical rotatory dispersion measurements (ORD) were made with a Cary Model 60 spectropolarimeter. Cells selected for minimal birefringence had a light path of 0.01 decimeters. All runs were made using a ten second pen period, 18 A° spectral band width and 1 A°/sec scan rate at 27° C. The concentration of each sample i 48 along with the refractive indices of the solvents were used to convert observed rotation to specific residue rotation by means of the following relations: The specific rotations [a]x were obtained from [a] - -where L is the path length in decimeters and c is the concentration in grams per 100 ml. The specific residue rotations [4»1 ]^ were calculated from: - , MRW . 3 [♦'.]* “ [<*]\ ioo n2+ 2 where MRW, the mean residue weight, was taken as 109, n the refractive indices of the solvents, approached 1.0. All ORD measurements are expressed as degrees U!lx . The helicity calculations were made from the coefficients A (a,p)i93 and A(a,p )225 Pf modified two term Drude equation (MTTDE) (10, 12, 49, 50): [frij. A(ot,p)193 X2193 j. A(a ,p >225^225 x 2 -X2 x2 -X2 193 225 The MTTDE can be written in the form: 2 2 ^ ^ 193 2 2 2 [$i] _ A ( a, P)193 + A(a,P )225 X 225- X 193. X 225 iqo X2 X2 A 2 193 “ 225 in which form it was plotted and/or solved for slope and intercept on the Programma 101 desk top computer. If [^i] j?-x2 /X 2 was plotted against X 2 ,X2- J? 193 193 225 225 the intercept was A(a,p)^g3 arid the sloPe was given by A(o, p)225. ^2225 - *^193/ ^193 from which the two coeffi- j cients were determined directly. j * i l . ' The percent helicities were determined i from the following empirical relationship derived by Schecter and Blout (12,50). H(%) .A, - A + 650 ! MTTDE (oi,p )193 ( a p)225 The primary ORD data were also treated according! to the Moffett Yang equation (62): j C4. . . . a ° . \ ° - 2. + b°x°4 X 2 - X 2 (X 2 -X 2 ) 2 2 2 2 when [$•] , X -X o is plotted against X Xo o ° the slope gives bQ and the intercept aQ . Linear plots were obtained with the data obtained at higher wavelength using. XQ = 212 nm. The percentage o-helix contents (H^-) of the fractions were calculated from the Moffett and Yang bQ relationship: H(%) = -100 bo/750 MY Circular Dichroism The circular dichroism (CD) measurements were 50 made with a Cary Model 60 spectropolarimeter equipped with a Cary Model 6001 circular dichroism accessory. The cells selected for ORD were used for CD. The wavelength range of the CD studies extended from 2100 to 2700 A°. The circular dichroism ( 1 - r) or is calculated using the relationship: AE = ( E1 - f r) z (Kc-Kr) c'l' where (Kc- Kr) is the observed difference in absorption between the left and right circularly polarized light, c' is the molar concentration and l 1 is the light path in decimeters. All CD results have been expressed in terms of molar ellipticity [®]x which has the same units as The molar ellipticity is expressed in degrees and is related to the decadic circular dichroism in liters per mole centimeter as follows: [®1* 58 2.303 . 4500 (e 1 _ er) 3305 JI RESULTS Kinetics of The Keratin DTDGA Exchange Reaction Keratin disulfide exchange reactions for all ratios of TA:DTDGA followed first order kinetics for the first 8 to 24 hours and second order kinetics for the subsequent period (Figures 5 to 9). Rate constants and r values were computed fox’ these periods and the latter gave values greater than 0.97 in all instances (Table IIlX Optimal levels of thiol catalyst would appear to lie between 1 and 2% of the DTDGA concentration, since the second order rate constants are greatest at TA:DTDGA ratios of 0.01 to 0 .0 2 . Final yields of soluble peptides were greater when the concentration of TA and or DTDGA was increased (Table IV). In the 0.1 ratio of TA:DTDGA the final yields of the 100 mM TA— 1000 mM DTDGA were 2 1/2 times those from the 10 mM TA— lOOmM DTDGA experiments. In spite of the strong 2 M tris HC1 buffer, pH 7.50, small pH changes occurred in all of the systems studied (Table IV). The maximum pH of 8.75 for all of the reactant mixtures studied was less than 1.4 pH units greater than the original pH 7.50 51 Table III. First And Second Order Rate Constants For The Hair Keratin DTDGA Disulfide Exchange Reaction. First order rate constants (kist) were computed geometrically on a Programma 101 from the slope of the common logarithm of concentration vs time, expressed in units of deciliters g_1hr“ . Second order rate constants (k2n(j) were computed geometrically on the Programma 101 from the slope of the reciprocal of peptide concen tration vs time, expressed in units hr“l. The correlation coefficients, and r2n(j were computed simultaneously as described in the text. TA/DTGA Ratio TAmM DTDGA mM klst x 10~2 (dlg-lhrl ) rlst k2nd x 10“5 (hr-1 ) r2nd 1.0 50 50 2.87 .99 4.54 .97 100 100 2.82 .98 4.57 .98 0.1 10 100 2.65 .98 2.45 .99 50 500 2.67 .98 2.45 .99 100 1000 2.64 .99 2.43 .98 0.02 1 50 1.38 .98 7.02 .99 10 500 1.37 .99 7.01 .99 0.01 1 100 1.46 .98 3.42 .98 10 1000 1.48 .98 3.38 .98 0.002 1 500 1.23 .98 3.74 .97 0.001 1 1000 1.05 .99 3.68 .97 cn t s 3 53 Fig. 5— Kinetics of keratin disulfide exchange^ with equivalent ratios of TA:DTDGA. (A) Keratin peptide solubilization. o= 50 mM TA:50 mM DTDGA; □ = 100 mM TA: 100 mM DTDGA. The reaction was carried out anaerobically at 50° C, initial pH 7.50 with 2.0 M tris HC1 buffer and 6GM urea. A slight pH increase paralelled the release of peptide. (B) First order rate plots of data in A. The solubilization was expressed in grams per deciliter, de termined from the composite extinction coefficient of the mixture. The reaction is linear for the first 18 hours: The first order rate constants over the linear period were: O = 2.87 x 10-2 gdl_1hr” , □ = 2.82 x 10"2 gdl^hr-1. Note non-linearity after 18 hours. (C) Second order rate plots of data in A. Note non-linearity for first 18 hours. The second order rate constants for the linear period (after 24 hours) were O = 4.57 x 10"5/hr. and □ = 4.54 x 10“5/hr. Absorbancy L og c 0 .0 5 (C) 0.04 0.03 0.02 o s 0.01 (B) 2.6 2.2 (A) 336 \ Time (Hours) t t 55 Figure 6— -Kinetics of keratin disulfide exchange with 1:10 ratios of TA:DTDGA. (A) Keratin peptide solubilization O = 10 mM TA:100 mM DTDGA, A -• 50 mM TA:500 mM DTDGA, □ = 100 mM TA:1000 mM DTDGA. The reaction was carried out anaerobically at 50° C, initial pH 7.50 with 2.0 M tris HC1 buffer and 6 M urea. A slight pH increase parallelled the release of peptides. (B) First order rate plots of data in A. The solubiliza tion was expressed in grams per deciliter, determined from the composite extinction coefficient of the mixture. The plot is linear for the first 24 hours. The first order rate constants over the linear period were: O = 2.65 x 10"2 d ig-1 hr"1 , A = 2.67 x 10“2 dig"1 h r-1, □ = 2.64 x 10 “” dlg-1 h r-1 . (C) Second order ra te p lo ts of data in A. The second order rate constants for the lin ea r period (a fte r 24 hours) were: 0 = 2.45 x 10-5 hr-1, A = 2.45 x 10-5 h r-1, □ = 2.43 x 10-5 hr-1. Absorbancy 0.05 0.04 0.03 o s 0.02 0. 01 2.6 2.2 o CP 3 i.o (A) 336 Time (Hours) 57 F ig. 7—K in etics of k eratin d is u lfid e exchange w ith 1:50 r a tio s of TA:DTDGA. (A) Keratin peptide so lu b iliz a t i o n . O- 1 mM TA: 50 mM DTDGA, A= 10 mM TA: 500 mM DTDGA. The reaction was carried out an aerobically at 500 c, i n i t i a l pH of 7.50 w ith 2.0 M t r i s HC1 b u ffer and 6 M urea. A s lig h t pH in crease p a r a le lle d the r e le a se of p ep tid e. (B) F ir s t order ra te p lo ts o f data in A. The s o lu b iliz a tio n was expressed in grams per d e c i l i t e r , determined from the composite e x tin c tio n c o e f f ic ie n t of the m ixture. The p lo t i s lin ea r for the f i r s t 24 hours. The f i r s t order rate constants over the lin ea r period were: 0= 1.38 x 10-2 d lg-1 hr-1, A = 1.37 x 10-2 d lg-1 h r-1. (c) Second order rate p lo ts of data in A. The second order rate con stants for the lin e a r period (a fte r 24 hours) were: A = 7.02 x 10-5 h r-1, o= 7.01 x 10-5 h r-1. Absorbancy L og c l / c 0 .0 5 0.04 0.03 0.02 0.01 (B) 2.6 2.2 (A) 336 Time (Hours) \ 59 Fig. 8— Kinetics of keratin disulfide exchange with 1:100 ratios of TA:DTDGA. (A) Keratin peptide solubilization. O = 1 mM TA:100 mM DTDGA, A = 10 mM TA:1000 mM DTDGA. The reaction was carried out anaer obically at 50°C. Initial pH 7.50 with 2.0 M tris HC1 buffer and 6 M urea. A slight increase in pH paralelled the release of peptide. (B) First order rate plots of data in A. The solubilization was expressed in grams per deciliter, determined from the composite extinction coefficient of the peptide mixture. The solution is linear for the first 24 hours. The first order rate constants over the linear period were: 0= 1.46 x 10“2 dlg-1 hr-1, A = 1.48 x 10-2 dlg-1 hr-1. (C) Second order rate plots for data in A. The second order rate constants for the linear period (after 24 hours) were: O = 3.42 x 10-5 h r-1, A = 3.38 x 10-5 h r-1. Absorbancy ° - 5H77T O (C) 0.4 -O 0.3 - ~ 0.2 (B) 2.6 ° 2.2 o» (A) - O 10 20 60 9 0 120 150 180 336 Time (Hours) 61 Fig. 9— Kinetics of keratin disulfide exchange with 1:1000 ratios of TA:DTDGA. (A) Keratin peptide solubilization with 1:500 and 1:1000 ratios of TA:DTDGA. O a 1 mM TA: 500 mM DTDGA, .A = T*mM TA:1000 mM DTDGA. The reaction was carried out anaerobically at 50° C, initial pH 7.50 with 2.0 M tris HC1 buffer and 6 M urea. A slight increase in pH paralelled the release of peptide. (B) First order rate plots of data in A. The solubility was expressed in grams per deciliter, determined from the composite extinction coefficient of the peptide mixture. The plot is linear for the first 24 hours. The first order rate constants over the linear period were: O = 1.23 x 10-2 dlg-1 h r-1, and A= 1.046 x 10-2 d lg-1 h r-1 . (C) Second order rate plots for data in A. The second order rate constants for the linear period were: O = 3.75 x 10-5 hr-1 and A = 3.68 x 10-5 hr Absorbancy 0.5 (C) 0.4 0.3 o - 0.2 (B) 2.6 ° 2.2 o> o -I 1.4 (A) S - 336 Time (Hours) Table IV. Initial And Final Conditions Of The 1% Keratin DTDGA Exchange Reaction. All series contained 6 M urea and were performed under anaerobic conditions in polypro pylene disposable syringes with the needles bent to deliver the samples into Auto analyzer cups and were analyzed immediately, as described in the text. The initial pH 7.50 for all series was established with 2.0 M tris HC1 buffer. Percentages solubilized were obtained by difference from dry weight of washed residue after 326 hours at 50° C. Series DTDGA (mM) Initial SH (mM) Final SH (mM) Final pH % Solubilized % Decrease Of Thiol After 326 Hours 1 50 1.5 0.5 8.62 18.14 33 2 100 1.5 0.5 8.66 24.65 3 200 1.5 0.5 8.68 36.89 4 500 1.5 0.5 8.72 41.56 5 1000 1.5 0.5 8.75 57.33 6 50 10.0 7.2 8.68 21.69 35 7 100 10.0 6.9 8.70 25*89 8 200 10.0 7.0 8.72 44.16 9 500 10.0 6.7 8.74 52.78 10 1000 10.0 7.2 8.78 61.59 11 50 51.0 32.5 8.70 24.84 29 12 100 51.0 34.0 8.74 32.84 13 200 51.0 32.8 8.78 56.22 14 500 51.0 32.7 8.82 61.58 15 1000 51.0 33.6 8.84 75.90 16 50 106 67.5 8.70 27.37 38 17 100 106 62.0 8.74 36.89 18 200 106 64.3 8.82 58.76 19 500 106 64.3 8.84 65.74 20 1000 106 67.5 8.86 78.58 < 3 1 CO 64 The decay of reagent thiol with time was very gradual (Figure 10). The greatest decline occurred during the first 24 hours. The subsequent slopes were very small so that final thiol concentrations were approximately two thirds of the original levels. The highest reagent thiol studies (100 mM) gave the highest absolute decrease, but when the relative or percentage decreases were considered, the differences in relative thiol decay for all levels varied only between 29 and 38%. The percentage solubilized after 326 hours at 50° C increased both with increasing TA and with DTDGA concentrations (Figure 11). The maximum yield obtained was 78.6% with the highest concentration of the 0.1 ratio of TA:DTDGA for which the maximum pH and -SH changes were found to occur (Series 20, Table IV). Fractionation of Derivatives After reaction with the disulfide exchange reagents for 14 days, the peptides solubilized were separated by a combination of pH fractionation and molecular sieve chromatography (Figure 12). Initial Fractionation When the disulfide exchange reaction was terminated, the insoluble hair residues were removed, washed and saved for stoichiometric studies. The pH 65 Fig. 10— Decay of thiol concentration for the keratin DTDGA exchange reactions. The original TA con centrations were: 0=1.5, A =10 mM, 0 51 mM and #=106 mM. Thiol analyses performed by the Ellman method. o H 3' (D o ov o (0 o ; 5 8 c _ < n U > — O CO o OJ OJ O) t!> ft u SH Concentration (mM) oj o CJl o o ~T~ T i I T ./ / 67 Eig. 11— Keratin solubilization as a function of reagent concentration. The percentage of hair solu bilized at the end of the experiment (336 hours) is given as a function of DTDGA concentration for each of the catalytic thiol concentrations used. The pH was 7.50 with 20 M tris HC1 and 6 M urea in all instances. Exper iments were conducted anaerobically at 50° C as described in the text. 0 = 1 mM TA, A=10 mM TA, #=50 mM TA, A =100 mM TA. Percent Solubilized ] i 9 0 7 0 5 0 - 3 0 8 0 0 1000 4 0 0 6 0 0 200 0 DTDGA Concentration (mM) 69 Fig; . 12— Fractionation scheme in the isolation of keratin DTDGA exchange derivatives. The 1% hair deriv atives were chromatographed on the Sephadex Gels and the 5% hairnderivatives were chromatographed on the Bio Gels indicated. Otherwise the two concentrations of hair were treated identically. The molecular weights given are the average values for the 1% and 5% hair derivatives. 70 Petroleum Ether Washed Hair 1% or 5% X — : --------- Supernatant Solubilized Keratin Derivative 0.5 M DTDGA 0.01 M TA 6 M Urea 2 M Tris HC1 pH 7.50 500 c 14 Days — 1 Amorphous Residue Adjust to pH 4.3 I --- Acid Soluble Fraction Bio Gel Peak #1 M.W. 13,600 (P-100) Peak #2 M.W. 6,000 1 Acid Insoluble Fraction Bio Gel (P-100) I I Peak #1 Peak #2 Peak #3 M.W. M.W-. Excluded 13,600 6,000] Bio Gel (P-300) Peak #1 M.W. 120,000 71 of the supernatant which varied from 8.4 to 8.8 was brought to pH 4.3 with 5 N acetic acid. The ionic strength was slowly reduced to 10“3 M acetate buffer, pH 4.3. As the ionic strength was reduced to 10“3 - 10-3 at pH 4.3, a precipitate was formed. The superna tant was found to consist largely of low molecular weight, high sulfur derivatives, while the precipitate consisted mostly of higher molecular weight, low sulfur derivatives. The separation at this stage was nearly complete; but traces of contaminant, which coprecipitated with the molecular weight derivatives, were evident from disc electrophoretic patterns of the acid insoluble fractions (Figure 13). Molecular Sieve Chromatography The supernatant and pH 4.3 insoluble fractions from 1% hair were each chromatographed on Sephadex G-100 and G-200 (Figures 14 to 16 inclusive) and the corres ponding fractions from 5% hair experiments were chromato graphed on Bio Gel P-100 and P-300 (Figures 17 to 19 inclusive). The 120,000 MW, pH 4.3 insoluble major peak was excluded from Sephadex G-100 and Bio Gel P-100, but was chromatographed on Sephadex G-200 and Bio Gel P-300. The chromatography of the supernatant fraction gave two peaks (Figures 16 and 19) while the pH 4.3 insoluble fraction gave three distinct peaks (Figures 17 and 18). 72 This fraction disappeared on reexchange and formed the 6.000 MW subunit. Additional Experiments On The Disulfide Exchange Reaction Effect of Hair Concentration When the disc electrophoretic properties of the major bands from 1% hair (Table V) were compared with those obtained from 5% hair, the differences in migration from the space gel were not significant, indicating that the fractions were similar with respect to molecular size and charge. They varied only in the relative amount of each fraction solubilized. Increasing the hair concen tration increased the overall yields of soluble peptides considerably, but the percentage yields were decreased for both the high and the low sulfur fractions. In the case of the 5% hair concentration, the overall yield of 2,250 milligrams was greater than four times the overall yield in the 1% hair experiments for the same relative volumes of reaction media, but 55% of the hair remained undissolved (Table V). The yield of 6,000 MW high sulfur peptides from 5% hair was about five times that obtained in the 1% hair experiments. The absolute amounts of the 120.000 MW, low sulfur fraction did not differ between the 10% and 5% hair levels. An additional slow moving band in the 5% hair experiments, comparable to that in 73 Fig. 13— Disc electrophoresis densitometer tracings of fractions from reexchange experiments with DTDGA. (A) Pool of principal fractions after first exchange reaction. (B) Acid soluble fraction after first exchange reaction. (C) Acid insoluble fraction after first exchange reaction. (D) Pool of fractions after second exchange reaction. (E) Acid soluble fraction after second exchange reaction. (F) Acid insoluble fraction after second exchange reaction. (G) Urea fraction, no exchange reaction. (H) Urea fraction after exchange with DTDGA. Conditions of the acrylamide gel electro phoresis are as described in the text and migration distances are given in Table VI. 75 -Fig. 14— Chromatography of acid insoluble exchange derivatives from 1% hair on Sephadex G-100. The column dimensions were 1.0 x 150 cm. The eluting buffer was 0.1 M tris HC1 pH 7.50 in 0.1 M NaCl. The flow rate was 5 ml per hour. Fractions were collected at 12 minute intervals. The mean VQ was 35.0 ml and the mean was 110.0 ml. The Lambda pump and Isco ultra violet monitor was employed. Absorbancy at 2 8 0 nm i 0 .5 0 .4 0.3 0.2 0 . 1 120 140 100 8 0 6 0 4 0 20 Effluent Volume (ml) 77 Fig. 1.5— Chromatography of acid insoluble exchange derivatives from 1% hair on Sephadex G-200. Column dimensions were 1.0 x 150 cm. The eluting buffer was 0.1 M tris HC1 pH 7.50 in 0.1 M NaCl. The flow rate was 5.0 ml per hour. Fractions were collected at 12 minute intervals. The mean Vo was 14.5 ml and the mean Vi was 47.2 ml. The Lambda pump and Isco ultra violet monitor were employed. Absorbancy at 280 nm 78 0 .5 0 .4 0 .3 0.2 7 0 6 0 5 0 4 0 3 0 E ffluent Volume (ml) i 79 Fig. 16— Chromatography of acid soluble exchange derivatives from 1% hair.on Sephadex G-100. Column dimensions were 1.0 x 150 cm. The eluting buffer was 0.1 M tris HC1 pH 7.50 in 0.1 M NaCl. The flow rate was 5 ml per hour. Fractions were collected at 12 minute intervals. The mean V0 was 35.0 ml and the mean V± was 110.0 ml. The Lambda pump and the Isco ultraviolet monitor were employed. Absorbancy at 2 8 0 nm 0.5 0 .4 0 .3 0.2 140 8 0 100 120 6 0 Effluc:w Volume (ml) 81 Fig. 17— Chromatography of acid insoluble exchange derivatives from 5% hair on Bio Gel P-100. Column dimensions were 2.5 x 45 cm. The eluting buffer was 0.1 M tris HC1 pH 7.50 in 0.1 M Na Cl. The flow rate was 5 ml per hour. Fractions were collected at 12 minute intervals. The mean Vo was 60 ml and the mean Vi was 153 ml. The Lambda pump and the Isco ultraviolet monitor were employed. Absorbancy at 280 nm i 1 . 0 0.8 0.6 0 .4 0.2 6 0 8 0 100 120 140 160 Effluent Volume (ml ) 83 Fig. 18s-Chromatography of acid insoluble exchange derivatives from 5% hair on Bio Gel P-300. Column dimensions were 2.5 x 45 cm. The eluting buffer was 0.1 M tris HC1 pH 7.50 in 0.1 M NaCl. The flow rate was 5 ml per hour. Fractions were collected at 12 minute intervals. The mean Vo was 48.0 ml and the mean Vi was 147 ml. The Lambda pump and Isco ultraviolet monitor were employed. Absorbancy at 280 nm 0.8 0.6 0.4 0.2 160 140 120 8 0 100 Effluent Volume (m l) \ 85 Fig. 19— Chromatography of acid soluble frac tion from 5% hair on Bio Gel P-100. Column dimensions were 2.5 x 45 cm. The eluting buffer was 0.1 M tris HC1 pH 7.50 in 0.1 M NaCl. The flow rate was 5 ml per hour. Fractions were collected at 12 minute intervals. The VQ was 60.0 ml and the was 147 ml. The Lambda pump and Isco ultraviolet monitor were employed. Absorbancy at 280nm 86 t I i ‘ i 0.8 0.6 0.4 0.2 160 100 120 140 8 0 Effluent Volume (ml ) Table V. Properties And Yields Of Fractions In Various Steps In The Isolation Of Keratin Disulfide Exchange Derivatives. The fractionation scheme is given in Figure 12 and yield measurements and electrophoretic data was obtained as described in the text. Step No. Operation Fraction M Yield (mgs) No. i of Bands Migration Dis tance from Space Gel (mm) *%Hair 5g%Sair lg%Hair 5g%Hair lg%Hair 5g%Hair 1 Exchange reaction Original 518 2,250 3 4 43 44 mixture 42 42 38 38 31 2 pH 4.3 low ionic Supernatant: 389 1,198 2 2 43 43 strength acid soluble 41 41 fraction Precipitate: 110 140 3 3 43 44 acid insol 42 42 uble fraction 38 38 3 Acid insoluble frac- Peak 1 76 _ 1 1 38 38 tion to Sephadex Peak 2 - - 1 1 41 41 G-100 or Bio Gel Peak 3 — - 1 1 43 43 P-100 4 Peak lofrom step 3 Peak 1 57 _ 1 1 38 39 to Sephadex G-200 Peak 2 - - 1 1 41 42 or Bio Gel P-300 Peak 3 — — 1 1 43 44 5 Acid soluble frac Peak 1 _ 1 1 41 42 tion to Sephadex Peak 2 353 - 1 1 43 44 G-100 or Bio Gel P-100 i the urea extracts was found in initial extracts. This fraction was found only when the peptides were removed immediately from the DTDGA exchange reagents and com plete exchange was not facilitated. Increasing the hair concentration did not have the effect of changing the character of the fractions generally, but resulted in an increase in the amount of 6,000 MW, high sulfur peptide which was depolymerized and solubilized. Six Molar Urea Extracts Almost no peptide was solubilized from hair in distilled water in the absence of other reagents, but when 6 M urea was used with tris HC1 buffer, pH 7.50, 15% of the finely cut hair dissolved and was recovered after 14 days at 50°C. A single peak was obtained on disc electrophoresis that migrated ca. 30 mm from the space gel into small pore gel (Table VI and Figure 13). On Bio Gel chromatography, the fraction had a MW of 48,000 (Table VII and Figure 20). The urea fraction was reduced on complete exchange to a single band correspond ing to the 6,000 MW high sulfur peptide. The use of 6 M urea in the disulfide exchange reaction medium increased the yield from 33 to 49%, when compared with the reaction carried out in its absence. Higher concentrations of urea were found to increase the yield only about 1% above that obtained with 6 M urea. Table VI. Summary Of The Number Of Bands And Migration Distances In The Keratin DTDGA Reexchange Experiments In Disc Electrophoresis. The lyophilized fractions were reduced in the 0.5 M DTDGA and 10 mM TA. All conditions of the reexchange reactions were iden tical with the original exchange reaction. The disc electrophoresis employed 7.5% acrylamide gel and was performed as described in the text. Fraction First Exchange Reexchange Bands Distance!from Space Gel (mm) Bands Distance from Space Gel (mm) Acid soluble (MW 6,000) (lg% and 5g% Hair) 2 44 42 1 44 Acid insoluble (MW 120,000) (lg% and 5g% Hair) 3 44 42 38 2 44 38 Pool of acid soluble and acid insoluble fractions 3 44 42 38 2 44 38 Urea 1 31 1 44 oo <0 90 Fig.— 20 Chromatography of urea extract of 1% hair on Bio Gel P-100. Column dimensions were 2.5 x 45 cm. The eluting buffer was 0.1 M tris HC1 pH 7.50 in 0.1 M NaCl. The flow rate was 5.0 ml per hour. Fractions were collected at 12 minute intervals. The mean VQ was 60 ml and the was 147 ml. The Lambda pump and Isco ultraviolet monitor were employed. 3 Absorbancy at 280nm 1.0 0.8 - 0.6 0.4 0.2 120 100 . 60 80 4 0 Effluent Volume (ml) \ Reexchange And Urea Fraction Exchange The two principal fractions did not show any change in disc electrophoretic properties after reex change, which indicated that the initial exchange of these fractions was complete (Table VI). Initial extracts contained traces of a 13,400 MW intermediate band, which disappeared after reexchange and added to the principal high sulfur, 6,000 MW fast moving band. In the case of the 5% hair concentration experiments, there was the appearance of an additional disc electro phoretic band which corresponded to the urea band. After exchange, the urea band was replaced by the 6,000 MW fast moving band (Figure 13). Influence of Temperature Initial yields of the disulfide exchange reactions carried out at 22° C were so small that no absorption at 280 nm was detectable during the first 18 to 24 hours. Total yields of 1% and 5% hair con centration experiments at 22° C did not exceed 2% of the original hair concentration. These results suggested the employment of the higher reaction temperature of 50° C. The nature of the disc electrophoretic bands obtained was identical at both temperatures, but the total yields of hair solubilized after 326 hours incu bation increased from less than 2% at 22° to 52% at 50PC. 93 Characterization of Derivatives Disc Electrophoresis Three principal disc electrophoretic bands, migrating 44, 42 and 38 mm from the space gel into the the small pore gel, were found in 1% hair exchange derivatives, using 7.5% acrylamide gel. These bands corresponded to the 6,000, 13,000 and 120,000 MW frac tions respectively. In 5% hair experiments there appeared a band which migrated with that found in urea extracts of hair and was found to be capable of further reduction in size from 48,000 to 6,000 MW by undergoing exchange. Effect of Varying Pore Size Using 5% acrylamide gel i.e. larger pore size than that of 7.5% acrylamide, obscured the 13,000 MW fraction so that it was indistinguishable from the 6,000 MW band. On the other hand, the use of smaller pore size e.g. 15% acrylamide which increased the sep aration of the 6,000 and 13,000 MW fraction, prevented the 120,000 MW fraction from entering the small pore gel. Molecular Weight by Molecular Sieve Chromatography A molecular weight of 120,300 was obtained for the major peak of the acid insoluble fraction in the 1% hair experiments and the 5% hair experiments gave effec- 94 tively the same values. The second and third peaks from the acid insoluble fraction gave molecular weights of 13,400 and 5,900. Values similar to these were obtained when the same determinations were made with Sephadex G-200 or Bio Gel P-300 on the 1% and 5% hair concentra tion derivatives, respectively (Table VII). Mean molecular weights of the principal fractions for both the 1% and 5% preparations were found to be 6,000 for the smallest acid soluble, high sulfur fraction, 13,000 J for the peak in this fraction and 120,000 for the prin cipal acid insoluble, low sulfur fraction. Stokes Radius from Molecular Sieve Chromatography The mean Stokes radius determined from Ackers solution (2) to the Renkin equation (47) for the acid soluble fraction of 13,000 MW was 15.0 A° for both the 1% and 5% hair preparations. The 6,000 MW acid soluble fraction gave a mean value of 6.4 A° for both the hair concentrations, while the 120,000 MW fractions had a mean Stokes radius of 46.7 A° for both hair preparations (Table VIII). Stokes radius for peak number three on Sephadex G-200 or Bio Gel P-300 could-not be determined from the column data, since the retardation was greater than 100%. The Renkin relationship cannot be employed when retardations are greater than two logarithms. Table VII. Molecular Weights of Keratin DTDGA Fractions From Molecular Sieve Chromatography. The distribution coefficient (Kd - Ve/Vo)for each standard was plotted against the logarithm of the concentration as described in the text. The molecular Weight of each peak was obtained from its corresponding Kd from Figures 1 to 4 inclusive. DTDGA FRACTION Designation Gel No. of Peaks Peak No. Ve/Vo (Kd) Molecular Weight Acid insoluble, lg% Hair Sephadex ; 3 1 1.37 Excluded (Figure 14) G-100 2 2.66 13,400 3 3.18 5,900 Acid insoluble, lg% Hair Sephadex 3 1 1.64 120,300 (Figure 15) G-200 2 3.59 13,200 3 4.32 6,000 Acid soluble, lg% Hair Sephadex 2 1 2.66 13,400 (Figure 16) G-100 2 3.19 5,800 Acid insoluble, 5g% Hair Bio Gel 3 1 1.14 Excluded (Figure 17) P-100 2 1.89 13,800 3 2.26 6,700 Acid insoluble, 5g% Hair Bio Gel 3 1 1.95 124,000 (Figure 18) P-300 2 2.61 14,000 3 2.84 6,800 Acid soluble, 5g% Hair Bio Gel 2 1 1.90 13,600 (Figure 19) P-100 2 2.29 5,400 Urea, lg% Hair Bio Gel 1 2.38 48,000 (Figure 20) P-100 Table VIII. Stokes Radius Determination Of Peaks Of Keratin DTDGA Exchange Derivatives From Molecular Sieve Chromatography. The Stokes radius from column data was computed from the Ackers solution of the Renkin equation, as described in the text. Where the retardation was greater than 100% i.e. for particles with Stokes radii less than 10 A°, on columns with pore sizes greater than 100 A°, the Renkin equation does not hold up and the column data will not give a Stokes radius. Retardation = Ve-V0/Vi. The calculated or reference Stokes radii, determined from the diffusion coefficient, were used in the estimation of the mean pore size (r). The calculated pore size or mean pore size was used in the calculation of the experimental Stokes radius (a). Protein Species Or Fraction Retardation (%) a/r(%) r(A°) a(A°)- Part A— Sephadex G-100 Data (1 g% hair) Bovine serum albumin 14.3 35.3 98 34.7 Soybean trypsin inhibitor 54.5 13.2 42 5.5 Peak No. 1 from acid soluble fraction 52.7 14.2 70 10.0 Peak No. 2 from acid soluble fraction 68.2 8.5 70 5.9 Part B— Sephadex G-200 Data (1 g% hair) Bovine gamma globulin 12.4 37.6 145 54.4 Bovine serum albumin 34.0 23.0 152L 34.7 Peak No. 1 from acid insoluble fraction 20.2 31.0 149 46.4 Peak No. 2 from acid insoluble fraction 60.0 8.4 149 12.6 Peak No. 3 from acid insoluble fraction 100 - - - Part C— Bio Gel P-100 Data (5 g% hair) Bovine serum albumin 8.0 43.2 81.0 34.7 Soybean trypsin inhibitor 26.6 24.5 22.4 5.5 Peak No. 1 from acid soluble fraction 11.7 38.3 52.0 19.4 Peak No. 2 from acid soluble fraction 54.2 13.3 52.0 6.9 <o Cl Table VIII— Continued Protein Species Or Fraction Retardation (%) a/r (%) r(A°) a(A°) Part D— Bio Gel P-300 Data (5g% hair) Bovine gamma globulin 31.0 23.5 232 54.4 Bovine serum albumin 39.6 19.5 178 34.7 Peak No. 1 acid insoluble fraction 32.6 22.8 205 47.0 Peak No. 2 acid insoluble fraction 59.8 11.6 205 23.8 Peak No. 3 acid insoluble fraction 100 — — \ L CD -a 98 Ultraviolet Spectrophotometry And Absorptivity Both major fractions have no absorption in the visible spectrum. In the ultraviolet (UV) both samples began absorbing at about 340 nm, increasing in absorp tivity as wavelength decreased to 278 nm. Initial slopes of absorptivity of the acid insoluble fraction were somewhat greater than for the acid soluble fraction. The former spectra reached a maximum at 278 nm and declined to a minimum at 260 nm. The absorption spectrum of cystine roughly paralelled both fractions (Figure 21). Concentrations, corrected for moisture content, selected for the spectra were 1.0 mg/ml for the 6,000 MW acid soluble fraction and 0.3 mg/ml for the 120,000 MW acid insoluble fraction. These were consis tent with the absorptivity coefficients at 278 nm of 6.5 for the former and 20.3 for the latter. It was possible to determine the concentrations of the purified preparation directly from the absorbancy in mgs per ml by the use of the conversion factors (Table IX). Infrared Spectrophotometry Infrared (IR) spectra of both major fractions showed two broad water bands between 3680 to 2800 cm"1 and 1750 cm-1 to 1480 cm”1-. The spectrum of the 6,000 MW acid soluble fraction (Figure 22) had three carbonyl stretching modes. Amide I and II absorptions in both 99 Fig. 21— Ultraviolet absorption spectra of disulfide exchange derivatives and cystine. Cary Model 15 spectrophotometer. ---120,000 MW acid insoluble peptide (0.3 mg/ml). ---6,000 MW acid soluble peptide (1.0 mg/ml). ...cystine (1.0 mg/ml). Absorbancy 1 . 0 0.8 0.6 0 .4 0.2 3 0 0 3 4 0 2 6 0 220 X (nm) Table IX— Summary Of Chemical And Physical Properties Of Principal Peaks Of Keratin DTDGA Exchange Fractions From Sephadex Chromatography. Properties of Sample Acid Insoluble Fraction Peak No. 1 From Sephadex G-200 Acid Soluble Fraction Peak No. 2 From Sephadex G-100 A*% (at 278 nm) 1 cm 20.3% 6,5% Conversion factor (absorbancy to mg/ml) 0.49 1.54 Nitrogen 1 1.6% 5.7% Molecular Weight 120,000 6, 060 Stokes Radius 46.4 A° 5.9 A° Helicity in 10 M tris HC1 buffer, pH 7.5 from ORD data 70.5% 15.0% Sulfur By amino acid analysis By chemical analysis 1 .68% 3.94% 5.32% 8 .88% 101 102 Fig. 22— Infrared spectrum of 6,000 MW acid soluble keratin DTDGA fraction. KBr pellet prepared from purified derivative after dehydration over P2O5 at 500 c. The Beckman Model 7 infrared spectrophotometer was employed. Percent Transmittance 103 i 8 0 7 0 6 0 5 0 4 0 3 0 20 1000 4 0 0 2000 4 0 0 0 3 0 0 0 Wave number (cm "1 ) » 104 major fractions were obscured by the water bands. A broad band extending from 600-400 cm"1 was assigned as a disulfide stretching frequency (Table II). The IR spectrum of the 120,000 MW acid insol uble fraction (Figure 23) had a peak at 2920 cm"1 corres ponding to a CHg stretching mode, which according to Bellamy corresponded to the in-phase vibrations of the hydrogen atoms (7). C=0 and C-0 stretching frequencies were distinct in this fraction and were assigned (Table II). Disulfide stretching absorptions were also present in the 120,000 MW low sulfur acid insoluble fraction, but the band was not as broad as in 6,000 MW high sulfur acid soluble fraction. Absence of -S=0 and -SH absorptions was notable in both spectra. Nitrogen, Sulfur And Amino Acid Analyses Kjeldahl nitrogen content of the 120,000 MW, pH 4.3 insoluble fraction was 11.2%, while the value for the 6,000 MW fraction was 5.7%. Amino acid analyses did not reflect the thioglycollate sulfur contents which were lost during the hydrolysis procedure. Chemical analyses on the other hand, reflected the total sulfur contents (Table IX). In addition to the apparent differences in molecular size of the two fractions, they differed con siderably in amino acid composition (Table IV). Most 105 Fig. 23— Infrared spectrum of 120,000 MW acid insoluble keratin DTDGA fraction. KBr pellet prepared from purified derivative after dehydration over P2O5 50° C. The Beckman Model 7 infrared spectrophotometer was employed. Percent Transmittance 9 0 8 0 7 0 6 0 5 0 4 0 3 0 20 4 0 0 0 3 0 0 0 2000 1000 4 0 0 Wave number (cm-i) notable are the differences in 1/2 cystine residues between the two fractions. The 6,000 MW fraction con tained a threefold greater quantity of this residue than the 120,000 MW fraction. Marked differences also occurred in arginine, glutamic acid, glycine, histidine, isoleucine, leucine and tyrosine. Concentrations of aspartic acid, leucine,.lysine, proline and valine were qualitatively similar in the two fractions. No correc tions were made for the labile amino acids or those released more slowly from the peptides. The presence of tryptophan is indicated in the 120,000 MW UV spectrum. While values for the exchange derivatives were consis tent with those for the amino acid composition of human hair, they differed more from comparable derivatives of Merino wool than can be explained on the basis of the differences in amino acid composition of Merino wool and human hair. Optical Rotatory Dispersion (ORD) Far UV Cotton effects for both the 120,000 MW and the 6,000 MW high sulfur fraction in 8 M urea were incomplete i.e. they did not cross over to a maximum. The ORD spectrum of the 10”4 M tris buffer solution of 6,000 MW high sulfur fraction gave a far UV Cotton effect similar to those produced in 8 M urea. The 6,000 MW fraction, however, was induced to give a complete 108 Fig. 24— Far ultraviolet optical rotatory dispersion of the 6,000 MW acid soluble DTDGA fraction in various solvents. ..,2-chloroethanol, ---8 M urea and —— 10"4 M tris HC1 buffer pH 7.50. A Cary Model 60 spectropolarimeter was employed. T09H i i i < s > o CD o» CD • o ID I O X CD 3 0 0 34 0 260 220 180 X (nm) \ 110 helical Cotton effect in 2-chloroethanol with a minimum at 234 nm, crossover at 216 nm and a maximum at 216 nm (Figure 24). Rotatory dispersion of the 120,000 MW low sulfur fraction in 2-chloroethanol and 10”^ M tris HC1 buffer, pH 7.5, gave typical far UV Cotton effects associated with the a-helix (Figure 25). The helix induced 2-chloroethanol Cotton effect had a minimum at 233 nm, a crossover at 222 nm and a maximum at 202 nm, while the Cotton effect obtained in the dilute aqueous buffer pH 7.50, gave values of 231, 216 and 204 nm for the minimum, inflection point and maximum respectively (Table X). Dispersion data was used to obtain coefficients of the MTTDE and the Moffett and Yang parameters, aQ and bQ_. These were used to compute the helix contents of the two major fractions in each of the solvents used. In the dilute aqueous buffer the coefficients of the MTTDE gave helicities of 75.0% and 25.1% for the 120,000 and the 6,000 MW fraction. The helicities computed from the bQ values gave values of 70.5% and 15.0% for the same respective fractions. In 8 M urea the helicities approached zero, while in 2-chloroethanol they approached 100% (Table XI). Ill Fig. 25— -Far ultraviolet optical rotatory dispersion of the 120,000 MW acid insoluble keratin DTDGA fraction in various solvents. ...2-chloroethanol, 8 M urea and ___10"'* M tris HC1 buffer pH 7.50. A Cary Model 60 §pectropolarimeter was employed. 112 i I C / > CD CD cn CD X > lO J O X 3 0 0 3 4 0 180 220 260 X (nm) Table X— Far UV Cotton Effect Characteristics From Optical Rotatory Dispersion Data Of Principal Peaks Of Keratin DTDGA Exchange Derivatives. The rotatory dispersion was studied on a Cary Model 60 Spectropolarimeter, as described in the text. The specific residue rotations [$’]were computed using a means residue weight (MRW) of 109 from U 1] = [a]^ (MRW/100) (3/ n + 2) where ta]^ is the observed rotation and n the refrac tive index of the solvent. The wavelength (X) is expressed in nanometers (nm). Peak Condition Minimum Crossover Maximum % nm Degrees nm nm Degrees Acid insoluble (MW 120,000) 8 M urea 2-chloroethanol 10~4 M tris HC1, pH 7.50 202 233 231 -4360 -3996 -3815 222 216 202 204 +9810 +7085 +62.7 +695.0 +488 Acid soluble (MW 6,000) 8 M urea 2-chloroethanol 10-4 M tris HC1, pH 7.50 217 234 214 -4033 -2725 -3348 216 216 +1090 86.3 474.0 108.4 113 Table XI— Optical Rotatory Dispersion Constants For Keratin-DTDGA Exchange Derivatives. Coefficients Of The MTTDE And Moffett Yang Equation And Corresponding Helicity Estima tions. The percentage helicity from the modified two term Drude Equation (MTTDE) was determined from Hmttde = A(a,p)l93 - A(«,p )225 + 650/55.8. The MTTDE and M¥ constants were obtained as described in the text. The helicity from the Moffett Yang (MY) bo . coefficient was from Hmst = -100 bo/750. Peak Solvent A193 A225 HMTTDE^ ao bo HMy(%> Acid insoluble 8 M urea -146 -264 13.7 +328 -18 2.4 (MW 120,000) 2-chloroethanol +2848 -2065 99.8 +369 -751 100.0 10"4 M tris HC1, pH 7.50 +2028 -1431 74.0 +320 -529 70.5 Acid soluble 8 M urea -461j -181 6.7 +567 -43 5.7 (MW 6,000) 2-chloroethanol +1963 -1388 72.0 +311 -513 68.4 lO"4 M tris HC1, pH 7.50 +346 -391 25.1 -113 -113 15.0 114 Circular Dichroism (CD) The 6,000 MW fraction gave similar spectra in 6 M urea and 10"^ tris HC1 buffer (Figure 26). Ellip- ticity spectrum of the 6,000 MW fraction in 2-chloro ethanol presented greater slopes and reached higher molecular ellipticities at the peak in this solvent, reflecting the induced helicity. Values of intensity for the negative band of the 6,000 MW fraction do not show as great a difference between helix favoring and disrupting solvents. The molecular ellipticity of the 6,000 MW fraction is 10“^ M trisS'HCl buffer, pH 7.50 was closer to the value obtained in 8 M urea than to the [0] of the peak in 2-chloroethanol, which reflected a natural tendency of this fraction to assume random configuration. CD spectra provided additional insight into the conformation of the principal fractions. The CD of the 120,000 MW fraction under various conditions presented marked variations in the intensities of the peak of the negative band (Figure 27). The position of the peak of the negative band of both fractions in all solvents was at 210 nm, except urea (Table XII). The molecular ellipticity [0] at the peak for the pH 4.3 insoluble 120,000 MW fraction was greatest in 2-chloro- ethanol, while in 8 M urea and 10”^ M^ tris HC1 buffer, 116 Fig. 26— Ultraviolet circular dichroism of the 6,000 MW acid soluble keratin DTDGA fraction in various solvents. ...2-chloroethano, ---8 M urea and 10“4 M tris HC1 buffer pH 7.50. A Cary Model 60 spectropolar- imeter equipped with a Cary Model 6001 Circular dichro- ism accessory was employed. (S09J60P) ^_0! X [ 0 ] -2 -4 - 5 20 0 220 240 X(nm) 260 280 • i • 118 ■Fig. 27— Ultraviolet circular dichroism of the 120,000 MW acid insoluble keratin DTDGA fraction in various solvents, ..,2-chloroethanol, ---8 M urea and ---10-4 m tris HC1 buffer pH 7.50. A Cary Model 60 spectropolarimeter equipped with a Cary Model 6001 circular dichroism accessory was employed. i to o o -I o>

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Schlesinger, Richard Joseph, 1925- (author)

Core Title Disulfide exchange derivatives of human hair keratins

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1969-06

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor McNall, Earl (committee chair), Bavetta, Lucien A. (committee member), Kharasch, Norman (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-707177

Unique identifier UC11362278

Identifier 6917890.pdf (filename),usctheses-c18-707177 (legacy record id)

Legacy Identifier 6917890.pdf

Dmrecord 707177

Document Type Dissertation

Rights Schlesinger, Richard Joseph

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