A T W The raw. adsorption data was then graphed in a log P/P0 vs. 1/T plot (Figures 21, 22, and 23) and after drawing a smooth curve through the points,, the appropriate P/P0 values were obtained by visual interpolation. These smoothed P/P0 vs. W values for various temperatures with water adsorption on denatured egg albumin are collected in Table I, The slope, A(P/P0)/AT, was then obtained in the following manner: T(°K.) 298 .313 328. 3^3 .358 373 (P/Pp) 0.125 .170 • 21C .265 .315 .375 A(P/P0)./AT (x 103) 2.b 2 2.81* 3 .1 8 3.50 3 .6 6 3.85 Aln(P/P0)/AT (x 10.3) 19.1 +0 16.70 L5.10 13.20 Ll.60 10.25 Qs - 3.15 3. 2b 3.21 * 3.36 3 .2 2 3 .0 8 The slope, at 328°K. equals (0.265-0.170)/(3l f3-313) or 3 .1 8 x 10~3. However, the first (298°K.) and last slopes 11 . J ' 55 ton oo i.o TCC) 09 01 0.6 OS 0.4 0.3 WATER ADSORPTION ON DENATURED EfrG ALBUM/N (FIGURES 3,7,9,11,12,13,14) 0! 3.2 3.1 2 9 3 3 2.8 2 7 FIGURE 25 100 , 90 30 70 55 10 0 9 W ■ - 1 6 05 04 0 5 0.2 WATER ADSORPTION ON . DENATURED EGG ALBUMIN (FIGURES 5t ?t 9, H, /2 ,13' M ) O T . 31 3 2 2 3 2 7 LOG ~ j a "d /d O O' 1 xj n-l* ^ Ya P/E^ « . J ar/p^V / W ^ ^AT Jn*l ^AT |n \AT /n**l whereby a third slope at point n ♦ 1 is obtained from know­ ing two other consecutive slopes at equidistant points, n, and n - 1, obtained in the manner Just illustrated* All of these slopes were then divided by the appro­ priate P/P0 to yield Aln(P/P0)/AT which in this case, W =5» has the values given on page 3 6. These slopes are 2 finally multiplied by the appropriate RT to yield the values of Qs - A H V tabulated on page 3 6. These values may be compared with those on page 35 to illustrate the smoothness of slopes computed in this fashion. A complete table of Qg - AHy for all the adsorption data on denatured egg albumin calculated by the method just described, follows as Table II* Included with this data are values of the heat of vaporization of water, AHV, 5 taken from the International Critical Tables. Selected W-values (Figure 21 *) from the desorption curves of this same denatured egg albumin have also been treated similarly Stf. E. Milne, Numerical Calculus (Princeton Univer­ sity Press, Princeton, N* J•, 19^9)> p. 135 ^International Critical Tables (McGraw-Hill Book Co., New York, 1928), V, 138. TABLE II W 5 6 I 9 10 II 12 a 11 17 19 20 21 NET HEAT OF ADSORPTION (Q3- AHV> KCAL./MOLE) OF WATER ON DENATURED EGG ALBUMIN 25°c. 40 _ . 55 _ — ZQ ----- 85— _ 3.15 3.24 3.24 1.1 + 6 2.1+1 3.03 2.22 3.11 2.63 1.62 1.94 2.13 1 .3 2 1.28 1.58 1.19 1.33 1A1 0.522 0.830 1.10 0.505 0.740 0.935 1.48 I.08 0.702 1.42 1.03 0 .5 5 8 0.360 0.396 0.396 0.432 0.378 0.270 0.468 0.505 0.576 0.32*+ 0.390 0.450 0.252 0.316 0.32*+ 0.216 0.288 O.36O A Hv(kcal./mole) 10.50 . 10.35 10.18. 3.36 3 .2 2 3 .5 2 4.07 3 4 5 2.92 2.80 3.03 2.36 2.56 1.80 2.11 1.46 1.75 1.10 1.26 0.810 1.06 0.485 0*702 0.505 0.650 O .306 0.611 0.685 0.468 0.505 0.432 0.234 ° 0 - M 10.00 9.90 3.08 4.50 2.94 3.71- 2.95 2.36 2.27 1.35 . 1.32 0.900 0.790 0.126 0.486 0.540 .8:121 9.70 '■r ro 55 80 ICC 0 9 00 07 0 6 05 0.3 02 WATER DE50QPTI0M ON DENATURED E&& ALBUMIN (FIGURED 5,7,9,/// E II3II4) Ol 3 2 2 9 3 .1 3.3 2 8 2 7 "d /d NO T ' k b In order to yield Table III. This 3arae method of comput­ ing slopes was also tried on the four-point native egg albumin isotherms givejp in Figure 25, but.because there were only four points for each W, the results turned out poor. Instead, therefore, other formulas developed by 6 Milne were used to evaluate the appropriate slopes from the'values, of Tabie iy. Milne's formulas compute deriva­ tives algebraically by evaluating the coefficients of equations that pass, through all of the experimental points. The slope, {AP/PQ/AT), obtained in this manner and mul­ tiplied by the appropriate RT^/(P/P0), yields the values of Qa - A Hv listed in Table V. The various Qs - values for Increasing quantities of sorbed water on denatured egg albumin given in Tables II and III have also been graphed in Figures 26 and 27* As will be made more evident in the discussion that follows, it is of great interest to calculate the entropy and enthalpy changes that water undergoes in the process of sorption. These parameters may then be compared with the thermodynamic changes that result upon the condensa­ tion of water vapor to form the liquid or even ice, in order to give some idea as to the physical properties of sorbed water. . It is further to be expected that such a ^Milne, op. cit.. p. 97. TABLE III RELATIVE HUMIDITY (P/P0) AND NET HEAT OF DESORPTION (Qg - KCAL./MOLE) OF WATER ON DENATURED EGG ALBUMIN X T ■ : 2 5 . _ C. 40 5 5 20 8 5 1 Q O w 5 P/Pn 0.050 o.loo -0.130 0.165 0.210 0.300 .58 6.>» ; 6 .8r Qj -AH, 5 . * t O 5.18 3.58 * 4 . 11 * 6.1(0 6.85 10 P / P o 0.285 0.345 0 . 4 4 0 0 . 5 5 0 0 . 6 5 0 0.680 Q g - & H / 2 . 4 3 2 . 9 2 3 * 2 2 . 3 * 2 6 . 1 . 8 4 . 2 . 3 6 15 p / p _ 0 . 5 7 5 0.690 0.760 0.800 O .830 0.870 Q g 2 A h v 2 . 1 6 1 . 7 3 1 . 0 3 0.738 0 . 6 5 0 0 . 7 9 5 2 0 P / P Q 0 . 7 6 0 0 . 8 6 0 0 . 8 9 0 0 . 9 0 0 0 . 9 2 0 0 . 9 6 0 Q s - A H y 1 . 4 2 0 . 9 5 5 0 . 3 2 4 O .432 0 . 3 9 1 0 . 3 7 8 -r FI6URE 2.5 naW£ F / ’ JTaia-)- (nr.uPLJ 4' " . -oeso2p l TABLE IV RELATIVE HUMIDITY OF WATER (P/PQ) VS. WATER SORBED (W) ON NATIVE EGG ALBUMIN OVER THE TEMPERATURE RANGE 25 TO 70°C. 0.060 .100 0.150 .200 .270 0.100 .UK) .185 .250 • 325 270 .200 . 250 •315 • 515 • 590 530 590 690 10 • 585 .690 0 .8 60 610 700 .V* 860 .670 0.790 0.900 0.900 A = Adsorption D = Desorption TABLE V NET HEAT OF SORPTION (Qg - KCAL./MOLE) OF WATER ON NATIVE EGG ALBUMIN \ T 25°C • i +0 55 70 w ■ . ■ A .■ D A D A D A. ■ D b 2 .h i 12.1 0.6if7 if.32 2.60 0.660 7.38 —0*8l 5 0.775 5.05 1.2*f 3-33 2.7if 1.9^ 5.22 ♦0.J7 6 l.Uo 0.905 l.Oif 3.27 1.78 2.21 4.6 7 2.6i f 7 2.59 2.91 l.Uo 2.85 1.78 2.86 3.36 3.16 8 1.2 1 if. 12 1.51: 1.86 3*iZ 2.2»f 1.26 5.22 9 0.828 3.95 1 .7 2 3.0§ 2.02 2.36 4.12 10 3.69 0.828 1.53 1.08 1.59 1.33 3.55 : 12 O .828 3 .2 8 0.790 1.37 0.935 1.36 1.46 3.32 V* °*5ft 3.38 O.i+68 1.26 1.32 0.925 1.26 2.03 16 0.288 2 .52 0> 1V 1.10 0.595 0.635 0.720 0.890 20 0.126 1.30 0.396 0.780 0.505 0.if98 0 .6 1 2 0.595 A = Adsorption D = Desorption KCAL./MOLE 'KCAL./MOLE F IG U R E E6 NE T HE A T OF 50RPT/OM O N. DENATURED E & & A L B U M IN AT 2 5 / 4 0 , 5 5 °C. a D E 30R P T I0N AD 5Q R PTI0N A ^ ^1 -J ! kj 7 FIG U R E 2 7 • . MET HEAT OF 50RPT!ON O N ' D EN A TUPED; ECrCr A i Q UM JN. . AT 10, Q5,1 (X)°C. • AD 5Q R PT/0N GDE5QRPT/OAJ 6 £ 4 2 / /a?°c. 4 N . 3 2. / ■ 7 0 ' £ . £ 10 12 14- 16 IE 2 0 6 4 Q 2 comparison may somehow contribute to our better understand­ ing the peculiarities of protein-water sorption. For convenience* these calculations are limited to W-values of 5, 10, 15 and 20 gm. of water/100 gm. of egg albumin over the 25 to 100°C. temperature rahge. These calculations have also been restricted to the steam- denatured egg albumin data because of the greater precision of these sorption data. Writing: F - H - TS and F = F° ♦ RT In P/P0 the following formulas can be written, with the subscripts, s, and v, referring to ' ‘sorbed" and "vapor": Fs■= F°„ ♦ RT In Ps/P0 Hs H°v * 9,- !since the free energy of the sorbed water (Fs) is equal to that of the vapor (Fv) and the enthalpy (H°v) of the water vapor is assumed only temperature-dependent. Therefore, since ' 52 ' . - TS° ♦ HI In P3/Po = 4 - Ss - TSa and, therefore, Sg - - - S$ f R: In P0/P3 - Qs/T To calculate S , the partial molar entropy of the 7 sorbed water, we note that Glatt finds that the spectro­ scopic entropy of water vapor, . S^, at 25°C. i» ^5-10^5 cal./mole/°K, together with a table of other entropy values at still higher temperatures. Prom these values together with entropy of vaporization data from the International 8 Critical Tables an absolute entropy table for both gaseous (v) and liquid (1) water has been constructed in Table VI. Taking the enthalpy of liquid water at 0°C. as zero kcal./ mole, an enthalpy table, Table VII, has also been con­ structed for both the gaseous and liquid states of water, 9 the data for which has been taken from steam tables. Com­ bining the data of Tables II, III, VI, and VII, we have made tables of some values of both the partial molar entropy, SaJ and enthalpy, H , of adsorbed (A) and desorbed s s ?L. Glatt, J. Adams, H. L. Johnston, Technical Report ^16-8 (Ohio State University, Cleveland, Ohio, 1953), p. 19. ^International Critical Tables, loo, clt. *0. A. Hougan and K. M. Watson, Industrial Chemical Calculations (J. Wiley and Sons, Inc.,. New York, I9*f6) , p. l*+3. . 53 TABLE VI ENTROPY OF WATER Temperature (°C.) . S° (cal./mole/°K.) qO 1 2 5 ^ 5 . 1 0 ^ 5 9 . 9 0 hO ^ 5.5066 12.50 5 5 ^ 5 . 8 7 7 ^ 11+.88 7 0 1 +6 . 21+88 ■ 17.05 8 5 1 + 6 . 5 8 9 1 1 8 . 9 9 1 0 0 1 +6 . 931+3 20.83 TABLE VII ENTHALPY OF WATER Temperature (°C.) (kcal./mole) 0 10.70 0.00 25 10.95 .1+5 1 + 0 11.09 .72 55 11.18 0 .9 9 70 11.28 lc 26 85 . ll.l+O 1. 51+ 100 11c 50 lo80 (D) water on denatured egg albumin (Tables VIII and IX). From the data of these Tables VIII and XX it i3 con­ cluded that desorbed water is bound more strongly than water adsorbed at the same W and.temperature, T. This.is, of course, indicated by the lower vapor pressure of the desorption isotherm for any given W and T. But from Table VIII it can be seen that the partial molar entropy of the bound water on desorption is lower than it is upon adsorption, indicating that desorbed water is also less free to move than the adsorbed water. This has also been mad© graphical in Figure 28. 10,11,12,13 Current theories of sorption hysteresis attribute its occurrence to the existence of two distinct domains in the solid adsorbent. Hysteresis supposedly results because the partition of sorbed molecules between the two different domains is a property beyond the control of the experimentor, i.e., an additional degree of freedom is present. While the thermodynamic calculations presented herein seem to imply that desorbed water is more strongly ^D. H. Everett and W. I, Whitton, Trans. Faraday Soc., i*8, 7*f9 (1952). ■^D. H. Everett and F.W. Smith, Trans. Faraday Soc., 18? (195^). 12D. H. ^J. A. Enderby, Trans. Faraday Soc.., £1, 835 (1955). 12D. H. Everett,: Trans. Faraday Soc., 1077 (195^). TABLE VIII ENTROPY (S , CAL./MOLE °K.) OF SORBED WATER (W) ON DENATURED EGG ALBUMIN V 25°C HO 11 JO_ m w 5 10 15 20 A 3.6 5.7 7.9 D -2.1* 0.5 7.9 A 7.5 9.5 D H. 2 5*2 6.5 A 9.5 11.9 1^.1 D if.2 7.7 12.2 A 9.5 11.8 1H.1 D 5.7 . 9.9 1H.1 *Heat of sorption error. 2*8 8.6 12.9 8 A 15.8 15.2 15.8 15.8 12. H >.1 12.0 1H.8 17.6 17.6 18.0 18.0 1H.6 . H,8 15 .2 15.5 19.1 19.1 20.0 20.0 vn '»n ENTHALPY (H , KCAL./MOLE) 9 \ X , 25°C . Uo w 5 A -2.70 -2.52 D -3.15 -3.^+6 10 A -0.71 * -0.63 D -1.98 -2.20 15 A ♦0.09 ♦0.32^ D -1.71 -1.61 20 A ♦0.20 ♦0.4-11 * - D -0 • 97 -0.23 TABLE IX OF SORBED WATER (W) ON DENATURED EGG ALBUMIN . 5 5 _______70 85 100 -2.25 -2.11 ■-I.69 -1.28 -2.59 -2.88 -4.87 -5.0^- -C.l+l1 * -0.5^ -0.575 -0 .5 8 -2.25 -2.0 -C.306 .-0.56- ♦0.591 * ♦0.755 ♦0.8 81 ♦l.oi -C.036 ♦0.522 ♦0.8 81 ♦1.01 ♦0 .6 7 ♦0 .8 3 ♦1.13 ♦1A 2 ♦0 .6 7 ♦0.8 3 ♦1.13 . On f ig u r e e e ■NET E N T R O P Y OF, S O R P T I O N O N . DENATURED EGO ■ AL BUM IN • ADSORPTION a DESORPTION . 70 'C 5 • i i ao bound to the protein.than Is adsorbed water, these compu­ tations, however, offer no.direct evidence to support Everett's explanation of thi3 intriguing phenomenon# ■CHAPTmR V- MA INTAILING CON^TalJT- VnPOR PHBSSUhE IN ' .THE PROT^lN-PANuTUHnTJON APPARATUS Preliminary work on determining the effect of rela­ tive humidity upon the denaturation-rate was begun by trying to use salt hydrates as the method of maintaining constant humidity. Tables of those salts useful in the temperature range 80 to 100°G. appear in the International .Critical Tables,^ Landolt-Bornstein Physikalisch-Chemlsche 2 ^ Tabellen, Lange's Handbook of Chemistry, Handbook of I x . Chemistry and Physics, and the Chemical Engineering Hand­ e d book. Gulphuric acid-water mixtures are ruled out since data for this system has been determined only in the room temperature region.^ 1International Critical Tables (McGraw-Hill Book Co., New York, 1926), I, 67. " ^Landolt-Bornstein Physlkallsch-Chemlsche Tabellen, 6th Auflage (Julius Borlnger. Berlin, Germany. 1928!), I2S0, 1907. ■ ' : -^Lange's Handbook of Chemistry (Handbook Publishers, Inc., Bandusky, Ohio, 1992), Bth edition, p, l^B* ^Handbook of Chemistry and Physics (Chemical Rubber Publishing Co. , Cleveland, Ohio., i960) , 38th edition, p. 2315. . ^Chemical Engineering Handbook (McGraw-Hill Book Co. , New York, I960), 3rd edition, p. 797* ^R. ii. Jtokes, 2nd. mng. Chem. , Vl, 2013 (19‘ +9) . 6o From preliminary work using hydrates or- saturated • solutions of NaCl, Nal, Kbr and KI,- it was soon found that the rate of uenaturation Was very sensitive to changing vapor pressure. but while internal reproducibility (i.e., v pulling two samples simultaneously) was good to -2 per cent, the entire run could never be reproduced again at the same temperature and vapor pressure. duch problems inducea a further literature search in the journals themselves wherein there appear such state­ ments as: For maintaining a controlled constant mois­ ture in experiments the usual sulfuric-acid- vater or saturated salt solutions have the disadvantage of rather long time perious before the vapor pressure is established con­ stant. A rapid change in temperature is accompanied only by a sluggish establishment of the equilibrium after about 3 hours.7 When saturated salt solutions are employed for humidity .control, experience has shown that certain precautions must be observed in order that the theoretical values may be ■used without the need of measurement. It is necessary to enclose the saturated salt solu­ tion in a sealed chamber. The chamber and the fixtures therein must be made of non- hygroscopic materials, preferably metal or glass, .else the time required for humidity equilibrium to be achieved may be Very great, sometimes of the order of days.or weeks. . . . It is desirable for the salt solution to occupy as large a,surface area as possible and for some means of air ven­ tilation or circulation to be provided. . . . . ideal conditions are rarely obtained in practice, it is probable that- the 7p. LeClerc, billc. Ind., 237 (195*+); Chem. Abstr. 131861 ' ' '61 theoretical values of relative humidity are • seldom reached. In general use, saturated salt solutions should not be expected to con- .trol the relative humidity to closer than about one percent relative humidity of the theoretical values. o Nexler and Hasegawa, furthermore, compared the vupor-pressure results they obtained with other literature- data on the same saturates. They found that these in general fell within a band-width of ^1 1/2 per cent rela­ tive humidity using their NBG results as the reference. This is more clearly shown .in the following copies of graphs of their KNO^, K^bO^, and NaCl data (lines) against other sources (points) mentioned in the journal article itself (Figure 29). Jince it became apparent that saturated solutions were not a very good method of maintaining constant vapor pressure in this proposed kinetic system, a further litera­ ture search was made to find out if some other method had ever been considered. Parkas and Melville"^ describe a '‘manometer and source for hot vapors" developed by G. T. ..aim.11 . ■ ^A. Wexler and b. Hasegawa, J. he s. - Natl. Bur.' btds., l_i, 19 (195‘ 0. 9 Ibid.. l^A.. Parkas and H. U. Melville, Bxperlmental Methods in Gas Reactions (r-:a Chilian and Co., Ltd., London, Lngland, ■ ■ 1939) , p. 102. He. T. laiin, Hev. bd. Instr., 1, 299 (1930) . RELATIVE HUM ID! TV FSOU RE 19 'RELATIVE HUMIDITY O F ' S E V E R A L S A T U R A T E D SOL JT/OH5 k n o 5 & RESEARCHERS 97 95 95 8 9 67 4 0 5 0 20 30 10 % RELATIVE HUMIDITY Kz 5 0 * 6 RESEARCHERS 20 30 40 50 S is N a C l 10 researchers IQ 76 74 T(°C.) 20 30. ■ 40 50 10 This Is a device for supplying or maintain­ ing the pressure of a vapor at any desired . . value up to the temperature of the boiling point. . . » The normal procedure is simply to use a mercury manometer but in the present instance . . . the liquid itself . . . is the balancing manometer. . . . The mode of opera­ tion will be clear from the figure below: AIR A MANOMETER PUMP Liquid is run in at E Into D and distilled over to C. The apparatus is evacuated and the connecting tubing, wound with nlchrome wire, heated to the desired temperature. Air is passed through F until the desired pressure is reached, as indicated by the mer­ cury manometer. As a result, liquid rises in A until it meets the hot tube, thus vaporiz­ ing until the pressure in the apparatus is equal to that of the air and vapor in CED, The capillary. B. prevents violent oscilla­ tion of the liquid in the U-tube. • ' . U ‘ f ^ahn^ recommendea that the liquid in D be freed of. air by freezing and pumping, then thawing and repeating the freezing procedure, before distilling the liquio into C. however, this was found unfeasible with water since repeated freezing often caused the container to crack due to the expansion of the ice upon its warming-up in the 13 thawing process. Therefore, a distillatton-prccedure to free the water in b from dissolved air was developed. outline of this procedure is, however, deferred until the actual instrument Used is described later. A copy of dahn's instrument was built ana tested for use with water. It was found that In prolonged use (e.g., b hours), some air was transferred through the water, C, ■all the way to the gas-side, a. however, if part A was sealed off to prevent the escape of water vapor, no air moved through the water. This was shown by turning off the current used to heat up the nichrome wire whereupon the hoc, cjt. : "^N. mrnest horsey, Properties Of Ordinary Water- hubstance (Heinhold Publishing Corp.-, New fork, 1940) , p." TO. 65 pressure on A dropped to the vapor pressure of water at the ambient laboratory temperature. It was found, in many cases, that the transfer of air was due to leaky stopcocks on the A-side. On the other hand, bubbles of air were also seen In the capillary and this could only be due to some sort of streaming movement of water sufficient to entrap air from side C. Such laminar movement was perhaps induced by the rapid boiling of water in slde-A in tho early stages of a denaturation run; the boiling ceased once the pressure reached a steady state. The problem wa3 finally solved by the subsequent discovery of literature on the diffusion of gases into lk water contained in thin capillary tubes. Smith, et al. 15 discuss Stefan's equation: ( - = r * 3 £ ) ' * \ \A ♦ * I in connection with the diffusion of nitrogen In water. In this equation, D is the diffusion coefficient, of a partic*- ular gas in water, A V Is the volume of gas that moves into ^R. E. Smith, E. Frless, and M. R. Morales, J. Phys. Chem., £2, 382 (1955)* ■^J. stefan, K. Akad. Wi3sensch. Wiener Ber.. 77. 37 (1873).. o6 the water in the time interval, At, while q is the inter­ facial area, and a is related to the Bunsen solubility coefficient, by the formula: a = ^ (t5o) Landolt-Bornsteln1^ has a table of Bunsen water-solubility coefficients for several gases at 25°C*» und a table of these Is given for those gases for which the diffusion coefficient, D, in water is also known. Gas °^25°C DxlO^Ccm.2/sec.) airr/ 0.01762 ----- oxygen18* ,028b-5 1.860 (16°C.) nitrogen1' * ’ ^ o01b3b- 1*62 (l8°C.) 2.2b-6 (25°C.) argon21 .03^72 l.»+63 (25°C.) ^ LandQlt-Bornsteln. op,. cj,£, . > I» 763- 17m . n. 198. l8i m . ^W. Jost. Diffusion (Academic Press, Inc,, New York, 1952), p. b75. ^Smlth. et al. T loc. cl,t. 67 helium22 .0 1 0 0 order of 10^ (25°c.) carbon dioxide2^ .759 1.595 U k 6 (I6°c.) (18°C.) ?5 acetylene ^ .93 1 .1 0 (0°C.) hydrogen2^1'2^ 0.3**72 3.59 3-37 (18°C.) (25°c.) Because air has an average molecular weight close to that of nitrogen (2 8 .8 5 9 for air, 2 8 .0 1 6 for nitrogen), and the diffusion coefficient for oxygen in water is only slightly greater than that of nitrogen, it seems that the use of air as the driver-gas in Zahn's apparatus is about p as good as using nitrogen since oC^D is of the same order of magnitude for both gases, that for nitrogen, however, being the lowest in the list. The most important term in Stefan's equation is the lnterfacial area, q, thereby implying that the best way of preventing the movement of gas into the water is to reduce this area to a minimum. This was done and was an immediate success in stopping the transfer of air into the water. 22Smlth. et al.. loo, clt. 2lfJost, ^hanflolt-Bornstein, i lg.Cj u.slfc. ^ j o 5tf loc. clt. 2?Dorsey, o p. clt.. p. 556. ' ' ' 68' A schema of the instrument.finally used is presented in . Figure 30 followed by a detailed description of its operating procedure. I. OPKBATIONAL PROCEDURE 1. Stopcocks A, B, F, R, and X shut off; A and X opened to pump out mercury manometer, M. 2. Stopcocks A and E (capillary leak) shut off; C (three-way T-capillary stopcock) turned into h position; B and H opened to vacuum line, V, to evacuate CGN section. 3* Container, Z, filled with distilled water and connected to C; stopcocks B and H turned off and then C turned counter-clockwise 90° to 1_ position; B opened to vacuum gently, and air pumped out of ZCB by alternate torching and intermittent pumping for about an hour, until water in Z thought free of air. 4-. B cut off, and vapor pressure of water measured with manometer, M, together with the temperature of the surrounding air 3pace. If the raanometric reading fell within one millimeter of that rated F/OURE 3D VAPOR PRE33U RE MAD03 TAT V ACUUM LINE NICE P O M E A WIRE w r a p p e d CONTINUED ON FIGURE 3 / ' 70 28 for pure water at the measured temperature, the water in vessel Z was then judged air free. . C turned clockwise 90° to position and H opened again to V; ' Z turned upside down on ball-joint and C turned again clockwise 90° to T position; water run into CGN until above bulb G in capil­ lary tubing on both sides. 6, C turned 90° counterclockwise back to | — posi­ tion and JB opened to vacuum until water level in both capillaries the same. 7. B and H cut off. 28 International Critical Tables. op. clt.. Ill, 212. CHAPTER VI LENATURATION VELOCITY EXPERIMENTAL TECHNIQUE The powdered egg albumin used in all these experi­ ments was obtained from; 1. General Biochemicals, Inc., Chagrin Falls, Ohio, Lots 35537, 35^51, 33059 of 10, 10, and 15 grams each. 2. Mann Research Lab3., New York, N. Y., Lot 533 of 15 grams. 3. Armour Research Division, Chicago, 111., Lot E-81116 of 15 grams. In most of the experiments, It was used "as is*" However, In some of the earlier experiments, the Armour material was lyophillzed to create a finer powder. It was found, how­ ever, that the particle-size distribution depended upon the initial protein concentration of the lyophilizing solu­ tion. For instance, from one 10 per cent protein lyophiliz- ing solution, the dry powder after being sieved showed the following particle-3lzd distribution: Pasig 6o-mesh 72 0 .5 per cent Pass 30-mesh blit not pass 60-mesh 31*8 Pass 10-mesh but not pass 30-mesh 19.2 Not pass 10-mesh U8.5 On the other hand, the lyophilized powder obtained from a one per cent protein solution had the following particle- size distributions Comparing both sets of results, we see that the pro­ tein lyophilized from the 10 per cent solution Is finer than that produced from the one per cent solution since 87 per cent of the powdered produce recovered from the more dilute solution (i.e., one per cent) will not pass the 30 mesh while only 67*7 per cent of the 10 per cent produce falls in this class. Further investigation also found that the finer material tended to sorb slightly more water at the same relative humidity than did the coarser powder. Equilibra­ tion of both the IO-3O cut and.the' 30-60 cut with a Pass 60-mesh 0 .0 per cent Pass 30-icesh but not pass 60-mesh 13.0 Pass 10-mesh but not pass 30-mesh 39.0 Not pass 10-mesh M5.0 ■^Mesh Number Hole size (inches) 10 0.078 .0232 0.0097 ' • . • • 73 saturated solution of 3odlum chromate for twenty-four hours at room temperature produced the following results: Hun I Run IT 10-30 30 -6 0 10-30 3O-6O Sorption weight gain 0.03?6 0.0286 0.0270 O.OS^l Dry protein weight 0.1821 0.1257 0.1535 0.1316 Per cent weight gain 20.8 22.8 17*6 18A Sodium chromate (Na^CrC^) was used because its saturated solution has a relative humidity of about 85 per cent. Using saturated Na^Cr^Oy, which ha3 a relative humidity of about 50 per cent, we find: 1 .0-30 30-60 Per cent weight gain 8.85 11.*+- Experiments somewhat analogous to these have been ? carried out by Benson et al. They report that two samples of egg albumin, where the surface areas differ by a factor of 2, show no measurable difference in water uptake. In two other samples, having areas in the ratio of 3*5 to 1, the difference is 2.5 per cent which i3 stated to be within experimental error in thi3 particular case. However, in the latter case, the. reported 25°C. value is some ? S. W. Benson, D. A. Ellis, and R. W. Zwanzig, J. Am. Chem, Soc., 22j .2102 (1950). 25 per cent higher than that of this author, Richardson, or Bull. They, therefore, do not confirm this author's observation that particle size can affect the extent of water sorption at constant relative humidity. It is hence felt that this matter is still an open question awaiting a more extensive reinvestigation. The author's experiments were carried out in support of several experimental observations that the 10 per cent material denatured slightly more rapidly than the coarser one per cont. However, this problem was permanently side­ stepped by the cessation of further lyophilization and the elimination of all results using lyophilized protein, i.e., all the Armour and most of the Mann Results. Instead all of the General Biochemical material was mixed together as it was received and used as it came from the bottle, each new bottle being mixed together with what was left over from the previous supply., Tills, of course, did not solve the problem of particle-size variation, nor was It intended to do so. How the dry protein itself was used in the denaturatlon apparatus (Figure 31) will now be described. Eight male 12/30 ground joints were sealed off about three Inches from the neck; each one being numerically marked to identify it (Figure 31j part A). A particular tube was oven-dried, cooled and.weighed; some powdered 'protein put in and the total weighed again. The protein /AT/AC Bi Z F/£U/$£ 3/ /-/FAT DFNA TUFA T/OAf APPARATUS & C 'FTTC ) ® D 0,1 ere) C-0 A/T/A/l/T0 F-: 31/FT 30 //£,/ A VtTW A 76- weight was of course determined by difference and suffi­ cient protein was put in so that the difference amounted to about a docigrara. But since this protein already contained water adsorbed from the air, a further weight-correction wa3 required. A large amount of this same protein taken from the same bottle was put into a small test-tube, weighed before and after (with a glass-wool plug) and pumped out overnight on the vacuum line, then weighed again next morning. It was found that the air-adsorbed water gener­ ally amounted to about 5 por cent of the initial "wet" pro­ toin weight and this same procedure was repeated with each new run. The loss of weight once determined was applied to each of the samples in a given run as follows: The protein-containing tube, A, was plugged with glas3 wool, G, and connected to another 1 2 /3 0 female joint of semi-circular shape, 13, wrapped with nichrome wire. The protein tube, A, was attached to the semi-circular part, water loss (from 6 .1 5 per cent evacuation-detormination) Empty tube weight (A) plus protein (P) "wet" protein 6.6091 * 0.0003 gm, 6.71^3 * .0003 0.1057 - .OOOlf -0 .0 0 6 5 dry protein 0,0992 * 0.000‘ f Br with sealing wax because it had been found that the glyceride beating-bath fluid often succeeded in creeping into silicone high-vacuum grease, the other sealing mate­ rial tested. The glass cane-line tube, AB, was then sealed at F with this silicone groase to one of a series of stopcocks, D, connected through the bottom to a horseshoe-shaped glass tube, C, having a vacuum outlet, W, at one end. This whole set-up, about eighteen inches in diameter and six inches high was placed in a glass cylindrical trough, T, contain­ ing a glycerine-ethylene glycol-water mixture which served as the heat-transferring agent from centrally-immersed Cenco knife heaters. The fluid was sufficiently agitated by an air-driven stirrer so as to keep the bath-temperature in the 80 to 100 degree range isothermal to well within one degree. The whole apparatus was connected to the water- vapor apparatus at stopcock F, as described in Figure 3^j through ball-joint P and operated in the following manner. After the ABCD set-up was placed in the bath, T, stopcocks D and F were opened to the vacuum line, V, and the protein samples pumped upon for at least twelve hours, generally for twenty-four hours. Upon completion of this evacuation, a run was ready to be performed. 1. The heaters and stirrer, were started in bath, T, to heat up the tube, A, to say 90°C., stopcocks D and F still open to the vacuum line. 2. The series of eight nlchrome resisters (Bl, B2 .,.B8) we, 3 connected serially to another eight-lead vari­ able reslster, R, both connected through a Variac to supply heating electricity generally of about 20 volts, the pur­ pose of which was to prevent the condensation of water in B. 3. Nichrorae wire, NHFP, (Figure 30), was simultane­ ously electrified to heat up the glass and, therefore, the water in capillary N. At the sarnie time, air leak, E, was opened and sufficient air was admitted to obtain an arbi­ trary pressure in the mercury manometer, M. This forced the water up higher into the heat«d capillary, N, and it boiled and receded until the vapor pressure of the water wa 3 equal to that of the air in the manometer, M. If the water level in both capillaries was not the same in this steady state, the hot wire at N was loose enough to be moved slightly either up or down to obtain, an equal water level in both tubes. The air pressure read by the mercury manometer under these conditions was taken as the vapor pressure of water for the run to follow and this air pres­ sure wa3 read at successive time Intervals during the run. •These pressures were found to vary within plus or minus one or two millimeters but because the total pressures were always about half an atmosphere, the variation in 79 pressure only produced a very small error. The run itself was ready to start, once the bath was in a thermal steady state, $ay 90 - 0.1°C., read on the bath thermometer. *+♦ Water vapor at the pressure of interest was admitted to the protein samples by turning stopcock F counterclockwise l8o degrees, the timer simultaneously being started. 5. At a series of arbitrary time intervals, an AB- cane was removed from the bath in the following manner, Stopcock b (Dl, D2, et cetera) vas turned 180 degrees so as to cut off the water vapor source from the protein, once a water vapor pressure reading had been taken. The resist­ ance winding, B (Bl, B2, et cetera), was taken out of the series, and the variable re3ister, R, adjusted so as to maintain the same current and heating effect a3 before. Then the AB set-up was pulled out of stopcock D (Dl, D2, et cetera), the timer read, part B removed from A, and the protein sample made ready for analysis. 6. Since the initial protein weight was known, the analytical problem was to determine the weight of Insoluble protein produced as an effect of heat and water vapor. Distilled water, itself, was the solvent used to separate the native protein from the denatured portion. It had been previously found that the use of a sodium acetate- acetic acid buffer solution to hold the pH of the wash to that of the l3oelectric point, pH >+,8', produced results (i.e., per cent denatured) no different then the experimen­ tal errors allowed. But a potassium acid phthalate- phthalic acid buffer solution at the same pH did produce somewhat higher results. This appears to be due to the adsorption of phthalic acid by the denatured protein since repeated washing with distilled water yielded the NaAc-HAc results. For analysis, the powder in the tube was removed by adding distilled water, containing a wotting agent, Tween 2 0, and scraping the solid-liquid mixture out into a 100 ml. beaker by the use of a wooden policeman. More water was added to tube A and the process repeated until no more protein was left in A. Enough water was added to the beaker, similarly numerically marked, to immerse the solid in plenty of water. The beaker was left standing for several hours, covered by a watch glass, and the finally- remaining solid protein was filtered in the following manner. 7* it wa3 found extremely difficult to filter these solutions through conventional Gooch crucibles or sintered glass crucibles. Therefore, a suggestion of Dr. Ryden Richardson was used to develop a glass wool type of Gooch crucible which worked well. A 2 1/2M piece of,10 mm. pyiex . tubing, .H (Figure 3 1), was narrowed at one end and a glass . '1 wool plug pushed down Into this narrowed end. ' The whole set-up was then oven-dried and weighed. The protein con­ tents of the beaker were drained Into this filter by suc­ tion through a specially built funnel. After all the water passed through the glass wool, more water was added and suo- tioned in order to remove any soluble protein adsorbed by the denatured protein from the solution. This whole set-up, H, plus glass wool plus insoluble protein, was then evacu­ ated overnight on the vacuum line, V , to remove the water and twenty-four hours later the tube was weighed again to determine the weight of insoluble dry protein produced by denaturation. For example, the results of denaturing a protein sample of 0 .0 9 9 2 - 0.0Q01 * gm. dry weight were: Weight of glass tube, H, with glass wool weight with dry denatured protein added dry weight of denature:d protein per cent denatured (0 .0 1 5 1 * 0.0 0 0V 0.0992 - C.QOO‘ 4- x 100) But before thi.s weighing technique hud finally been developed, ultra-violet analysis of the solution hud also been tried. However, dissolving the partially-denatured protein in a fixed small amount of water proved unfeasible 5.0007 1 0 .0 0 0 3 5.0158 - 0.0003 0 .0 1 5 1 - 0.000*f 1 5 .2 i o A and, therefore, thin method of analysis was replaced by. direct weighing as described. From these weighing figures it can readily be seen that weighing errors prevented the determination of the per cent denaturation to much better than one per cent since so little egg albumin was used per determination. In the foilowing graphs (Figures 32 to 67) are semi­ log plots of per cent denaturation vs. time at various constant relative humidities and temperatures. A semi-log representation was chosen both for theoretical and experi­ mental reasons. It is firstly postulated that the denatur­ ation proce-se in the solid state takes place and progresses "molecule by molecule." Evidence for this postulate is the relative reproducibility of two samples pulled simul­ taneously from the bath, each one containing a different initial amount of protein. Secondly, it was found possible to represent the experimental data, after the initial time lag due presumably to the sorption lag, reasonably by first-order plots (i.e., semi-log). The initial induction period can be explained, at least In theory, as follows. Denaturation nucleation sets in at the sites where a cer­ tain minimum number of water molecules have sorbed. As the number of such "saturated" sites grows until sorption equilibrium is attained, one would expect a similar increase in the denaturation rate until this equilibrium is - 1 P /SU R F 32 PATS O f O f NATL/OAT/ON O f POO A l S O if/N A r SO V. . p 22 C m.n\ p/n 0.6 35 tv . / o . o ' t'/i \ S. 8 5 S/25 ,i i i i i 25 50 7 5 /OO J 2 5 1S0 ?o 770 v f f 33 PATS O f DfNATOPAT/DN fio OF FOG A /8 U M /N A T SO ‘O. 7* - i-o- S O- F 2 4 5 rrlrn. A O - P /P, 0.070 ~ ~— _ W //.2 P /2 1 2 .8 FAS i i i i . i 25 SO 75 /OO 125 J50 f/mf (AttA/t/rrs) F /S u R F 3 4 50 RATF F f 7)fNA7Z/RA77ON OF FS6 A13FM/AJ A F 6 0 ‘O. 4o- P 252 mm 30 ' P/Po ° rP ° IV //. 7 1 t i/z /• / BPS 1 ' .. 1 . . . . 1 ! % ' 25 50 .7 5 /OO T/ ' MS (jMf/VUTFS) ETP05A7T SOLUBLE ■■ ' PFPOFNf SOLUBLF *0 ■ 4 0 30 IX. F/ 6 O'P5 3 5 PATE O F DONATURAT/ON - OF 000 AL B U M IN A T 6 O e 0. p> I , 263m m ' 7/Fo- 0.735 Nv i> // ' 72 .5 Z//2- 1 /• O F FPS i i 1 ■15 ' 52 75 100 725 / /■'ME (M/NUTfS) ■ 4 D 3 0 20 JO P ' - 2 7 5 m m P/P0 0.775 IP 73.5 Z //2 0 .5 0 MS. F/SNPE 3 & PAT5 O f D P P A T op ATJOP OF 506 A LB U M J77 AT80*0. 6 0 75 700 T/M5 (M/NO 735) FFPoEpr o o u jo lo F/OUPf 3; P A TE O F 00O'ATOP A J/ON O F 536 ALBUMJN A T 8 O cC. 286 m m PJPg o . 810 A/ / A ,5 h/2 C .P O P 3. i . l ---- 1 -- 30 D O L O 7 / A f f c '/W N U T F S ) I,/2 51 U K 'S sat V / tJ//AU S /S 'J ) £2602X39 M r s o s d s n a t m a t m O S £ 6 6 A lS U M /N A T f( > Y ■}/ r 32'A m /r\. F/r'c C-i,/5 1 tv 8 2 . S t/-> ■ 5 0 s 5 S 2 2 6 2 / 6 M S 3 / S M S 4 / 5 5 2 5 /•u\fe i ■(///•. -S i3 ) ' / / ,‘j 4 '\/A'AJ. 0 \ ii.SJMU, 41 0 4 -'I m ■ . 200 7/A/S v A f/M ’Jf .• / /. ' U * ■ -'/ 4 - •A rt- o > oox^r.jOA:..:.' • i \ .„ J / tfi/M /K l A 1 iO . A f. \ . ' . T V t 0 m2 £ 42 " o y 0 / O W A 7 0 4 4 7 7 0 A/ ' ■ J/Ji/A s/A r / < / ■ io'o .80 7 4 0 -O -V 0t2 ) 420 ■ . > k f: 43 \';v t ,y i (tvAiuh'Ahrpj AT. 3.’ ' *r _ Af/M 'Pt -jJ //,'rV/AV 4 4 A.A I f . y ~ UfA A TOa'AT/TA/ PA i*P . ALAi/M/fiJ AT O T . 3 0 ■ /' - f ^ P ’Ti rrt C (.Sf » \ 4 .e > l 1 .8 H A 'S 0 r,Aip , ,u/Aui //l»t/AV~ a1 A 'M lO A l .V A lO (,’ M /A i AT 4 0 ' ‘ V \ 'r ' f4 ■ AVi// , - / ■ i,v.,\/Ar;/KA7 / H£ F /F u F /F SO Ra T £ O F OFA/A TUFA T/ON O F F 0 6 A i ROW Kf A 7 70*0. S O /OO . /S O 7/m£ SM/M/FFs) V } k § $ ?o 8 0 70 So so *0 30 F 1 G /P F £ / FA FF O F DFT/ATtJFAnCH OF £66 A18UM/H A T 70 ‘O . S’ 3 7 ? m m P / P t 0 .7 0 6 ' iV 10.4 It/2 / HA-. i i \ /S O 200 2 S O p m f C oiTA/urss) . //..7,aV 52 i u . - / ■ / , - . / } toa'at/oa O' / > ' • < • AlriUMlM AT io\:. I J t r /.if Af SJ F’ ATi Of DFAATUFAT/C* Cf £i.-6 AIB/Jflf/.V AT ''fO”C. i ) } J T H .-rim . O.lii) / H4 T/Mf iA f/A '('T£B ) fi/iuxt: 54 L’ A/f OF OFNA!UFfll/OH OF F66 AlfW.U/A AT FO'C 1 I Mr . M/AC f t . 4 t ■< ' -,a / ■ a, />f ' . v / v * / ■ 7/ / * ; , ' / A / / v A T . ' < S . ) f/f/ff 5 (. k ' A T f O f M \ A T o A ' A n C k / O f f 44 A t S I M / H A T /tX> V / • ’ 4/4oi/" f ' / r , 4. ‘ ,40 ; r j i O A t ' fiMt ( . ' f/f f i 5/ X J I f r f C f A / A T / J k ' A T / M O f f.‘ f AifcJAHA/ A1 / O O V PFPC fA /r S0/L/8LF PFFCF//T S O iO B /F ? £ > 80 70 60 P/6C/P 5 S 8 PA7 F OF SfA/ATOPAT/OA/ OF F66 ALBUM/A/ AT / 00° C . - p F62 mm. P/Po 0.635 ’ w 7 5 f ' / p 3 PFS. , , 60 /2 0 /S O t / m f C V ///U T /7 S ) F /6 D £ £ £ 9 PATF OP D£A/ATU£Ar/ON OF £ 6 0 ALBVM/N A T /00°C P A ^ 6 mm, P /P D 0.C3S W 7 .5 r i/2 2.5 m s . 60 n o is o TIM F (M lN U r e s ) P F P C F A / T SOLUBLE . P E P C E A J T SOLUBLE FIGURE 60 PATE O F GENA TOR A T/OfJ OF EGG ALBUM/A/ A T /0 0 °E . ~ P 4BE m o t P /P , 0 .6 4 0 200 too r/M E ( Ai/MUTES ) FIGURE 6 / PATE OF DEMATORAT/OH OF E 66 ALBUM/M AT /00*E So /O O 400 F t M E CM/A/UTES) P FR C S N T S0LU8Le • PSPCfM T S O iU S L S 90 80 70 G O 5 0 700 /SO T/M S (M /N U TS S ) r \ F /G U R S 6 2 PA TS OF DSNATUAATW ■ \Q F £66 ALBUM/HAT /0 0 ° C . - > SOOmnx.. r \ _ P/Pa 0.660 " w 8 .0 t j/z / . 5 HRS: i --- 1 ----------- A ---------- i— --- f t 80 70 G O ■ F(S U P S 6 3 RAT S OF Df NATURAT/ ON .O f £66 ALBUMI N AT /O O V . 50 Y p 500 mm. 40 Y P /P , 0.660 W 8.0 , T //2 /.3NRS: /5 0 200 ZSO Ti m s C m ///u r s s ) p ig n io s j n j o & j h F /6 //P S 6 4 PATS O F DSA/ ATURAT/ ON O f £66 ALBUM ///ATM *£m fo 80 70 60 SO 40 P 5//mm P/P. 0 . 672 50 100 /SO 200 TIM S (M ///U T S S ) P££0£3/r sosasitr P 'C U P F 6S FATF 0 ? D FFA tu'RATIOF OF £0 6 ALBUM/M A T ,00 *0 P -5 7 5 m m P!P„ 6. (> 7 5 tV 8.4 Z,,1 /• 4 P PS. t / m f ( m m o r e s ) AM PS/vr Fcvt& or / f p u f f / uooosls £ ... \ F AC ■ FA OF OF O F F A r_ PAT. O', C~ £6 6 A LB U M ifJ A T /OS'S ■ F> £ 3 3 m m A P/P, : 702 . iV U j 7 .0 !■ SSPR S ■ s o JSC T/M F. C.'.'/MUFFS') . Fi 06 < £ 0 7 ■ T 2ATF O f CFNATU8AT/OF i OF cOS ALBUM/M AT i OC’O p S3 6 jk,m ■ p/ p. o 7C6 /V ? P ‘ ‘L - \ c 42 P P f. > i \ so ICO T/MF CMWUTFS) » ~ n achieved* Once this occurs, the rate of nucleation would then become dependent only on particle size* It has been observed that the rate of conversion of monoclinic to rhombic sulfur, at room temperature, becomes more and more first-order the more the starting material has been pulverized.^ Hume and Colvin believe these results are du& to the homogeneous rate of nucleation being the rate determining step, for with fine particles propaga­ tion of such nuclei is practically instantaneous* Thus first-order kinetics result not because the reaction Is particularly unJLmolecular, but because the observed rate equals a constant rate of nucleation times the number of unreacted particles* An additional reason for treating the denaturation data of thi3 dissertation as first-order is that such treatment conforms with the great majority of solution-donaturation data so presented, even though 6 there is literature to the contrary. Following these graphs also are log-log plots of the denaturation half-life (assuming a first-order 3j. Hume and J. Colvin, Phil. Mag., 8, 590 (1929). **1*. J. Gibbs, Arch. Biochem* and Biophys*, 3l2» 216 (1952). ^H. Chick and C. M. Martin, J. Physiol., 1 (19H). ■ ^1. N. Bulanaki and N. A. Shabanova. Ukrain. Biochem. Zhur., £6, 235 (195^)Chem. Abstr., it2» 9705h (1955). ■ ■. • • 97 reaction) V3. 'partial pressure,-P/PQ (Figures 68, 69 and 70),and vs. W, the weight of adsorbed water, (Figures 71» 72 and 73). These weight-plots were computed from the adsorption isotherms on denatured egg albumin (Figures 12, 13 and 1*0 because this is the only sorption data at these temperatures. The data of Figures 71* 72, 73 can also be repre­ sented by equations of the form: log I ' 1/2 ■ ■ = A (logW) B specifically: T(°C.) A B (root-mean- square-error) 80 - 9.^ 10.21 - 0.21 * hr. 90 -16.7 16.90 t 1 .3 2 " 100 -11.8 10.92 - I.33 • ' Because it has been assumed that- the denaturation reac­ tion is first-order in native egg albumin (NEA), we may write: -d In (NEA) , ' ■ ■ ---------- = k - 0.693/^ dt and, therefore, the reaction rate constant, k, at these three temperatures is: a 96 ’ ‘ rso one ,w OF7VA7 UMAT/OH HALF-L /HP AT VAR/OUS PAQHAL PRFSSURCS' A T 8 0 V. 0.4 70 r/P * P A L F -l/F f fa e i/ffS ) . *99 F/6(J#£ 6? OFA/ATiff!ATION A ACT-£/££ AT VA0/OUS f a p t /a l PMSSUPFS AT ?(?V. i.O 0.2 ts 10 0.75 P /P , H A L F -L /F £ C F C U F S ) D i r>o F /S U P F 70 OFNATURATIOH HALF - LiFF AT VARIOUS PART/AL ' PRCSSURfS AT 100 ° C. . 8 0 SO JO 0.1 0,8 0 1 O S 0.4 p/p. 0 FtAuHC 7/ denatuhat/ oaj HALF-LIFE W ITH VARIOUS AMOUNTS OF ADS0F5E0 WATCH (W ) AT 8 0 ' 0. 0 4 n 15 n a « 0 o 102 Ft&V/SiF 72 DE T J 1 \ TUfiA T/07J NAlFHEE W/TH VAQ/OuS AM O tfurS OF AOSdXBfC WATER (W ) A T 90° C . So so so 2 0 ai 0.7 8.0 1 0 0 W f ja u O F 73 DfAMTUXAr/OU tfA ir -U /F W /TH vaf/o vi amoua/73 a r 40 sorbfd YYATfF (W ) /\T /o o 'c . 40 to to Of OS 0 ( . os 0.4 0 . 2 /■ P 6.0 IV' ” • • • T ( C.) k ‘ ’ 80' if. 27 x 10' " 11 (W)9^ ■ ■ • 90 .8.71 X 10"18 .(W)16’7 • 100 8.32 X 10"1 2 (W)11,8 These values suggest that the denaturation of egg albumin by water vapor, If first order in protein, is some tenth . to fifteenth order In water, W. Examining Figure 7^» it is apparent that the same weight of adsorbed water induces a greater increase in denaturation velocity for each ten degree temperature rise. We note, for example, that a water weight, W, of 10 grams of water per 100 grams of egg albumin yields a denaturation half-life of about six hours at 8o°C., which then decreases to 1.5 hours at 90°C. and finally to about 0.1 hour at 100°C. But because there is so much scatter and so little overlap of weight regions over the whole temperature range, little can be gained by a plot of log ^"1/2 (half-life) vs. 1/T over the entire weight-sorbed range in order to obtain the activation energy. Therefore, a semi-algebraic method of estimating this quantity has been used since this was possible even with the data-spread already noted. Starting with the definition of activation energy, A Ha, we haves HALF-LIFE {h o u r s) -9 • F/SU FF 74 OFUA TUH A T/ON H A L F -lift AS A FUNCTtofi OF AMQUNT O F tVATFF C/VJ AT S O ; 9 0 ’ t 700‘ C. So 30 0.1 0.0 O L 0.4 7 8 lo // n /j /5 w 106 d'ln k\ p ' - <4 Ha/RT a-1 ,w * * As the .data is fir3t-order plotted, d in k - - -d In ^1/2* But d(l/T) = (- ‘ l/T2 dT), therefore, <9 In 7 1/2 Also: . <5 In y i/2 5 (1/T) - <9 (1/T.) /W <^(1/T)yri/2 V J 1/T Therefore: A Ha/R (-_Aln -| 1/ 2N lA(1/I)/ f W 1 A1" # 'VT Each of the two last partial derivatives has to be evaluated over that part of the temperature and half-life region Judged most reliable, as follows: T = 90°C • T 1/2 .1*0 2.0 3.0 M-.O - A In 7'1 /2 0 .6932 0.1+05^ .0.2877 In W 2 .31 2.27 2,25. 2.23 A In W 0.0*f 0.02 0.02 - Ain 7*!/^ . 17.3 20.3 Ilf.»f A In W • Averaging .these values we obtain 17.3 as the approximate .value of (-Ain T1/2/ A In W)T at 90°C. The other deriva­ tive (Ain W/A[l/T]- )fjL/2 ls obtained as follows; • T(°C.) 80 90 100 T(°K.) 353 / 363 373 . 1/T x lO*3 . 2.833 2.755 ■ 2.681 -Al/T X. 10*^ 7.8 7 A 'f 1 /2 (hours) 1 1 1 In W . 2.50 2.31 2.1h -Ain W 0.19 0.17 A in W/A ( 1 /T ) 2MtO 2290 A Ha (kcal./mole) 83 78 T 1 /2 (hours) 2 2 2 In W 2 .^3 2.27 2.07 -Aln W 0.16 0.21 A In W/A(l/T) 2050 2700 A H (kcal./mole) c l 70 92 T 1/2 (hours) 3 3 3 In W 2,38 2.25 2.0^- -Aln W 0.13 0.21 A In W/A (1/T) 1670 281+0 A H a (kcal./mole) 57 97 These activation energy figures, in the more reliable half- life range of one to three hours range in value from about ’60 to 100 kilocalorles per mole. They may” be compared with various values of 128 ,to 13^-, 96 and 87 kcal./mole for the * • activation energy of the solution denaturation of egg albu** 7 min compiled by Eyring and Steam. Lastly,' the results of these experiments lead to a possible explanation of why the presence of sugars in protein-water solutions reduces the rate of denaturation of these proteins.^,^,' * ’ < ‘ > It is known that sugar added to an aqueous virus suspension withdraws water osmotically 11 12 from inside the virus globules. ’ Similar effects occur when wet protein crystals are immersed in sugar solu- 1 3, 1^ tions. Therefore, in the light of these experimental results it is suggested that the presence of sugar reduces ^H. Eyring and A. Steam, Chem. Rev., 2ha 253 (1939)< 8A. Beilinson, Blochem. 2.j 399 (1929). 9C. D. Ball, J. Biol. Chem.T I5lt 163 (19^3). . l^c. R, Hardt, I. F. Huddleson, C, D. Ball, J. Biol. Chem., 163. 211 ('19^6) • 1XJ . E. Smadel, J. Expt. Med., 68» 607 (1938). 12D. G. Sharp, J. Biol. Chem., 159, 29 (19*+5) • L. McMeekan, R. C. Warner, J. Am. Chem. Soc., £!i, 2393 (19^2). ik T. L. McMeekan, M. L. Groves, N. J. Hipp, J. Am. Chem. Soc., Z2» 3662 (1950). •"109 the denaturation rate by inducing partial internal dehydra- tion of the protein molecule. In support of this thesis, 15' we may look at Ball*s results more closely.. An 0.2 molar sugar solution protects a 10“^ molar egg albumin solution. about equally well if it is either sucrose or d-mannltol. These solutions turn, out to have practically identical osmotic pressures at 0°C,^ Glucose has a greater boiling point elevation, 0.53°C., than sucrose, 0.*f9°C., at this X7 18 same concentration. 1 Therefore, having the lower vapor pressure, glucose should be more effective than sucrose : which indeed Ball finds it to be. Finally, fructose, which is poorer than glucose a3 a protectant at equal concentrations, turns out to be slightly better at saturation than glucose. But a saturated solution of fructose is 5*0 molar while glucose is only M-.6 molar at saturation.1^ These facts all suggest that , a colligative property of these sugars is at the heart of l5C. D. Ball, J. Biol. Chem., I5lf 163 (19^3). 16 Landolt-Bornsteln Physlkallach-Chemische Tabellen. ge (Julius Springer, Berlin, Germany, 1923), Auflaee (Julius Springer, Berlin, Germany, 1923), II, o• 17International Critical Tables (McGraw-Hill Book Co., New York, 192b), IV, *+29. l8kftaa.9ltr9prqat&lQ loc* cit. ^A. Seidell, Solubilities of Inorganic and Organi< Compounds (D. Van Nostrand Co., New York, 19^6), I, 093. . the anti-clenaturation protection that their" presence affords; specifically their vapor pressure -lowering abil CHAPTER VII • SUMMARY Water sorption isotherms have been determined pn native egg albumin at 25, 55, and 70°C. and on steam denatured egg albumin at 25, **0 , 55 , 70 , 80 , 90 and 100°C. Sorption hysteresis decreased with increasing temperature and disappeared at the highest temperatures in the upper- most relative humidity range. Detailed computation has shown that the hysteresis phenomenon is related to a change in the thermodynamic properties of the sorbed water. Desorbed water is apparently more strongly bound to the protein substrate that is the adsorbed when water sorption hysteresis is present. It is found that the entropy and enthalpy of adsorbed water decrease when isothermal desorption to the same partial pressure takes place. The extent of water sorption on denatured egg plbumin was found to depend upon the method of denatura­ tion. This suggests that the end product of the denatura­ tion process is not a definite thermodynamic state but one dependent upon the method of denaturation. A study of the effect of wnter vapor, at constant e ■ o f , Q ° * . . • .112 relative humidity, upon the rate of heat denaturation of' solid egg albumin in the temperature range 80 to 100°C. has also been made* Solid egg albumin was denatured in the presence of water vapor, the vapor pressure of which was controlled by a special vapor pressure manostat. It has been observed that the denaturation of solid egg albumin seems to follow a first order rate law with respect to the protein. The experimental relationship found between the relative humidity of water and the denaturation velocity indicates that the denaturation reac­ tion is some tenth to fifteenth order in the amount of 1 water bound to the protein. It ha3 also been shown that the activation energy for this process lies in the range 60 to 100 kilocalories per mole. This high order with respect to water suggests that hydrophilic protein bond3 can be broken only after they have been saturated with many water molecules or that the role of water in the denaturation process is that of a "lubricant." By this is meant that though the water probably sorbs both between and within the protein mole­ cules, its importance lies in the fact that it be in such a physical state that the protein chains are made more free to uncoil under thermal excitation, them they would themselves be without the water. 113 Finally, these experimental findings have suggested a possible explanation for the rate reducing effect that sugars have upon denaturation in aqueous solutions* o BIBLIOGRAPHY o BIBLIOGRAPHY A, ARTICLES Ball, C. D. J. Biol. Chem., 151, 163 (19*+3). Barker, H. A. J. Gen. Physiol., 12, 21 (1933)• Beilinson, A. Biochem. Z., 2 H . 399 (1929) • Benson, Sidney W,, Ellis, David A., and Zwanzig, Robert W. J. Am. Chem. Soc., 2Z t 2102 (1950). Benson, Sidney W,. and Richardson, Ryden L. J. Am. Chem. Soc., 2Z> 2585 (1955). Bulanski, I. N., and Shabanova, N. A. Ukrain. Biochem. Zhur., 2 k 1 235 (195*0. Bull, Henry B. J. Am. Chem. Soc., ££, l*+99 (19MO. Chick, Harriette, and Martin, C. J. J. Physiol,, *jO, .(1910), ia, 1 (1911), h i t 61 (1912), b i t 261(1912) • Davis, S., and McLaren, A, D. J. Polymer Sci., 1, 16 (19W). Dawihl, W.', and Rlx, W, Z. Physik, 112. 65** (1939). Enderby, J. A. Trans. Faraday Soc., ^1, 835 (1955). Everett,’D. H. Trans. Faraday Soc., 1077 (195*0. Everett, D. H., and Smith, F. W. Trans. Faraday Soc., 50. 187 (195*0. Everett, D. H., and Whitton, W. I. Trans, Faraday Soc., M , 7*+9 (1952). Eyring, H., and Stearn, A. ’ Chenl. Rev., 253 (1939). Gibbs, R. J. Arch. Biochem. and Biophys., 35* 216 (1952). 116 Hardt, C. R., Huddleson, I* F., and Ball, C, D. J. Biol, Chem., i£l, 211 (W6). Hultin, T,, and Herne, R. Arkiv. Kemi. Min. Geol., 26A. 20 U 9 W . Hume, J., and Colvin, J* Phil. Mag., 8, 590 (1929). LeClerc, P. Silic. Ind., 12, 237 (195^). Lewith, S. Arch. Exp. Pathol. Phann., 2&» 3**1 (1890). McLaren, A. D., and Roven, J. W. J. Polymer Sci., 2a 289 (1951). McMeekan, T. L., Groves, M. L., and Hlpp, N. J. J, Am. Chem. Soc., 2Z> 36o2 (1950). McMeekan, T. L., and Warner, R. C. J. Am. Chem. Soc., 6ifc, 2393 (19^2). Mellon, Edward F., Korn, Alfred H., and Hoover, Sam R. J, Am. Chem. Soc., 2761 (19^9). Neurath, Hans, Greenstein, Jesse P., Putnam, Frank W., and Erickson, John 0. Chem. Rev., lit, 157 (19l +10. Schellman, J., Simpson, R. B., and Kauzman, W. J. Am. Chem. Soc., 21> 5152 (1953). Sharp, D. G. J. Biol. Chem., 159. 29 (19^5). Shaw, T. M. J. Chem. Phys., 12> 391 (19W. Simpson. R. S., and Kauzman, W* J. Am. Chem. Soc., 75, <1953). Smadel, J. E. J. Exp. Med., 68, 607 (1936). Smith, R. E., Friess, E., and Morales, M. R. J. Phys. Chem., 51> 382 (1955). Stefan, J. K. Akad. Wisgensch. Wiener Ber,, 2Z> 37 (I878). Stokes, R. H. Ind. Eng. Chem., ^1, 2013 (19W • Wexler, A., and Hasegawa, S. J. Res. Nat. Bur. Stand., 53 • 19 (195*0. Zahn‘ , C, T. Rev. Sci. Instr., 1, 299 (1930). B. BOOKS Brunauer, Stephen. The Adsorption of Gases and Vapors. Princeton University Press, Princeton, N. J., 1953, Vol. I. Chemical Engineering Handbook. McGraw-Hill Book Co., New York, 1950, 3rd edition, Dorsey, N. Ernest. Properties of Ordinary Water-Substance. Re inhold Publishing Co., New York, 19*+0. Parkas, A., and Melville, H.'W. Experimental Methods in Gas Reactions. MacMillan Ltd., London, England, 1939. Glatt, L.. Adams, J., and Johnston, H. L. Technical_Report ~5l6-o. Ohio State University, Cleveland, Ohio, 1953* Handbook of Chemistry and Phvslcs. Chemical Rubber Pub­ lishing Co., Cleveland, Ohio, 1956, 3®th edition. Hougan, 0. A., and Watson, K. M. Industrial Chemical Cal­ culations. J. Wiley and Sons, Inc., New York, 19^. International Critical Tables. McGraw-Hill Book Co., New York, 1927, Vols. I, II, III, IV, V. Jost, W. Diffusion. Academic Press, Inc., New York, 1952. Landolt-Bornsteln Phyalkali3ch-ChemlschSLlabeUen. lih Auflage. J. Springer, Berlin, Germany, 1923, Vols. I, II. Lange’s Handbook of Chemistry. Handbook Publishers Inc., Sandusky, Ohio, 1952, oth edition. Milne, W. E. Numerical Calculations. Princeton Univer­ sity Press, Princeton, N. J., 19^9. • Neurath, Hans, and Bailey, Kenneth. The Proteins. Academic Press, New York, 1953, Vol. II. Seidell, A. Solubilities, of Inorganic and.Organic Com­ pounds. D. Van Nostrand Co., New York, 19*+o, Vol. I. Sosman, Robert B. The Properties of Silica. Chemical Catalog Co., New York, 1927. o 0 • * « 118 Steinbach. o; F., and King, C. V, Experiments In Physical Chemistry. American Book Co., New York, 1950. C. UNPUBLISHED MATERIAL Richardson, Ryden L. Ph.D. Thesi3, University of Southern California, 195^» o