Sept,., 1960
SOlWl‘IOX O F
HYDROGEN CHLORIDE
regard very little can be inferred from the bulk structural and thermodynamic properties of solids. In other words. the Statement that “a solid is in its standard state” has little or no significance with respect to its surface properties. To be specific, “the immersional heat of a substance” a t the present time is a meaningless assemblage of words unless one can describe completely all the parameters of the surface, adsorbent, and adsorbate phases. Acknowledgments.-This work is a contribution from the American Petroleum Institute, Project 47d and the authors thank them for their continued support and interest. Appreciation is expressed to A h . R. L. Ekery aiid Dr. L. Slutsky and hIr. C. L. \Tilliarns, Jr., for their aqsistance in various phases of the study. I~ISCUSSIOX L. ,4.R o m (E. 1. du Pont de Semours & Co.).-Siiice surface crystdlinity appears t o he an important factor, I would suggest that electron diffraction be used to determine variations. Do the srirfacp areas stay constant as a function of degassing temperature? K h a t is the evidence you have for the presence of free vibrating hydroxyl groups on anhydrous &03surfaces? IV, H. WADE-For all samples, the BET arms were found to be independent of outgassing temperature. There arc’ several infrared studies u hich shox discwte surface OH bands. -4. C. ZsTimxouER (IJ(-high I-iiivcrsity) -The question of double layer formation terms not to h a w been considered. Thr sniall amount s of impiiritics prewnt may posbibly product, quite difftwiit dumina-a ater interfaces
BY
DRY LYOPHILIZED p-LACTOGLOBULIN
1199
from sample to sample. In t,liis laboratory such heat effects have been detected easily for the modd system of graphite immersed into surfactant solutions; even trac: calcium (a few parts per million) gives differences i n heats of immersion. Do you believe such effects might be contributing? IT. H. WADE.-I would say that the regularity of the data is very surprising if your hypothesis is corrwt. GEORGER. LESTER (Universal Oil Products).-You have stated that the calcinations were done by the manufacturer. The outgassing experiments might he very susceptjble t o time and storage between calcination and outgassing. Secondly, my o m calculations, based on a spinel structure for a-alumina, indicate that the averape number of 8- ions in the 100, 110, 111 planes is about 11 ions per 100 L$.2. This may explain the lovier OH loss for the two samples of a-;U,Os. If the “amorphous” sample is really a mixture of true ~~-41203 and amorphous .1120a, as often has been srigyest,ed,this explanation may apply also.
n.J. C. YATES(Columbia Uiiivereity).-I wish t o congratulate Dr. Wade on his statement that “little can be inferred from the bulk structural and t’hermodynamic properties of solids.” While as to your general stat’ement that the low area materials are more crystalline than high area mat’erials, I agree, but I t’hink t,hat there may be exceptions. For instance, the spectra of the OH groups on .1lon C are quite different from t’hose on the higher area alumina gel (Peri, ACS meeting, Sept., 1959). The peaks on illon C are smeared together, while the bettercrystallized alumina gel showed three distinct peaks. This is what one would expect from the flame process used in manufacturing Alon C. IV. H. \TauE.--ht the present time, I liave found 110 correlation between extent of surface OH coverage and crystallinity.
T H E SORPTIOK OF GASEOUS HYDROGEN CHLORIDE BY DRY LYOPHILIZED 8-LACTOGLOBULIN’ BY WASYLS. HNOJEWYJ AND LLOYD H. REYERSON School o j Chemistry, University of Minnesota, Minneapolis, Minn. Receiued March 7, 1960
The sorption of gaseous HCI by dry lyophilized /%lactoglobulin was studied at 27”. HCl is held so firmly on some of the sorption sites that it cannot be removed by pumping to a high vacuum. The results show that the amount remaining sorbed on the protein is some reciprocal function of the temperature.
Earlier work in this Laboratory2 showed that gaseous HC1 was very strongly sorbed by insulin at -78.9 and 20”. The protein used in that study was the zinc crystalline insulin. I n that work it was not possible to determine the total effect of the zinc in the sorption process. However, the amounts of HC1 bound more or less permanently depended on the temperature of the sample during the desorption process. As the temperature was lowered, the protein bound more aiid more HC1. About five times as much was bound at -78.9” as was held a t room temperature. It was felt desirable to repeat the sorption studies of €IC1 by a protein having no metal ions in the structure. Qualitative studies on the sorption of HCl by lyo( 1 ) f3upportt.d by a grant from U.8 P. Health.
(2) (lY3Li)
L H Reyerson and Lowell Peterson, THISJ O U R V I L 69, 1117
philized p-lactoglobulin, carried out in the Carlsberg Laboratorium by one of the authors (L.H.R.) indicated that the amounts sorbed were relatively large. This preliminary study also indicated that the protein was quite stable in that the protein containing adsorbed HC1 was not appreciably hydrolyzed when put into water. This is in contrast, to the marked hydrolysis of the peptide-like bond in nylon when it contained adsorbed HC1. S‘ ince a great deal is known about lyophilized p-lnctoglobulin, it was decided to determine quantitatively the sorption of HC1 by this protein. -4 complete adsorption isotherm was obtained a t 27”. Following desorption the amounts of HC1 retained at I Oe6 mm. pressure were determined a t 27,30 and GO”. Experimental The j3-lactoglobulin used in this study was a fresh sample of the same protein, obtained from the Carlsberg Labors-
1200
WASYL
s. KNOJEWPJ A N D LLOTJ)€1. REYERSON
Yol. G4
t = 27°C I before HCL- treatment
2 after HCL-treatment 3 probable
Comparison adsorption isotherms of H20- vapor
on P-lactoglobulin i -
2
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Pressure mm Fig. 1. torium, on which sorptions of H20 and DzO were ~ b t a i n e d . ~ TABLE I 125.8 mg. of this protein was weighed into a glass bucket RESIDUAL AMOUNTS O F HCl HELD B Y THE PROTEIN A S which was suspended from a McBain uartz spiral balance FUNCTION O F THE TEMPERATURE AT A which had a sensitivity of 2.285 mg.qmm. Extension of SOME RECIPROCAL the spiral was measured with a traveling microscope having PRESSURE OF 10-6 mm. a sensitivity of =k0.003 mm. The sample and the quartz Time of desorption, J l m o l c s of EICl boiind Tcnip., "C. hr. per g. of protein spiral were thermostated as previously described. Equilibrium pressures in the system were measured by a mercury 27 156 1.5404 manometer using a cathetometer reading to h0.02 mm. 40 50 1.4630 Prior to admitting gaseous HCl to the system, a complete 60 118 1.IT63 adsorption-desorption isotherm of H20 vapor was determined for this sample at 27". The protein weighed exactly mm. When the same a t the completion of the desorption of H20 as system pumped down to a pressure of it did before HtO vapor was admitted to the sample. The equilibrium was finally reached, 1.540 mmoles/g. of HCl adsorption part of this isotherm is shown as the upper remained sorbed on the protein. The protein was then curve in Fig. 1. The results were identical Kith those rc- heated to 40' again and evacuated to 10-6 mm. Some HCl was removed during this process. The protein was then ported in the previous study.3 Following this, dry gaseous HCI was admitted to the heated t o 60" and the evacuation repeated. At this highly evacuated system containing the protein and a temperature the protein still retained 1.176 mmoles/g. complete sorption isotherm was determined in the same man- HC1. These three desorptions took about 10 days to ner as previously described.* Liquid HCl of 99.87y0 purity complete and the results are given in Table I. With the was redistilled several times and the liquid from the last residue of adsorbed HC1 still on the surface of the protein, distillation was vaporized t o provide the gas used in the a second adsorption of water vapor was carried out a t 27'. sorption. Amounts of HCI adsorbed were determined up to Curve 2 in Fig. 1 gives the isotherm for this second sorption 5.484 mmoles per gram of protein a t an rquilibrium prcssurcl of HzO, based on the original weight of the protein. of 325.3 mm. The adsorption isotherm is given in Fig. 2. Results and Discussion The first equilibrium point (1.9 mmoles/g.) was reached in :-tboiit 3 hours, the equilibrium HCI pressure being 4.515 It is evident from these results that this protein mm. Following this first rather rapid adsorption, each has nowhere near the number of sitcs available for successive point required from two to three days for equilibrium to be reached. This is from six to ten times longer binding HCl as it has for HzO and DzO. Except than mas required for the sorption of HSO. This very slow for the first isotherm point which was reached in a process of adsorption indicates that a sizable energy or few hours, all other points required days to reach ,xtivation is involved equilibrium, indicating a high energy of activation The RC1 mas thrn desorbed :IS rapidly RS possible and the and some probable form of chemisorption. The iso(3) L. H. Rryerson and W. S. Hnojewyj, Tars J o u n s a ~ 6, 4 , S l l ( 1960).
therm appears to be flattening out as it passed a x-npor pressure of more than 300 mm. At the
/ ~~
~
Adsorption isotherm of HCL-vapor on p -LACTOGLOBULIN at 27°C 1
I
I
I
200
100
I
1
300
Pressure mm. Fig. 2.
highest point reached, 204 molecules of HC1 were bound per molecule of protein. This is about half as many as the number of D20molecules which had to be adsorbed for the maximum exchange of deuterium observed in our previous While all of the HzO and D20 molecules are released by the protein during complete desorption a t lo4 mm.p., only slightly more than two thirds of the adsorbed HCX molecules can be removed by a high vacuiiin at E " . Heating the protein to 40 and then t o 63" resulted in slight additional dcsorptions as shown in Table I. It is interesting to observe that the number of IlCl molecules remaining adsorbed a t 27" just equals the number of active amino groups in the side chains of this protein. Prolonged heating and evacuation a t 40 and 60" only removes one fourth of these molecules, indicating a stroiig chemical bonding. IIowever, the presence of these strongly bonded HC1 molecules hac: only a minor (+feeton the adsorption of HZO as is shown as curve 2 in Fig. 1. If each HC1 bonded to an actire side-chain amino group prevented an €120 molecule from being adsorbed, then it was estimated that the isotherm for HzO on the protein permanen1 ly lioldiiig HC1 molecules ought to be similar to the dotted line 3 in Fig. 1. Isotherm 2 i n Fig. 1 lies slightlr below isotherm I , indicating that the residual IlCl molecules do h a z ~some slight
effect on the relaxation of the structure of the protein insofar as the adsorption of HzO is concerned. Since the numbers and kinds of amino-acid residues are well known for P-lact~globulin~ and completely established by SangerS for insulin, it mas decided to determine the ratio of the number of HC1 molecules bound a t zero pressure to the number of active amino groups in the side chains of these two proteins (57 for @-lactoglobulinand 14 for insulin) a t the several temperatures studied. The results are shown in Fig. 3. I n the case of insulin, where the data a t lower temperatures were available,2the ratio is greater than 1.5 a t 0" but at 27" the ratio for both proteins is nearly unity and it falls slightly thereafter, reaching about 0.75 at GO", the highest temperature studied. These results seem to be a reasonable proof that the adsorbed HC1 molecules are most tightly bound by the amino groups on the side chains of the proteins. This binding may well be of a chemical nature. It seems likely from these results that the adsorption of the HC1 on the side chains prevents the opening up of the protein molecule and it does not seem probable that the backbone peptide links get a chancc to adsorb mnriv HCl inolo~dcs,a n d if nd( $ 1 K I.inderstr@in-Lang. Souvenir, J. SOC.B i d . ('hemrsts, I n d t a , 181 (1955). ( 5 ) F. Sangor, Bull. SOC. c h i i n bzol., 37, 23 (1955).
WASYLS.HNOJEWYJ AND LLOYD H. REYERSON
1202
-.n
no
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T'ol. 64
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Temperature
O C
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Fig. :J
sorbed the bonding appears to be weak. Qualitative experiments by one of us (L.H.R.) at the Carlsberg Laboratorium definitely showed that this same protein with a maximum amount of HC1 adsorbed was not hydrolyzed a t the peptide links when placed in water. The CO-SH bond in nylon does hydrolyze under similar conditions, as is shown in our earlier work in this Laboratory.6 Thus there seems to be no question but that the same dry lyophilized protein behaves very differently toward HzO and DzO than it does toward dry ( 6 ) L. H. Reyerson and L. Peterson, THISJOURXAL,60, 1172 (1956).
gaseous HC1, and this seems only reasonable. The number of molecules of DzO needed for the niaximum exchange3 shows that H2O and D 2 0 must, in part, be adsorbed on the peptide bonds in the backbone of the protein. The maximum number of HCl molecules found adsorbed in this study slightly exceeds the number of side chains of the amino-acid residues which have active groups on which adsorption could occur. Certainly the very strong bonding of the HC1, which remains on the protein a t essentially zero pressure, can be accounted for by its being bonded to the amino groups on the side chains of the protein.
