The Sorption of Water Vapor by Lyophilized Ribonuclease - The

Chem. , 1958, 62 (10), pp 1214–1216. DOI: 10.1021/j150568a013. Publication Date: October 1958. ACS Legacy Archive. Note: In lieu of an abstract, thi...
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JOHN G. Foss AND LLOYDH. REYERSON

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with surface coverage in Figs. 4 and 5 and a sample BET plot in Fig. 6. Free energy changes and work of adhesion are shown in Tables I and 11. TABLE I 15’ Ya0

System

- YWO

ergs/crn.s’

HzO-annealed Quartz HAO-unannealed Quartz HZO-calcite

yao

- Yell

w,

ergs/cm. 2

ergs/cm. *

318 283 281

391 357 355

244 209 208

TABLE I1 25 O %O

-

yavo,

System

ergdcm.2

HzO-annealed Quartz HzO-unannealed Quartz HZO-calcite

231 203 186

yao

- YSl.

w,

ergdcm.2

ergs/cm.l

303 275 258

375 347 330

Discussion The areas per water molecule on the adsorbent surface are: 13.72 A.z on unannealed quartz silica, 11.73 on annealed quartz silica and 14.30 A.2 on calcite. The difference between the values for annealed and unannealed quartz may be explained

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as follows. Hydroxyl groups, bound to silicon atoms are known to be the species active in the physical adsorption of water vapor on quartz.6 When quartz is crushed Si+4ions are replaced, in the surface, by the readily polarizable 0- ions and an amorphous layer is produced.’ Annealing, which restores the crystal structure,* results in an increase in the number of silicon atoms, and therefore in an increased number of adsorption sites per unit surface. The values calculated and shown in Tables I and I1 tehd to confirm the validity of this explanation. Similar work was done on the system waterkaolinite but adsorption hysteresis precluded an accurate interpretation of the data in the foregoing terms. Acknowledgment.-The authors are pleased to express their appreciation to the American Petroleum Institute for its financial support of this work. (6) V. A. Dzis’ko, A. A. Vishneyskaya and V. A. Chesalova, Zhur. F i z . Rhim., 2 4 , 1416 (1950). (7) W. A. Weyl, “Structure and Properties of Solid Surfaces,” edited by R. Gomer and C. S. Smith, The University of Chicago Press, 1953. (8) D. D’Eustachio and 8. Greenwald, Phys. Rev., 69,532 (1946).

T H E SORPTION OF WATER VAPOR BY LYOPHILIZED RIBONUCLEASE1 JOHN G. Foss AND LLOYDH. REYERBON The School of Chemistry, University of Minnesota, Minneapolis 14, Minnesota Received March 3, lot8

Isotherm data for the sorption of water on ribonuclease are reported. Heats and entropies of adsorption are calculated and discussed briefly.

Because of the important role played by water in determining the properties of proteins it would be desirable to learn more about the binding forces and mechanism of binding between these molecules. Since the biologically important properties of proteins manifest themselves in solution the water-protein interactions in solution are of greatest interest. However, there are also good reasons for studying the very much simpler case of water sorption on dry proteins. Amberg2 has commented on some of these in a paper published while the present investigation was being carried out. This study was initiated for several reasons. First it was hoped that entropy changes on sorption might be used together with model calculations to determine any marked structural changes which might take place on hydrating the protein. If such entropy changes could be measured starting with the dry protein and progressing till the protein was in solution, something might be learned of what important changes occur on going into solution. A second general goal of this work was to determine experimentally whether any real confidence can be placed in using isotherm data to determine heats of sorption when compared with calorimetrically determined values. Finally it was ’

(1) This work was supported in part by a Grant-in-Aid from the E. I. du Pout de Nemours Company and from the National Institutes of Health. (2) C. H. Amberg, J . A m . Chsm. S o c . , 79, 3980 (1957).

hoped to learn whether sorption isokherms or calorimetric heats are affected by marked changes such as denaturation. Amberg’s2 recent paper on the calorimetrically determined heat of sorption of bovine serum albumin already has provided a partial answer t o the second question as will be discussed below. However, calorimetric studies will be continued using a diphenyl ether phase change calorimeter similar to that described by G i g ~ e r e . ~ This paper will deal only with the isotherm data obtained for ribonuclease. Experimental Sorption isotherms were measured gravimetrically using a quartz spring helix having a sensitivity of 11.9 mg./cm. Displacements were measured with a traveling microscope to +0.001 cm. The protein was held in a light glass or aluminum bucket and thermostated with a water jacket. A silicone oil manometer was used to measure the lower y s u r e s (up to 7 mm.) while higher values were determined y equilibrating a thermostated water sample with the protein. This method was almost essential a t pressures close to saturation for room temperature since temperature fluctuations would then cause marked changes in the pressure. The water used for the adsorbate was distilled and deionized. It was outgassed by a series of freezings, thawings and evacuations. The ribonuclease was purchased from the Armour Company (Lot 381-059) and lyophilized from a one per cent. water solution. The protein was outgassed by pumping (3) P. A. Giguere, B. G. Morissette and A. W. Olmos, Can. J . Chem., 33, 657 (1955).

Oct., 1958

SORPTION OF WATERVAPORBY LYOPHILIZED RIBONUCLEASE

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at the temperature to be used for the highest isotherm (in this case 30.0"). Pumping was continued till the pressure reached less than 0.001 p . This would usually take two days and by that time there would be no noticeable change in weight during the course of a day.

Results and Discussion Figure 1 shows the differential heats and entropies of sorption of water on lyophilized ribonuclease. These values were calculated from interpolated points on a large scale graph using the equations given by Bettelheim and V01man.~ The isotherms and free energy values are not presented as these are smooth monotonic functions very similar to those reported by other workers. (If desired, the 20" isotherm easily could be calculated from the entropies and heats.) As is well known it is extremely difficult to calculate reliable heats in the region of low coverage 80 little weight can be placed on values below 2%. An error of only 0.01 mm. in the isotherms could 0.5 to lead t o errors in the heats ranging from =t4 cal./mole in going from 2.5 to 0.5% sorbed. Since the standard state is water vapor a t 20" and 1 atm. the changes indicated are for one mole of water being transferred from this state to the surface. The water vapor is assumed to be ideal. These heat and entropy values are of little interest unless they can be related to the molecular properties of the protein. Benson and Ellis'6 work is extremely important in this regard since they showed that water was adsorbed on proteins in the same manlier regardless of the state of subdivision. If this were not so it would not be possible to compare this work with that of other investigators unless identical samples were used. (Areas determined using nitrogen differ in a way dependent on the state of subdivision.) * A B E T plot from the 20" isotherm is linear up to p / p ~= 0.15 and gives a BET monolayer at 5.8%. (Both the 20 and 30" BET plots consist of two straight segments with a change in slope at approximately 5.5%.) On comparing this with Fig. 1 it is seen that the break in the heat and entropy curves at 5.5% corresponds to the formation of a monolayer. However, it is probably not a monolayer in the usual BET sense since as mentioned above a different monolayer would be obtained with nitrogen. We will follow Pauling's6 suggestion and interpret this monolayer value as indicating a saturation of the polar side groups on the protein. Ribonuclease has a total of some 39 polar side groups, which corresponds to 5.1% on saturation (assuming 1 molecule of water per polar group and a molecular weight of 13,900). This is in close agreement with the observed BET monolayer. Work on ribonuclease in neutral aqueous solutions a t room temperature has shown that it is normally in a folded configuration. It seems reasonable to suppose that on lyophilizing the solution this configuration is a t least partially maintained. This would suggest that if the protein is in a state, approximating an a-helix, the potentially available polar peptide sites are tied. If the ribonuclease

*

.

(4) F. A. Bettclheim and D. H. Volman, J . Poly. Sci., 24,445 (1957). (5) 6 . W. Benson, D. A. Ellis and R. W. Zwaneip, J. A m . ChenL. Sor., 72, 2102 (1950). ( 6 ) L. Pauling, i b z d . , 67, 555 (1945).

2

4 G Weight % ' sorbed.

8

Fig. 1.-Differential heats and entropies of adsor tion of water on ribonuclease 0 ; heats of adsorption on 8 S A - -

-

were to be lyophilized from a solvent in which it is known to be in an open configuration, such as formamide, this might cause a marked increase in the number of polar sites and a consequent increase in the BET area. It is planned to carry out such an experiment in this Laboratory. I n order to evaluate the change in integral entropy for the water going from the gas to the surface phase it is necessary to know the surface pressure cp for both isotherms.' Knowing this the change in the integral entropy is found from '

To find

cp

we use 'P =

KkT

sop

adlnp

(2)

where p is the equilibrium pressure when a percent. of water is sorbed. K is a constant which (7) T. L. Hill, P. H. Emmett and L. G. Joyner, i b i d . , 73, 5102 (1951).

JOHN G. Foss AND LLOYD H. REYERSON

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includes the specific area of the sample and will presumably be the same for both isotherms. For an accurate value of cp it is necessary to make measurements into the region of very low pressure and then use graphical methods or an empirical equation of state to evaluate the integral. We shall use the latter procedure. It was found that the data for water sorption on RNase fit a Freundlich isotherm down t o about 2y0. (Experimental errors are too large below 2% to tell if the Freundlich isotherm is still valid.) Still remaining approximately parallel both isotherms then deviated from a straight line on the log a vs. log p plot. Since it is not necessary to know the absolute value of cp to use (1) we can use the Freundlich isotherm in (2) assuming a similar error will be made for both calculations. Using a =

Cplln

(3)

(where c and n are constants dependent on temperature) p may be eliminated from the integral in (2) giving (a

= nKkTa

There is both experimental and theoretical justification8 for assuming n = blkT (where b is a constant) so that (p

=

bKa

From this we see that

(where & is the differential entropy of the adsorbed water molecules). Comparing with (1) 8s = 3 8

Thus, subject to the above mentioned approximations, we can compare the entropy of the water vapor with that of t,he sorbed water using the measured differential entropies. There is one final (8) B. M. W. Trapnell, “Chemisorption.” Butterworth Scientific Publications, London, 1955.

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qualification which must be made about the above discussion. All of the equations used were derived for the case of an adsorbate on an inert solid and in the case of the protein water system this will certainly be a poor assumptioii a t high coverage^.^ It may even be a poor assumption a t these low coverages. The entropy of water vapor a t 293°K. is approximately 45 e.u. of which 35 are translational and 10 rotational. From the figure it can be seen at 0.5% almost 39 e.u. have been lost suggesting the water has lost all of its translational and perhaps some of its rotational freedom. The energy of binding to this site is 17 kcal. and the heat and entropy fall as the high energy sites become saturated. When the high energy sites are filled, other sites having unfavorable entropies but comparable energies are filled-perhaps two point sites in which the water’s rotation is hindered. If there are two point sites the binding energy could be distributed between two bonds to the protein. When these low entropy sites are filled the water will add to sites having a lower binding energy but a higher entropy. As mentioned above the break at 5.5y0 is interpreted as meaning that all of the polar sites are filled and now a new group of sites begin to be filled. These differ from the polar sites in a t least one respect. They have a rather small spread of energies (approximately 1 kcal. vs. 5 kcal. for the polar sites). For comparison, AmbergV data for his run number 2 are indicated by a dotted line. Though two different proteins were being studied, there is reason to expect similar results since both have a similar number of polar side groups and give almost identical monolaye values (5.8 for the ribonuclease vs. 5.8 to 6.1 or the bovine serum albumin). This apparent agreement for the heat data strongly suggests that the use of calculated heats is justified in spite of the irreversibility of the adsorption isotherms.

k

(9) T. L. Hill, J. Chevi. Phys., 18, 24G (1950).