Heat of Solution of Lyophilized Solid Ribonuclease A
The Journal of Physical Chemistry, Vol. 82, No. 15, 1978
1701
The Dependence of the Heat of Solution of Lyophilized Solid Ribonuclease A on Water Content Rami Almog and Eugene E. Schrier* Department of Chemistty, State University of New York at Binghamton, Binghamton, New York 13901 (Received November 23, 1977; Revised Manuscript Received March 20, 1978) Publication costs assisted by the U.S. Public Health Service
The enthalpy of solution of lyophilized ribonuclease A in dilute buffer has been measured as a function of the water content of the solid at 25 "C. The negative of the enthalpy of solution, -AHsolnB, approaches 400 kcal mol-l at very low water content, is 200 kcal mol-1 at 60 mol of H20per mol of solid ribonuclease A, and approaches zero asymptotically as the water to protein ratio in the solid becomes infinite. About six water molecules are bound to the protein with a net enthalpy of solvation of -8 kcal mol-' of water added to a mole of protein while the net solvation enthalpy provided by the next hundred waters is -2 kcal mol-I of water added to the solid protein. The latter value is in agreement with estimates of the enthalpy of adsorption of liquid water on serum albumin.
Introduction The hydration of proteins continues to be an area of interest and contention. A recent review1 has pointed out the scarcity of data concerning the enthalpy of interaction of water with proteins. In the course of a determination of the enthalpies of transfer of ribonuclease A from dilute buffer to solutions of various cosolutes which is described in the accompanying paper,2 we observed a marked dependence of the heat of solution of lyophilized ribonuclease A on the content of residual water in the solid protein. The possible relevance of this heretofore unreported relationship to the energetics of protein hydration will be developed below. Experimental Section Materials. Ribonuclease A was purchased from Sigma Chemical Co. (Type 11-A). Solutions of the protein were passed through a mixed bed ion-exchange resin to remove residual phosphate. The pH of a protein solution after passage through the column was 9.6-9.7 in agreement with the known isoelectric point.3 The protein solutions were lyophilized at concentrations below 1%to avoid aggregati~n.~ It was found that the water content of the lyophilizate could be varied between 16 and 90 mol of H 2 0 per mol of ribonuclease A by using longer or shorter periods of lyophilization. Higher water contents were achieved by placing the protein in a chamber containing a concentrated salt solution. Where a water-to-protein ratio less than 16 was desired the sample was dried over P205in vacuo at room temperature. By this method it was possible to achieve ratios as low as 4 mol of H 2 0 per mol of ribonuclease A. Water contents for the solid, lyophilized protein were determined by drying samples to constant weight at 105 "C. These samples were discarded after the determination. Usually a series of ampoules was filled at one time and samples for the dry weight measurements were taken at the beginning, middle, and end of the series. The deliquescent nature of the lyophilized solid having a water content below 16 mol of water per mol of protein made it necessary to fill the ampoules with these samples in a drybox. Method. The calorimeter and general procedure used for making the heat of solution measurements have been described p r e v i ~ u s l y . ~Solutions ,~ in which the contents of the ampoules were dissolved contained 0.025 m tris0022-3654/78/2082-1701$01 .OO/O
(hydroxymethy1)aminomethane and 0.15 m KC1 at pH 7.0. The ribonuclease A concentrations in the solutions after to 1.1X m. ampoule breakage ranged from 5.8 X No dependence of the heat on the final ribonuclease A concentration in solution was observed. Measurements were carried out so that 25.0 " C was the mean temperature. A check on the calorimeter and procedure was provided by measuring the heat of solution of tristhydroxymethy1)aminomethane in 0.1000 M HC1. The mean value of six measurements, -7.120 f 0.005 kcal mol-l, is in agreement with -7.120 f 0.001 kcal mol-' obtained by Burnetti, Prosen, and G ~ l d b e r g . ~ Results and Discussion Figure 1gives the dependence of the negative of the heat of solution of lyophilized ribonuclease A in buffer solution, -AHsolsolnB, on the molar ratio of water to ribonuclease A in the original solid phase. The estimated uncertainty of a given value is f 2 kcal mol-l. The large exothermic heat of solution of lyophilized ribonuclease A is primarily a manifestation of the strong solvation of charged and other polar surface sites by water upon dissolution of the protein. This solvation opposes the weaker protein-protein interactions in the solid phase. We can account for a value of -AHsohBapproaching 400 kcal mol-l as the water content of the lyophilized solid approaches zero as follows. Nemethys suggested that the enthalpy change for the formation of a hydrogen bond in aqueous solution is approximately -2.5 kcal mol-'. Kuntzg determined the number of water molecules associated with each amino acid residue in a protein. Assuming (a) that only the side chains and the peptide backbone units of the residues of aspartic and glutamic acid, arginine, lysine, histidine, and three of the six tyrosines as well as the terminal amino and carboxyl groups are exposed to water in the native state of the protein at pH 7.0, (b) that no other residues are exposed, and (c) that each water molecule which is part of the hydration layer as determined by Kuntz forms a new hydrogen bond with some part of the amino acid residue, we calculate that 400 kcal mol-' will be liberated when a mole of separated protein molecules is placed into water. Other exothermic contributions may come from the exposure of other polar but uncharged groups in the native state. In addition, it is likely that the conformation of the protein in the lyophilized solid is different from that in solution. In an investigation carried 0 1978 American Chemical Society
R. Almog and E. E. Schrier
The Journal of Physical Chemistry, Vol. 82, No. 15, 1978
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Figure 1. The dependence of the negative of the enthalpy of solution, -AHd:, of lyophilized solid ribonuclease A on the ratio of the number of moles of water contained in the lyophilized solid to the number of moles of ribonuclease A.
out using Raman spectroscopy, Yu and co-workers1° showed that there were local conformational changes in the tyrosine and disulfide regions of lyophilized solid ribonuclease A compared to the molecule in solution. Some modification of the spectrum of the amide groups in the backbone region was also noted. Complete unfolding in the solid state and subsequent refolding to the native molecule upon dissolution11J2would provide an additional exothermic contribution of 70 kcal mol-l. The sum of these exothermic contributions would be reduced by the unknown amount of energy necessary to separate the protein molecules from each other in the solid phase. Even so, there do seem to be ample exothermic contributions from the sources mentioned to yield the magnitude observed. The striking decrease of -AHsohBwith increasing water content is a reflection of presolvation of the solid protein, i.e., -AHsolnBdecreases because the enthalpy of the solid phase is becoming more negative as the water content increases. This change serves as an indicator of the energetics of the interaction of the protein surface with water. Figure 1 appears to be divided roughly into three regions. The first extends from 0 to about 6 mol of water per mol of ribonuclease A, The slope of the straight line drawn through this part of the curve yields a value of -8 kcal mol-' per mol of water added to the solid. Similarly, a straight line drawn through the region between 6 and about 100 mol of water gives -2 kcal mol-l per mol of water added, while the region between 160 and 200 mol gives -0.6 kcal mol-l per mol of water added. Since the slope of the overall curve changes continuously, these values cannot be taken too literally but they do suggest a division into stronger and weaker binding sites. We infer from the data that there are a few tightly held water molecules. Rao and Brym13 have reached the same conclusion using careful analytical techniques. They
suggest that these water molecules may be integral parts of the structure of the protein. Additional water molecules are likely to bind to polar sites on the outside of the protein. Water molecules in the third region probably bind to the already formed water monolayer. The curve should go to zero asymptotically since enough water must be absorbed to obtain a zero AHsolnB at infinite dilution. The net enthalpies of solvation obtained here may be compared to the enthalpy of adsorption, A H A , of water on serum albumin calculated from the data of Bull14by Kuntz and Kauzmann.l It is necessary to subtract out the enthalpy of vaporization of the pure liquid from their data in order to make the reference state comparable to what we are dealing with here. In the range of 0.04 g of H,O/g of protein to 0.10 g of H,O/g of protein, the average value of -AH*is 1.8 kcal mol-l for serum albumin while -AH of net solvation from the middle region of Figure 1was given above as 2 kcal mol-l. The value of - A H A decreases with increasing water content in a manner similar to that observed for -AH of net solvation. The initial region of very low water content was not investigated in the work of Bull14but there are free energy of adsorption data which resemble the low water content end of the curveal It appears that solution calorimetry has provided a novel way of obtaining interesting data regarding the interaction of a solid protein phase with pure water.
Acknowledgment. This research was supported in part by Grant No. GM 11762 from the Institute of General Medical Sciences, U.S. Public Health Service. References and Notes (1) I. D. Kuntz, Jr., and W. Kauzmann, Adv. Protein Chem., 28, 239 (1974). (2) R. Almog, M. Y. Schrier, and E. E. Schrier, J . Phys. Chem., following article in this issue.
(3) C. Tanford and J. D. Hauenstein, J . Am. Chem. Soc., 78, 5287 (1956). (4) A. M. Crestfieid, W. H. Stein, and S. Moore, J . Biol. Chem., 238, 618 (1963). (5) E. R. Stimson and E. E. Schrier, J. Chem. Eng. Data, 19, 354 (1974). (6) M. Y. Schrier, P. J. Turner, and E. E. Schrier, J . Phys. Chem., 79,
1391 (1975). (7) A. P. Burnetti, E. J. Prosen, and R. N. Goldberg, J. Res. Natl. Bur. Stand., Sect. A , 77, 599 (1973). (8) G. Nemethy, Ann. Inst. Super. Sanifa, 6, 487 (1970). (9) I. D. Kuntz, J. Am. Chem. SOC.,93, 514 (1971). (IO) N-T. Yu, B. H. Jo, and C. S. Liu, J. Am. Chem. Soc.,94, 7572 (1972). (11) In a subsequent study of lysozyme [N-T. Yu and 8. H. Jo, Arch. Biochem. Biophys., 156, 469 (1973)]it was shown that a dlssolved sample of the protein when lyophilized and redissolved gave a spectrum identical with that of the original dissolved protein. This suggests that the conformational changes in lysozyme are reversible. Since the thermodynamic quantities related to the unfolding of lysozyme and ribonuclease A are very similar, we infer that the conformational change in lyophilized ribonuclease A is reversed upon solution to give the native molecule. It is a well known fact that lyophilization does not alter the enzymatic activity of ordinary proteins. (12) N-T. Yu and B. H. Jo [ J . Am. Chem. Soc., 95, 5033 (1973)]have shown that the Raman spectra of lyophilized solid ribonuclease A does not change when the water content is changed from 0 to 100% implying that the conformation of the protein in the sold Is independent of'its water content. (13) (a) P. B. Rao and W. P. Bryan, J . Mol. Biol., 97, 119 (1975); (b) P. B. Rao and W. P. Bryai, Biopo/ymers, 16, 291 (1978). (14) H. B. Bull, J . Am. Chem. SOC.,66, 1499 (1944). ~
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