Thermodynamic and Related Studies of Water Interacting with

6

T h e r m o d y n a m i c a n d R e l a t e d S t u d i e s of W a t e r Interacting with Proteins

Downloaded by KTH ROYAL INST OF TECHNOLOGY on November 22, 2016 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch006

JOHN A. RUPLEY, P.-H. YANG, and GORDON TOLLIN Department of Biochemistry, University of Arizona, Tucson, AZ 85721

It is recognized that important protein processes are controlled by the environment. In their study of an enzyme model reaction, for example, Bruice and Turner (1) have shown that change from water to a dioxane-water solvent alters the rate of carboxylate ion attack on substituted phenyl esters by four to six orders of magnitude. In spite of agreement on the importance of solvent, there is a lack of understanding of the basis of solvent control. Our interest in this problem came through attempting to relate the structure of active sites determined by x-ray diffraction analysis to the thermodynamics of binding of substrates and substrate analogs. These efforts were not successful. The difficulty of understanding the thermodynamics of protein reactions is exemplified through comparing protein folding with binding of a substrate analog (Table I). These reactions differ more than ten-fold in area of contact but by less than a factor of two in free energy and enthalpy. Although the way in which solvent participates may not be the origin of the unusual chemistry, it appears to be the least well understood aspect of protein reactions. Investigations on protein-water interactions can be categorized according to whether protein solutions or protein films and powders were studied. Solid samples allow variation of water activity over a wide range. Because of this advantage, experiments on such samples are the focus of this paper. It is possible that the protein conformation may change with drying from the solution to the hydrated solid. Evidence given below indicates that the conformation in the dry state is not significantly different from that in dilute aqueous solution. The literature and experiments discussed below describe the process of protein hydration, which is addition of water to dry protein to obtain the solution state. An understanding of this process is expected to increase understanding of water-protein interactions in solution. The protein hydration process, which can be described unambiguously by experiment, should be distinguished from the water of hydration concept, which is viewed 0-8412-0559-0/80/47-127-111 $05.50/0 © 1980 American Chemical Society

Rowland; Water in Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


3

350

15,150

1

- 29710

- 33050

(J mol" )

AG° 1

- 57740

- 94720

( J mol" )

ΔΗ° 1

-

1

mol" )

AS°

92880

- 205010

( J K"

a

Area c a l c u l a t i o n s from Shrake and Rupley (17). Thermodynamic parameters f o r lysozyme u n f o l d i n g from Tanford and Aune (33) and f o r s u b s t r a t e b i n d i n g from Banerjee and Rupley (34).

Substrate B i n d i n g Lysozyme + ( G l c N a c )

Lysozyme

Protein Folding

Change i n Surface Area

Table I . Change i n Surface Compared w i t h Change i n Thermodynamic P a r a m e t e r s


6. RUPLEY ET AL.

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d i f f e r e n t l y a c c o r d i n g t o the i n t e r e s t s o f the i n v e s t i g a t o r s and the experimental approach.


Thermodynamics o f Protein-Water I n t e r a c t i o n s Free Energy. The f r e e energy o f h y d r a t i o n i s determined by s o r p t i o n isotherms ( 2 ) . These measurements show that the hydra t i o n process i s stepwise. The isotherm f o r a p r o t e i n t y p i c a l l y has a "knee" a t 0.05 _h (g of water/g o f p r o t e i n ) , o r 0.1 χ ( r e l a t i v e water vapor pressure) which r e p r e s e n t s b i n d i n g of s t r o n g l y i n t e r a c t i n g water t o charged groups ( 3 ) . Between 0.05 h and 0.3 h_ (0.1 and 0.9 x) there i s a p l a t e a u i n the isotherm. Above 0.9 x, which i s c l o s e t o the l i m i t of s o r p t i o n measurements, the amount of water bound i n c r e a s e s s h a r p l y w i t h increased p r e s s u r e . Some models used i n i n t e r p r e t i n g s o r p t i o n data a s s o c i a t e the p l a t e a u r e g i o n w i t h m u l t i l a y e r water. This cannot be c o r r e c t , because even 0.4 g o f water/g o f p r o t e i n i s b a r e l y enough t o cover the s u r f a c e . The p l a t e a u r e g i o n corresponds (see below) t o b i n d i n g of water a t p o l a r s i t e s , such as c a r b o n y l groups, and the r i s e a t h i g h r e l a t i v e pressure r e p r e s e n t s the s t a r t o f the l a s t stage i n completion of monolayer coverage. Enthalpy. The enthalpy o f h y d r a t i o n has been determined from the temperature dependence of the s o r p t i o n isotherm. The magnitude of the heat of s o r p t i o n i s about 80 kJ/mol of water a t low coverage (the "knee r e g i o n a t 0.05 _h) and decreases t o the heat of v a p o r i z a t i o n of water (44 kJ/mol) by 0.2 h. Because h y s t e r e s i s i s observed g e n e r a l l y i n s o r p t i o n isotherm measure ments, v a n t Hoff heats of s o r p t i o n , c a l c u l a t e d assuming thermo dynamic e q u i l i b r i u m , may be i n c o r r e c t . A c a l o r i m e t r i c study f o r c o l l a g e n ( 4 ) confirms the v a n t Hoff v a l u e s . A Monte C a r l o simu l a t i o n of the lysozyme-water system (5) a s s i g n s a p o r t i o n of the water a t the p r o t e i n surface an energy of i n t e r a c t i o n of 80 kJ/mol or g r e a t e r . Water a t the p r o t e i n surface d i f f e r s more i n enthalpy than i n f r e e energy from b u l k water (Table I I ) ; the magnitudes of both the heat and entropy o f s o r p t i o n decrease s t r o n g l y w i t h i n creased coverage of the s u r f a c e , i . e . w i t h decreased s t r e n g t h o f interaction. The dependence o f the heat of s o r p t i o n on the extent of cover age has been observed t o be i r r e g u l a r , w i t h an extremum i n the knee r e g i o n of the isotherm (6, _7)· A c a l o r i m e t r i c study (8) has demonstrated a s i m i l a r i r r e g u l a r i t y i n the h y d r a t i o n of p o l y saccharides. The extremum i n the heat of s o r p t i o n f o r lysozyme (6) corresponds w i t h one i n the heat c a p a c i t y (see below) that r e f l e c t s proton r e d i s t r i b u t i o n . 11

f

f

Volume. Volume has been measured as a f u n c t i o n of the extent of h y d r a t i o n f o r ovalbumin (9) and f o r $ - l a c t o g l o b u l i n i n the c r y s t a l (10). The l i n e a r dependence o f the volume on system comp o s i t i o n shows that the p a r t i a l s p e c i f i c volume of the solvent i n


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the s o l i d sample i s i d e n t i c a l t o that of bulk solvent at hydrat i o n l e v e l s above 0.2 h. Heat Capacity. Measurements of the heat c a p a c i t y of p r o t e i n systems are p a r t i c u l a r l y i n t e r e s t i n g f o r s e v e r a l reasons: 1) they r e f l e c t s o l v a t i o n of nonpolar elements i n a d d i t i o n t o other p a r t s of the p r o t e i n surface and thus can be viewed as the most comp l e t e thermodynamic probe f o r w a t e r - p r o t e i n i n t e r a c t i o n s . 2) Heat c a p a c i t y can be measured c o n v e n i e n t l y f o r both s o l u t i o n and s o l i d samples; thus the two c a t e g o r i e s o f p r o t e i n h y d r a t i o n s t u d i e s , those on s o l u t i o n s and those on s o l i d samples, can be c o r r e l a t e d . 3) There i s a s u b s t a n t i a l l i t e r a t u r e on the heat c a p a c i t i e s o f s m a l l molecules and on a d d i t i v i t y r e l a t i o n s h i p s , which appear t o be more accurate f o r heat c a p a c i t y than f o r other thermodynamic functions. Heat c a p a c i t y measurements f o r p r o t e i n s are made u s i n g d i f f e r e n t i a l scanning c a l o r i m e t e r s , which give the heat c a p a c i t y as a f u n c t i o n of temperature, and l e s s f r e q u e n t l y , by drop c a l o r imetry, which gives the heat c a p a c i t y a t a f i x e d mean temperature. Scanning c a l o r i m e t r i c measurements of l i g h t l y hydrated p r o t e i n samples over the temperature range centered on 0 °C show t h a t a t r a n s i t i o n heat and change i n heat c a p a c i t y a s s o c i a t e d w i t h m e l t i n g o f water i s observed only above a t h r e s h o l d l e v e l of h y d r a t i o n . For example, measurements f o r c o l l a g e n (11) show that there i s no t r a n s i t i o n f o r samples o f 0.35 ti; thus at l e a s t t h i s amount o f water i n t e r a c t s so s t r o n g l y w i t h the p r o t e i n that i t cannot f r e e z e . Estimates of the water of h y d r a t i o n , here defined as the amount o f nonfreezing water, can be made by a n a l y s i s o f data f o r samples of s u f f i c i e n t l y h i g h water content t o show a melting t r a n s i t i o n . Scanning c a l o r i m e t r i c measurements of the denaturation process as a f u n c t i o n of extent of h y d r a t i o n have been reported f o r s e v e r a l p r o t e i n s ( 3 - l a c t o g l o b u l i n : (12); lysozyme: (13)). The m e l t i n g temperature r i s e s and other parameters of the denaturat i o n t r a n s i t i o n change as the h y d r a t i o n l e v e l i s decreased below 0.75 h. The changes are l a r g e below 0.3 _h. Heat c a p a c i t i e s determined f o r a f i x e d mean temperature over the f u l l range o f system composition, d r y p r o t e i n t o d i l u t e s o l u t i o n , have been reported f o r ovalbumin (9) and lysozyme (14). Suurkuusk (15) has measured heat c a p a c i t i e s of s e v e r a l p r o t e i n s at extremes of the composition range. The fixed-temperature heat c a p a c i t i e s obtained u s i n g a drop c a l o r i m e t e r are p a r t i c u l a r l y accurate. Measurements o f t h i s k i n d made at 25 °C, a temperature between the f r e e z i n g point and the onset o f thermal d e n a t u r a t i o n , d e f i n e changes i n the thermal p r o p e r t i e s of the system that were not detected i n scanning c a l o r i m e t r i c measurements. A l s o , i n t e r p r e t a t i o n of the r e s u l t s i s not complicated by the changes i n s t a t e , a s s o c i a t e d w i t h f u s i o n o f the solvent o r denaturation o f the p r o t e i n , that are p a r t of the scanning measurements. Figure 1 shows the heat c a p a c i t y of the lysozyme-water


6.

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Table I I .

Water Interacting with

115

Proteins

Thermodynamic Parameters f o r Water I n t e r a c t i n g w i t h Lysozyme a


Hydration Range

A G

(g o f water/g of p r o t e i n ) I

II III IV

0.38 - ( 9

0.3 b < cr H0.2

0.1

_l

0

0.2

I

I

I

0.4

0.6

0.8

L

1.0

1.2

1.4

1.6

1.8 2.0

Z2

GRAM H 0/ GRAM PROTEIN 9

Figure 6. Values of τ for the variable Tempone environment as a function of hydration level. Error bar shows the range of values that gives acceptable simulated spectra. Fraction of Tempone in the variable environment is 0.5 ±0.2 at high hydration.

1

,

, , , , , ,

r

WEIGHT PERCENT WATER

Figure 7. Enzymatic activity of lysozyme as a function of water content, at pH 8, 9, and 10. Open symbols: measurements on powders hydrated by isopestic equilibra tion. Closed symbols: solvent added to powder.


WATER IN POLYMERS

126

In f a c t , i t i s remarkable that the agreement between the dynamic and s t a t i c measurements i s so c l o s e i n d e f i n i n g the sequence of events i n the h y d r a t i o n process: as noted above, NMR, d i e l e c t r i c r e l a x a t i o n , ESR, and enzymatic measurements each d e f i n e one or more of the steps i n the h y d r a t i o n process seen i n s t a t i c measurements.


Absence of Conformation Changes w i t h Hydration Most of the s t u d i e s described above were on p a r t i a l l y hydrated p r o t e i n powders or f i l m s . I t i s necessary to r e l a t e the conformation of the p r o t e i n i n t h i s s t a t e to that i n s o l u t i o n . There i s a s u b s t a n t i a l body of evidence supporting the c o n c l u s i o n that the conformation of the p r o t e i n does not change measurably between about 0.2 h and the d i l u t e s o l u t i o n : 1) enzymatic a c t i v i t y i s observable at 0.2 h. 2) The i n f r a r e d spectrum changes c o n t i n u o u s l y w i t h h y d r a t i o n above 0.1 h and i s c o n s i s t e n t w i t h surface group h y d r a t i o n without change i n p r o t e i n conformation. 3) The temperature of the thermal denaturation t r a n s i t i o n i n creases w i t h decreasing h y d r a t i o n below 0.7 h. 4) The p a r t i a l s p e c i f i c volumes of s e v e r a l p r o t e i n s are the same i n d i l u t e s o l u t i o n and i n the s o l i d at h y d r a t i o n l e v e l s above 0.2 h. 5) The c i r c u l a r d i c h r o i s m spectrum of lysozyme i n a f i l m i s c l o s e l y simi l a r t o that i n s o l u t i o n C3Q). In order to compare the conformation i n powders at very low h y d r a t i o n (0.02 h) w i t h that at higher l e v e l s , above 0.2 h, ESR measurements were made on samples of lysozyme c o v a l e n t l y l a b e l l e d with succinimidyl-2,2,5,5-tetramethyl-3-pyrrolin-l-oxyl-3-carb o x y l a t e . The m a t e r i a l contained two s p i n l a b e l s per p r o t e i n molecule, s u f f i c i e n t f o r s p i n - s p i n i n t e r a c t i o n to be observed. Spectra were measured at -160 °C as a f u n c t i o n of h y d r a t i o n . At t h i s temperature, molecular motion makes no c o n t r i b u t i o n to the spectrum, and an a n a l y s i s of spectrum shape can be used to e s t i mate the d i s t a n c e between s p i n centers (31). The l i n e shapes were i n v a r i a n t to change i n h y d r a t i o n (Table I V ) . The mean d i s tance between s p i n centers i s 26-28 Â . Thus to a r e s o l u t i o n of 1 A there i s no change i n conformation w i t h h y d r a t i o n . This conc l u s i o n holds s t r i c t l y only f o r those p o r t i o n s of the p r o t e i n that have reacted w i t h the l a b e l , and i t i s p o s s i b l e that there are changes g r e a t e r than 1 Â i n other p o r t i o n s of the molecule. However, we b e l i e v e t h a t t h i s p o s s i b i l i t y i s u n l i k e l y , because of the wide d i s t r i b u t i o n of amino groups about the lysozyme surface and because of the cooperative nature of changes i n conformation. It i s a l s o p o s s i b l e but, we b e l i e v e , unreasonable that conformations d i f f e r e n t at v a r i o u s h y d r a t i o n l e v e l s at 25 °C w i l l be brought at -160 °C to the same conformation. Thus the i n v a r i a n c e of the ESR l i n e shapes between low and high h y d r a t i o n l e v e l s i s strong support f o r the i n v a r i a n c e of the conformation. To summarize, v a r i o u s measurements i n d i c a t e t h a t the c o n f o r mation of the p r o t e i n i n the powder or f i l m s t a t e i s the same as


6. RUPLEY ET AL.

Water Interacting

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WEIGHT FRACTION


0.1

I

1.0

ι

ι

0.8

ι

WATER

0.15

ι

127

Proteins

ι

ι

1

0.6 0.4 -lnP/P

0.2

1

0.2

05

1

a 0

0

Figure 8. Logarithmic plot of the data of Figure 7, with arbitrary ordinate trans lations to bring curves for different pH into coincidence: (Α) ρ H 8; (Ο) pH 9; (D)PH10

Table IV.

Average Distance Between Spin Centers a t -160 ° C ^

Hydration (g of water/g o f p r o t e i n ) 0.02 0.21 0.22 0.30 0.41 a

1 d 0.54 0.54 0.53 0.53 0.53

a

Average Distance Between Spin Centers (&) 26 26 28 28 28

d^ i s the peak-to-peak height f o r the two outer extrema of the f i r s t d e r i v a t i v e l i n e s ; d_ i s the peak-to-peak height f o r the c e n t r a l f i e l d l i n e ; the average d i s t a n c e between s p i n centers corresponding t o the r a t i o d^/d was obtained from the curve given by L i k h t e n s h t e i n (31).


WATER IN POLYMERS


128

i n d i l u t e s o l u t i o n . The ESR and enzymatic p r o p e r t i e s are s e n s i t i v e t o small changes i n conformation (about 1 X ) . A p p a r e n t l y , removal of the solvent from about a p r o t e i n , l i k e i n c o r p o r a t i o n of a p r o t e i n molecule i n t o a c r y s t a l , does not a f f e c t the backbone conformation, although the arrangement of surface s i d e chain groups i s expected to be a l t e r e d s l i g h t l y . In view of the importance of solvent f o r p r o t e i n f o l d i n g , the absence of an e f f e c t of dehydration on conformation i s s u r p r i s i n g . I t i s p o s s i b l e that the s t a b i l i t y of the n a t i v e c o n f o r mation at reduced water content has a k i n e t i c b a s i s , owing to conformation changes being i n h i b i t e d . P i c t u r e of the H y d r a t i o n Process The correspondences noted above between v a r i o u s measurements of the h y d r a t i o n process suggest that the events i n the h y d r a t i o n of dry p r o t e i n f i t the f o l l o w i n g p i c t u r e , which f o l l o w s c l o s e l y a d e s c r i p t i o n by C a r e r i , G r a t t o n , Yang and Rupley (32) and which i s i l l u s t r a t e d i n Figure 9. A) The dry p r o t e i n molecule has a conformation s i m i l a r to that of the p r o t e i n i n s o l u t i o n , and i n f i l m s or powders i t makes few contacts w i t h n e i g h b o r i n g molecules. B) The f i r s t water added i n t e r a c t s predominantly w i t h i o n i z a b l e groups and r e s t o r e s the normal pK order among groups perturbed s t r o n g l y through dehydration. This s t r o n g l y bound water (Region IV) i s d i s p e r s e d about the p r o t e i n surface. I t c o n s t i t u t e s 25% of the water of h y d r a t i o n , which i s equal to the percentage of the surface c o n t r i b u t e d by i o n i z a b l e groups (17). C) At a conc e n t r a t i o n of near 0.1 g water/g p r o t e i n , c l u s t e r s develop, p r e sumably centered on p o l a r and charged p r o t e i n surface atoms. This change i s evidenced by d i s c o n t i n u i t i e s i n the i n f r a r e d prope r t i e s , i n the heat c a p a c i t y and the s o r p t i o n isotherm, and i n ESR s p e c t r a . The h i g h heat c a p a c i t y found f o r the bound water between 0.1 and 0.25 h (Region I I I ) i n d i c a t e s that the hydrogen bonding arrangements are v a r i a b l e , as they are i n b u l k water. Time-domain measurements (NMR, d i e l e c t r i c r e l a x a t i o n , and ESR) show that t h i s water i s r e s t r i c t e d i n motion r e l a t i v e to b u l k water. Presumably t h i s type of s t r u c t u r e f o r the water s h e l l (mobile, v a r i a b l e arrangements centered on p o l a r and charged surface s i t e s ) obtains a l s o i n the f u l l y hydrated molecule. D) Between 0.2 and 0.3 l i , h y d r a t i o n of the hydrogen-bonding s i t e s i s complete. E) Condensation of water over the weakest i n t e r a c t i n g p o r t i o n s of the s u r f a c e , the nonpolar r e g i o n s , leads to monolayer coverage at 0.4 tu There must be s p e c i a l l o c a l arrangements of water at the p r o t e i n s u r f a c e , i n order to o b t a i n h i g h coverage per water molecule adsorbed. The condensation i s a major event i n the h y d r a t i o n process; i t i s seen i n the heat c a p a c i t y , a s t a t i c measurement, and i s the point at which dynamic p r o p e r t i e s ( d i e l e c t r i c r e l a x a t i o n ; s p i n probe c o r r e l a t i o n time; enzymatic a c t i v i t y ) show l a r g e changes. The m o b i l i t y of the protein-water system i n c r e a s e s d r a m a t i c a l l y w i t h completion of


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6. RUPLEY ET AL.

Figure 9.

Representation of the events of the hydration process. Letters correspond to discussion in the text.



130

WATER IN POLYMERS

the monolayer. F) The arrangement of the water within the rnono^ layer is such that the protein at this hydration level meshes well with bulk water. In this regard, there are no changes in thermal properties with increase in hydration above 0.4 h. The heat capacity is a sensitive probe of hydrogen bonding, and thus would have detected changes produced by or upon the water shell. Consequently, the special local arrangements of water suggested in (E) above would have to be among those characteristic of l i q uid water. Thus it is not surprising that some multilayer water should be located by x-ray diffraction analysis and yet have the thermal properties of bulk water. G) Dynamic properties change as the hydration is increased above 0.4 h. It is significant, however, that these changes in motional properties appear to be small compared to those occuring during the condensation process at 0.25-0.4 h. Abstract The interaction of water with proteins can be studied effectively using solid samples, because it is possible to control water activity. Sorption isotherms have been reported for a wide variety of proteins and related molecules, defining the free energy and enthalpy as a function of hydration level. Heat capacity measurements, which can be carried out over the full range of system composition, serve to correlate the studies on partially hydrated solid samples with studies on the dilute solution state. The thermodynamic results distinguish stages in the hydration of dry protein and suggest a simple picture of the process. This picture accomodates results of other static measurements, such as infrared spectroscopic, and kinetic measurements, such as ESR and enzymatic activity. Enzymatic activity is observed before completion of monolayer coverage, and the development of it appears to be associated with a condensation event. Kinetic properties of the solid continue to change above a hydration level (ca. 0.4 g water/g protein) sufficient to establish the thermal properties characteristic of the dilute solution. The structure of the solid protein as a function of hydration level is discussed. Acknowledgement s We are grateful to Patricia Adams, for carrying out measurements of the enzymatic activity of lysozyme, and to Professor Walter Kauzmann for stimulation and critical discussions. This work was supported by NIH research grant GM-24760.


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Literature Cited 1. 2. 3. Downloaded by KTH ROYAL INST OF TECHNOLOGY on November 22, 2016 | http://pubs.acs.org Publication Date: August 19, 1980 | doi: 10.1021/bk-1980-0127.ch006

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Bruice, T . C . ; Turner, A. J . Amer. Chem. Soc., 1970, 92, 3422-3428. Kuntz, I.D.; Kauzmann, W. Adv. Protein Chem., 1974, 28, 239-345. Leeder, J.D.; Watt, I.C. J. Colloid Interface Science, 1974, 48, 339-344. Pineri, M.H.; Escoubes, M.; Roche, G. Biopolymers, 1978, 17, 2799-2815. Hagler, A.T.; Moult, J . Nature, 1978, 272, 222-226. Hnojewyj, W.S.; Reyerson, L.H. J . Phys. Chem., 1961, 65, 1694-1698. Luscher, M.; Ruegg, M. Biochim. Biophys. Acta, 1978, 533, 428-439. Bettelheim, F.A.; Block, Α.; Kaufman, L . J . Biopolymers, 1970, 9, 1531-1538. Bull, H.B.; Breese, K. Arch. Biochem. Biophys., 1968, 128, 497-502. Richards, F.M. Ann. Rev. Biophys. Bioeng., 1977, 6, 151-176. Mrevlishvili, G.M.; Privalov, P.L. "Water in Biological Systems," Kayushin, L . P . , Ed., Plenum: New York, 1969, 63-66. Ruegg, M.; Moor, U.; Blanc, B. Biochim. Biophys. Acta, 1975, 400, 334-342. Fujita, Y.; Noda, Y. Bull. Chem. Soc. (Japan), 1978, 51, 1567-1568. Yang, P.-H.; Rupley, J.A. Biochem., 1979, 12, 2654-2661. Suurkuusk, J . Acta Chem. Scand., 1974, B28, 409-417. Hutchens, J.O.; Cole, A.G.; Stout, J.W. J. Biol. Chem., 1969, 244, 26-32. Shrake, Α.; Rupley, J.A. J. Mol. B i o l . , 1973, 79, 351-371. Benson, S.W.; Buss, J.H. J. Chem. Phys., 1958, 29, 546-572. Guthrie, J.P. Can. J. Chem., 1977, 55, 3700-3706. Sturtevant, J.M. Proc. Natl. Acad. Sci. USA, 1977, 74, 2236-2240. Careri, G.; Giansanti, Α.; Gratton, E. Biopolymers, 1979, 18, 1187. H i l l , T.L. J. Chem. Phys., 1949, 17, 762-771. Lee, B.; Richards, F.M. J. Mol. B i o l . , 1971, 55, 379-400. Hilton, B.D.; Hsi, E.; Bryant, R.G. J. Amer. Chem. Soc., 1977, 99, 8483-8490. Finney, J . L . Phil. Trans. Roy. Soc. Lond., 1977, B278, 3-32. Watenpaugh, K.D.; Margulis, T.N.; Sieker, L . C . ; Jensen, L.H. J . Mol. Biol., 1978, 122, 175-190. Squire, P.G.; Himmel, M.E. Arch. Biochem. Biophys., 1979, 196, 165-177. Harvey, S.C.; Hoekstra, P. J . Phys. Chem., 1972, 76, 29872994.


132 29. 30. 31.


32.

33. 34.

WATER IN POLYMERS

Banerjee, S.K.; Holler, E . ; Hess, G.P.; Rupley, J.A. J. Biol. Chem., 1975, 250, 4355-4367. Chirgadze, Yu.N.; Ovsepyan, A.M. Mol. Biol., 1972, 6, 721726. Likhtenshtein, G.I. "Spin Labeling Methods in Molecular Biology," Wiley-Interscience: New York, 1974. Careri, G.C.; Gratton, E . ; Yang, P.-H.; Rupley, J.A. "Protein-Water Interactions. Correlation of Infrared Spectroscopic, Heat Capacity, Diamagnetic Susceptibility and Enzymatic Measurements on Lysozyme Powders," submitted for publication, 1979. Tanford, C.; Aune, K.C. Biochem, 1970, 9, 206-211. Banerjee, S.K.; Rupley, J.A. J. Biol. Chem., 1975, 250, 8267-8274.

RECEIVED January 4, 1980.


Thermodynamic and Related Studies of Water Interacting with

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