Denaturation of globular proteins

216

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 35720 D E N A T U R A T I O N OF GLOBULAR P R O T E I N S I I I . A COMPARATIVE STUDY OF T H E I N T E R A C T I O N OF U R E A W I T H SEVERAL PROTEINS

JOHN R. W A R R E N AND J U L I U S A. GORDON

Department of Pathology, University of Colorado School of Medicine, Denver, Colo., 80220 (U.S.A.) (Received August I7th, 197 o)

SUMMARY

I. The extent of urea interaction with bovine serum albumin, ovalbumin, and fl-lactoglobulin has been measured in aqueous 8 M urea solution b y our ultrafiltration technique 1. The ratio of urea to protein interaction calculated from the observed data is almost identical for all three proteins (about one urea molecule per three amino acid residues in each completely unfolded protein molecule) and is very similar to that previously reported for lysozyme 2. This observation strongly suggests that urea interacts with sites common to the four different proteins, such as components of the polypeptide backbone. 2. The m a x i m u m extent of urea interaction with ovalbumin occurred within minutes after addition of this protein to 8 M urea, remaining unchanged during the subsequent slow unfolding of the protein as followed by changes in optical rotation. The data suggest that a urea-protein complex is formed shortly after exposure of ovalbumin to concentrated urea, the protein then undergoing complete unfolding without quantitative change in hydration. 3. Additional studies showed that the interaction of acetamide with fl-lactoglobulin was accompanied b y an increase in disordered structure of this protein in contrast to albumin which retained its native conformation. The relative extent of acetamide interaction with albumin was found to be considerably smaller than that seen with fi-lactoglobulin. The hydrophobicity of acetamide might explain its increased effectiveness for fi-lactoglobulin.

INTRODUCTION

This laboratory is interested in the molecular mechanism by which urea and related amides denature proteins. Recently we reported 1,2 that the urea denaturation of bovine serum albumin and lysozyme is accompanied by stoichiometric interaction of urea with the denatured form of the proteins. However, the chemical nature of the interaction of urea with proteins remains unclear. I f urea molecules interact with sites Biochim. Biophys. Acta, 229 (1971) 216-225

D E N A T U R A T I O N OF G L O B U L A R P R O T E I N S .

III

217

on the proteins independent of amino acid composition, the extent of urea interaction would likely be similar for each of the proteins. Thus the interaction of urea with ovalbumin and fl-lactoglobulin was studied in 8 M urea and compared to that observed with albumin and lysozyme both in this and previous studies1, I. The experimental data reported in this paper show that with each of the four proteins studied, nearly one urea molecule interacts for every three amino acid residues of the completely unfolded protein in 8 M urea. From a comparison of our urea interaction data with the known amino acid composition of these proteins, we can now conclude that the number of urea interaction sites on the four proteins is independent of amino acid composition, the sites probably being similar if not identical in character for the different proteins. If such urea interaction sites are composed of specific chemical groups, the peptide bond would be a prominent example of such a group. A separate kinetic study of ovalbumin in 8 M urea revealed constant and maximal urea-ovalbumin interaction during unfolding of the protein, providing additional evidence for our earlier conclusion I that the hydration of a denatured protein is quantitatively similar to that found in the native state. MATERIALS AND METHODS

Materials

Bovine serum albumin (crystalline; Armour Pharmaceutical Co., Kankakee, Ill., Lots B-7o3o7, D-712o9, F-716oi ) was used as supplied by the manufacturer. Ovalbumin (crystalline; Immunology Inc., Glen Ellyn, Ill., Lot 469; Mann Research Laboratories, Inc., New York, N.Y., Lots R-2223, S-I636, S-I963; Nutritional Biochemicals Corp., Cleveland, Ohio, Lot 5613; Pentex, Inc., Kankakee, Ill., Lot PPo662; Sigma Chemical Co., St. Louis, Mo., Lot I7B-869I ) and fi-lactoglobulin (crystalline; Pentex, Inc., Kankakee, IlL, Lots 38, 39) were further purified by extensive dialysis against distilled water and lyophilized to dryness. Protein was stored over Drierite under a vacuum at 4 ° before use in experiments ; residual water of the protein as determined by exposure of the protein to lO5 ° for 12 h never exceeded 3% (w/w). Organic and inorganic reagents were the best available commercial products and were used without further purification. U ltrafiltr at ion

Our technique of ultrafiltration for the study of small organic molecule-protein interaction has been described in extensive detail previously 1. Briefly, the nearly dry protein under study was dissolved in a 4o-ml aliquot of solvent (aqueous urea or acetamide) to concentrations ranging from 1.3 to 5.2% (w/v) for albumin and 2.6 to 5.2% (w/v) for ovalbumin and fl-lactoglobulin. This solution was placed into a Diaflo Model 50 ultrafiltration cell (Amicon Corporation, Lexington, Mass.) faced b y an Amicon UM-I (for serum albumin or ovalbumin) or UM-2 (for fl-lactoglobulin) membrane, the membrane having been previously equilibrated with the appropriate solvent. 8-1o ultrafiltrates were obtained over periods of up to 3 h and the refractive index of each ultrafiltrate was determined on a Bellingham and Stanley Abb6 60 high accuracy refractometer. Any difference in refractive index of the ultrafiltrates following protein addition to the retentate was translated into change of molarity by Biochim. Biophys. Acta, 229 (1971) 216-225

218

j . R . WARREN, J. A. GORDON

comparison with standard molarity-refractive index curves a from o to 8 M for aqueous urea and acetamide. Variation between ultrafiltrates in a series from one experiment was always found to be less than -1- o.oo5 M. All ultrafiltration experiments were performed with non-buffered solutions without added salt to eliminate possible confusion from binding of buffer or salt molecules to protein. Refractive index changes in ultrafiltrates following protein addition are thus a relatively direct reflection of change of "free" urea or acetamide concentration and hence the extent of urea or acetamide interaction with protein. The interaction of urea with ovalbumin and albumin was also determined as a function of time. It was experimentally shown b y simple dilution t h a t change of urea concentration in the retentate comparable to those occurring in the presence of protein was detectable in the ultrafiltrates within 5 min. Thus for the kinetic s t u d y of slowly unfolding ovalbumin in 8 M urea, ultrafiltrates were collected at ten intervals over 5-I8O min following protein addition. As a control, a similar s t u d y was performed after the addition of serum albumin to 8 M urea, a protein which is known to instantaneously unfold in this solvenO.

Spectropolarimetry Relatively concentrated stock solutions of protein were diluted with precision microburettes in the appropriate solvents for polarimetry. Protein concentrations were determined b y spectrophotometry using a value of 9.60 for 827 ~%s of fl-lactoglobulin 5, of 6.67 and 7.50 for 8280 i% of serum albumin e and ovalbumin ~, respectively. Optical r o t a t o r y dispersion measurements were made at 9 wave lengths from 330 t h r o u g h 600 n m in a Io-cm water-jacketed cell at 25 ° with a modified P e r k i n - E l m e r Model 141 spectropolarimeter utilizing an x e n o n - m e r c u r y light source. Results are reported as the specific rotation, [a]A~5°, or the mean residue rotation, F~'125 L j~ o, where, looa

[m']~ 5° =

(1)

3 M0 [~]~5° lOO (~' + 2)

(2)

in which a represents the observed rotation, l the length of the polarimeter tube in decimeters, c the protein concentration in g per I0O ml solution, M 0 the mean residue weight of the protein (M o of serum albumin 114, of ovalbumin 122, of/5-1actoglobulin II7), and n th~ refractive index of water at each wave length. The dispersion of rotation with wave length was analyzed b y the Moffitt-Yang equation where [m']~ ~ ° -

a j r '0

P_

bo~,4o

~ + (~,~), 0

(3)

O"

with 2 o set at 212 nm. Values of a o and b 0 were obtained from tile intercept and slope of linear plots of [m'] (22 -- 2~o) versus (22 -- 2~o)-1. RESULTS

The extent of urea interaction with four different globular proteins is compared in aqueous 8 M urea solution (Table I). The magnitude of the decrease in urea concenBiochim. Biophys. Acta, 229 (1971) 216-225

DENATURATION OF GLOBULAR PROTEINS.

219

In

TABLE I INTERACTION OF UREA WITH GLOBULAR PROTEINS IN 8 IV/ UREA The v a l u e s for t h e c h a n g e in u r e a are t h e a v e r a g e of t h r e e or more s e p a r a t e u l t r a f i l t r a t i o n e x p e r i m e n t s a n d are a d j u s t e d for p r o t e i n h y d r a t i o n , a s s u m e d t o be 2o% , as d e s c r i b e d p r e v i o u s l y 1. T o t a l p r o t e i n r a t i o s for each p r o t e i n m o l e c u l e in s o l u t i o n (see t e x t ) . D e n a t u r e d p r o t e i n r a t i o s for e a c h d e n a t u r e d p r o t e i n m o l e c u l e in s o l u t i o n (see t e x t ) .

(z) Protein conch, × Io4(M)

(2) Change in urea (AM ~o.oo5)

Ratios of interaction with Total protein (3) Urea~protein (M/M)

Denatured protein (4) Urea/amino acid

(5) Urea/amino acid

(M/M)

(M/M)

Bovine serum albumin 1.8 3.7 4.3 5.6 7.3

--0.032 -- c/~°67 --0.072 --o. Ioo --o.121

178 181 167 179 161

0.31 o.31 0.29 0.31 0.28

0.34 0.34 0.32 0.34 o.31

--o.o64 --0.o74 --o.098 --o.132

lO9 lO9 iii 112

0.28 o.28 o.28 o.29

o.32 0.32 0.32 o.33

--o-o75 --o.IiO --o .164

95 lO 4 lO 3

o.3o 0.33 0.32

0.30 0.33 0.32

--o.128

--

Ovalbumin 5.9 6.8 8.8 11.8

fl-Lactoglobulin 7.9 IO.6 15. 9

Reduced lysozyme

(ref. 2) 28.0

0.36

tration observed for ultrafiltrates obtained from albumin-urea solutions increased by a factor of four with a 4-fold increase in albumin concentration (Columns I and 2, Table I). Likewise, doubling of the amount of ovalbumin or fl-lactoglobulin added to 8 M urea resulted in a 2-fold greater decrease of urea c~ncentration in the ultrafiltrates (maximum increase in protein concentration allowed before precipitation). Thus, the interaction of about 17o urea molecules with each albumin molecule, around I I I urea molecules with each ovalbumin molecule, and IOO urea molecules with each fl-lactoglobulin (Column 3, Table I) was independent of protein concentration over the concentration range studied. Furthermore the ratio of urea intelaction per amino acid residue for total protein added to 8 M urea was nearly identmal for all three proteins (Column 4, Table I). The marked time dependence shown by others for the unfolding of ovalbumin in 8 M urea s was confirmed under our experimental conditions (Fig. I). Nevertheless, we found that urea interaction with ovalbumin was maximal and remained unchanged during the period when the optical rotation of the protein went from that of a near native to the unfolded conformation. In contrast, bovine serum albumin also gave a Biochim. Biophys. Acta, 229 (1971) 216-225

220

J. R. WARREN, J. A. GORDON

[-L;--:

260

"A "E 2 4 0

~ 200

7

~

\

i

o

0 160

.~ 1 2 0

200

0

~ooo

o

o

~

o

160 120 60

o

~)

o

.~ 1 6 0

E 12o

O. (0

D

80 r

4

0/

i Time

810

i

i

120

i

8o

i

160

(minutes)

2

I

4 Molarity

I

i

6

6 of

110

12

Urea

Fig. i. E x t e n t of urea interaction with ovalbumin (C)) as a function of time in 8 M urea; protein unfolding followed b y change in specific rotation at 400 nm and 25 ° for 1.o% ovalbumin (A). Fig. 2. Mean residue rotation of three proteins a t 405 nm as a function of increasing urea concentration: 1.o% bovine serum albumin (C)), 1.o% o v a l b u m i n (A), a n d 0.5% fl-lactoglobulin ([7). Ovalbumin and fl-lactoglobulin solutions k e p t nonturbid b y additional presence of o.2 M NaC1. Equilibrium values for the rotation of the albumin solutions were reached i m m e d i a t e l y after preparation, of fl-lactoglobulin solutions 3 h after preparation, of ovalbumin solutions after 72 h. The curve for each protein is extrapolated b e y o n d the water solubility of urea ( - - - - - ) .

maximal interaction with urea within 5 min, but it is extensively unfolded at this time 4. Assuming a two state transition, one can easily calculate the extent of urea interaction with each completely unfolded albumin, ovalbumin, or fl-lactoglobulin molecule in 8 M urea 9. The equilibrium constant for the reaction native protein ~-fully denatured protein, K, in 8 M urea was found for albumin and ovalbumin from the relationship K =

[ m ' ] 4 0 5 - [m',~]405 [m~]t05-

(4)

[m'],05

in which Em'N]405 is the mean residue rotation of native protein and [m']ao5 the rotation in 8 M urea (Fig. 2). Since neither albumin nor ovalbumin is completely denatured at the highest urea concentration obtainable in water at 25 ° , the curve describing the mean rotation of each protein as a function of urea concentration was extrapolated to concentrations exceeding the water solubility of urea. This allowed us to obtain an estimate of the value for [m'DJaos, the rotation of completely unfolded protein (see Fig. 2). The fraction of denatured protein in 8 M urea, fD, was then calculated from the value of K using the relationship K =

[denatured protein]

fD

[native protein]

1 -- fD

(5)

Alternatively, values of fD were obtained using the Moffitt-Yang parameter b0 in Biochim. Biophys. Acta, 229 (1971) 216-225

DENATURATION OF GLOBULAR PROTEINS.

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221

Eqn. 4, with the value of b0 of each protein in 6 M guanidine hydrochloride* taken as an approximation of completely denatured protein 1°. The value of fD derived for albumin from values of Em']4o 5 or b0 was o.85 and 0.84, respectively, for ovalbumin 0.82 and 0.80. Since [m']4o5 of fl-lactoglobulin in 8 M urea was equal to ~m'D]405 (Fig. 2), f n of this protein was i.oo. Having calculated the fraction of denatured protein molecules, the extent of urea to amino acid interaction with only completely unfolded protein molecules, D, can be obtained from the equation 1 D

U -- o.12 - + o.I2

(6)

fD

in which U is the urea to amino acid ratio observed for total protein added to the solution (Column 4, Table I) and o.12 an approximate value for the urea to amino acid ratio observed at the upper limit of non-denaturing concentrations of urea as outlined previously ~*. Thus, for each of the three proteins studied here the urea to amino acid ratio for each completely denatured protein molecule in 8 M urea was found to be nearly identical (o.32 ~ 0.o2, Table I). Interaction of acetamide with serum albumin and fl-lactoglobulin was also studied. As seen in Table II, acetamide revealed 5o% less interaction with albumin when compared to urea at the same concentration. Furthermore, the optical rotatory dispersion parameters of albumin in 8 M acetamide were equal to those seen for the native protein (Table I I I ) n. In contrast, about one acetamide molecule was found to interact per four amino acid residues of each fl-lactoglobulin molecule in 8 M acetaTABLE II INTERACTION OF ACETAMIDE WITH GLOBULAR PROTEINS IN 8 M ACETAMIDE T h e c h a n g e s in a c e t a m i d e are a v e r a g e v a l u e s of t h r e e or m o r e s e p a r a t e u l t r a f i l t r a t i o n e x p e r i m e n t s , a d j u s t e d for 2 o % p r o t e i n h y d r a t i o n as d e s c r i b e d p r e v i o u s l y L R a t i o s of i n t e r a c t i o n are r a t i o s for each p r o t e i n m o l e c u l e in s o l u t i o n (see t e x t ) .

Protein

Bovine serum albumin fl-Lactoglobulin

Conch. × ~o 4 (M)

Change in acetamide ( A M :6 o.oo5)

Ratios of interaction Acetamide/ protein (M/M)

Acetamide/ amino acid (M]M)

5.7

--o.050

88

o.15

lO.6

--0.079

75

0.23

* T h e v a l u e for b 0 of n a t i v e a l b u m i n a n d o v a l b u m i n i n w a t e r w a s --319.9 a n d --I4O.O, r e s p e c t i v e l y , of a l b u m i n a n d o v a l b u m i n in 8 M u r e a --83.8 a n d --43.8, a n d of r a n d o m coil a l b u m i n a n d o v a l b u m i n in 6 M g u a n i d i n e h y d r o c h l o r i d e --39.4 a n d --20. 4. ** W e p r e v i o u s l y r e p o r t e d 1 t h a t a p p r o x . 6 0 - 8 o r e a g e n t m o l e c u l e s c a n i n t e r a c t w i t h e a c h a l b u m i n m o l e c u l e bef ore a n y c h a n g e in t h e o p t i c a l r o t a t i o n of t h e p r o t e i n occurs. K n o w i n g t h e n u m b e r of a m i n o a c i d r e s i d u e s in a l b u m i n , we c a n g e n e r a l i z e for p u r p o s e s of t h i s p a p e r t h a t a b o u t o.12 m o l e c u l e s of d e n a t u r a n t c a n i n t e r a c t p e r a m i n o acid r e s i d u e of a p r o t e i n p r i o r t o t h e occurr ence of d e n a t u r a t i o n . This a s s u m p t i o n is s u p p o r t e d b y t h e n e a r i d e n t i t y of v a l u e s r e s u l t i n g f r o m the subsequent calculation.

Biochim. Biophys. Acta, 229 (1971) 216-225

222 TABLE

J.R.

W A R R E N , J. A. GORDON

III

OPTICAL ROTATORY PARAMETERS OF SERUM ALBUMIN AND fl-LACTOGLOBULIN IN VARIOUS SOLVENTS S e r u m a l b u m i n is a d d e d t o 1 . 0 % (w/v). f l - L a c t o g l o b u l i n is a d d e d t o 0 . 5 % (w/v) ; 0.2 M NaC1 a d d e d to keep solutions non-turbid.

Solvent

/'m']40~ ~5°

a0

b0

--179. 3 --171. 5 --271. 4

--363.9 --338.7 --702.3

--319.9 --313.3 -- 83.8

- - 77.1 --lO9.O --291.4

--175.6 --253.6 --73o.7

-- 72.4 --lO7.3 -- 59.9

Serum albumin Water 8 M acetamide 8Murea

fl-Lactoglobulin Water 8 M acetamide 8Murea

mide, compared to the one acetamide per seven to eight amino acids detected with albumin (Table II). This relatively greater acetamide interaction with fl-lactoglobulin was accompanied by both an increased levorotation and a more negative value of a 0 (Table III). The optical rotatory dispersion changes of fl-lactoglobulin upon interaction with acetamide differed significantly from those seen upon urea denaturation of the protein in that b0 assumed a more rather than less negative value (Table III). The extensive gel formation seen upon addition of ovalbumin to 8 M acetamide prevented meaningful experiments with ovalbumin-acetamide solutions. DISCUSSION

The experimental design of the ultrafiltration experiments makes the absolute decrease in urea concentration observed in ultrafiltrates from protein-urea solutions, as discussed previously:, almost certainly due to the interaction of urea with added protein. Uniformly adjusting the observed data for 20% (w/w) protein hydration* leads to a progressively greater decrease of urea concentration upon the addition of serum albumin: or lysozyme ~ to increasingly concentrated urea solutions. The corrected data does assume no significant change in the number of water molecules binding to protein during denaturation. Most reports do suggest little or no quantitative change in hydration upon denaturation 12. In a recent report, however, it is suggested that protein hydration decreases upon denaturation with the number of denaturant molecules interacting with protein undergoing little or no change 13. To rule out this possibility here, ultrafiltrates were taken from ovalbumin-urea solutions at successive times during the slow unfolding of the protein in 8 M urea. As seen in Fig. I, the total decrease of urea concentration in these ultrafiltrates was constant during the conversion of ovalbumin to its denatured conformation. Thus we conclude that a continuous release of water from the hydration shell of ovalbumin into the "bulk" solvent during the period of protein unfolding does not occur under these conditions. The opposite situation, i.e., that hydration increases during protein un* T h e q u e s t i o n is n o t o n e o f m a k i n g a c o r r e c t i o n for h y d r a t i o n , s i n c e t h e r e m u s t b e s o m e , but how much of a correction to make. We chose 20% hydration to apply uniformly, but it should b e n o t e d t h a t 1 5 % or e v e n 2 5 % h y d r a t i o n w o u l d n o t a l t e r t h e b a s i c c o n c l u s i o n s d r a w n i n t h i s a n d previous papers.

Biochim. Biophys. Aeta, 229 (1971) 2 1 6 - 2 2 5

DENATURATION OF GLOBULAR PROTEINS.

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223

folding, is equally unlikely as the uptake of water by the denatured protein would have to be exactly counterbalanced b y an increased interaction with urea providing no net change of urea concentration in the bulk solvent. We also considered the possibility that the hydration shell is instantaneously and finally altered upon exposure of ovalbumin to concentrated urea, prior to any significant change in the protein's conformation. Even though such an event cannot be excluded b y the data presented in Fig. I, it seems unlikely that any quantitative change in the hydration of ovalbumin in 8 M urea would alone destabilize the native protein. Indeed, it is well established that the protein myoglobin assumes a similar ii not identical conformation whether studied in the relatively "dehydrated" crystalline state or more fully "hydrated" in aqueous solution 14. Thus we conclude that urea interacts with denatured in preference to native species of protein molecules, the greater decrease of urea concentration observed at high urea concentrations in our earlier studiesL2 best explained as the increased interaction of urea with denatured protein without significant change in the amount of bound water. In addition it appears that a urea-protein complex is formed very soon, if not immediately after the addition of ovalbumin to concentrated urea, leading to a slow unfolding of the molecule. TABLE IV COMPARISON OF THE AMINO ACID CONTENT OF DIFFERENT PROTEINS Each category defined b y c o m p o s i t i o n of a m i n o acid side-chain. Aliphatic includes alanine, valine, isoleucine, leucine, proline, and methionine; acidic or basic aspartic acid, glutamic acid, lysine, arginine, histidine ; polar asparagine, glutamine, serine, threonine ; a r o m a t i c t r y p t o p h a n , tyrosine, and phenylalanine; other glycine, cysteine, and cystine. The c o n t e n t of a category is reported as %, which represents moles of a m i n o acids per mole protein.

d mine acid category

z{ lbumin

Ovalbumin

fl-Lactoglobulin

Lysozyme

Aliphatic Acidic, basic Polar Aromatic Other

3° 37 15 7 II

34 3° 2o 8 8

23 36 18 IO 13

28 21 25 9 17

The content of amino acids with hydrocarbon, acidic or basic, polar, or aromatic side chains was calculated for each of the proteins studied 15-17. As shown in Table IV, the relative amount of such amino acids varies considerably from protein to protein, with the exception of the aromatic amino acids which are reasonably constant. Despite the differences in amino acid composition, the relative extent of urea interaction was found to be the same for all four proteins when completely unfolded (Table I). This constant value suggests that urea does not interact primarily with the alkane, charged, or polar side chains along the fully extended protein molecule, but rather with some group present in all of the proteins in the same relative amount. Interaction of urea with the relatively few aromatic amino acids in these four proteins, about lO% of total residues, leads to ratios of nearly four urea molecules per amino acid; this seems unlikely on both steric and mechanistic grounds. Another Biochim. Biophys. Acta, 229 (1971) 216-225

224

j . R . WARREN, J. A. GORDON

chemical group shared by all protein molecules in larger amounts is the peptide bond, more strongly suggesting that sites of urea interaction on protein molecules are composed primarily of peptide bonds. Other evidence for the interaction of urea with peptide bonds has been reported. The increased solubility of the model peptide acetyltetraglycine ethyl ester in urea solutions is probably due to the formation of a complex between one urea and two to three peptide bonds, possibly through bifunctional hydrogen bonds is. More recently, SKERJANC AND LAPANJE 19 observed a large negative transition enthalpy for the urea denaturation of chymotrypsinogen A. Even though data on urea interaction with chymotrypsinogen is unavailable, the presence of about 82 urea interaction sites on each unfolded chymotrypsinogen molecule 2° can be estimated from our investigation of other denatured proteins. I f the transition enthalpy from o to 8 M urea of 166800 cal/mole for chymotrypsinogen 19 is assumed to be the sum of the enthalpies for individual urea interaction sites, the enthalpy of interaction for one urea molecule with one urea interaction site would be --2034 cal/mole. This value is in close agreement with that given b y ROBINSON AND JENCKS 18 for the enthalpy of complex formation between i urea molecule and 2-3 peptide bonds (--2240 cal/mole at 25 °) in acetyltetraglycine ethyl ester. And recently, the binding of urea to the amide groups of polyacrylamide gels has been suggested 21. The interaction of acetamide with serum albumin or/3-1actoglobulin was smaller than that seen with urea (Table II). The lesser affinity of acetamide for proteins is perhaps due to the inability of this amide to act as a bifunctional hydrogen bond donor ~2. Somewhat surprisingly, the relative acetamide interaction with fl-lactoglobulin exceeded that observed with serum albumin and, unlike albumin, was accompanied b y change in the optical rotatory dispersion of the protein. The increased levorotation, more negative value of a 0, and more negative b0 (Table III) detected with/Sdactoglobulin upon aeetalnide interaction resemble closely the behavior of this protein in "hydrophobic" solvents 2~. This suggests that acetamide interacts primarily with the "hydrophobic" core of the fl-lactoglobulin molecule. Such a mode of interaction would be consistent with the replacement of an amino nitrogen by a methyl group in the acetamide molecule. We are presently attempting to quantitate the interaction of urea and related compounds with homopolymers of different amino acids. Hopefully certain amino acid residues can be finally eliminated as potential sites of urea interaction and the conclusion reached in this paper can be strengthened, i.e., that the peptide bond is the major site of urea interaction. ACKNOWLEDGEMENTS

This work was supported by grant GM-II345 from the National Institutes of Health. One of us (J.R.W.) was supported as postdoctoral fellow by Training Grant GM-977, National Institutes of Health. REFERENCES I J. A. GORDON AND J. R. WARREN, J. Biol. Chem., 243 (I968) 5663. 2 J. R. WARREN AND J. A. GORDON, J. Biol. Chem., 245 (i97 o) 4o97.

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