Biochimica et Biophysica Acta, 491 (1977) 93-103 © Elsevier/North-Holland Biomedical Press BBA 37613

P U R I F I C A T I O N A N D SOME P H Y S I C O C H E M I C A L PROPERTIES OF BOVINE K-CASEIN

HENK J. VREEMAN, PAULA BOTH, JAN A. BRINKHUIS and CORRY VAN DER SPEK

Netherlands Institute for Dairy Research (NIZO), Ede (The Netherlands) (Received August 10th, 1976)

SUMMARY

1. A description is given of the fractionation of K-casein on DEAE-cellulose using a pH gradient. With this method an improved separation of the x-casein components with a higher negative charge is obtained. 2. It is shown that at least one of the K-casein fractions has a second phosphate ester group. The heterogeneity of x-casein therefore is not exclusively caused by a varying N-acetylneuraminic acid content. 3. Ultracentrifuge experiments and exclusion gel chromatography show that the purified r-casein fraction having the lowest electrophoretic mobility exhibits a monomer-polymer association equilibrium. The free energy of association per mol monomer in 0.2 M NaC1 is approximately --36 kJ-mo1-1.

INTRODUCTION

K-Casein is part of the casein complex which constitutes about 8 0 ~ of the proteins in cow's milk. It is associated with the other casein components into micelles and it protects the latter against flocculation by calcium ions. Two genetic variants of bovine K-casein are known: A and B (see ref. 1), each showing seven zones on electrophoresis in a urea starch gel at p H 8.6 which contains mercaptoethanol. These components have been shown to differ in their N-acetylneuraminic acid (AcNeu) content [2, 3]. The major and slowest zone of the A- and the B-variant has a relative mobility of 0.60 and 0.52 respectively [3] and does not contain carbohydrate. From the amino acid sequence of bovine r-casein B, which has been established recently [4], the monomer molecular weight is found to be 19.023. r-Casein has a strong tendency to associate to large polymers with a uniform size; the polymer weight is insensitive to temperatures between 1 and 32 °C (see ref. 5). As yet almost all physical characterisation has been done with unfractionated K-casein preparations [5, 6, 7]. Such preparations, however, are usually contaminated by varying amounts of other caseins and also contain K-casein with varying amounts of carbohydrate. We therefore decided to use the main, carbohydrate free, fraction which can be obtained by column chromatography of r-casein from a cow homozygous for the A- or the B-variant. The nomenclature of the K-casein electrophoretic zones is still confusing. The American Committee on Nomenclature of the Proteins of Cow's Milk [8] suggests

94 the use of subscript numbers to designate the number of carbohydrate chains (i.e. AcNeu groups) bound to the peptide chain: A0, A~ etc. On the other hand Pujolle et al. [2] number the zones as they appear on the gel with subscript numbers starting with one for the slowest moving K-casein band (e.g. At, A2 • • ", A7). Since it has not been established that the seventh zone (the one with the highest mobility) contains as many as six AcNeu groups [3, 9] and that the difference in electrophoretic mobility of all the r-casein bands is exclusively due to difference in the number of AcNeu groups we use the numbering of the bands according to Pujolle et al. [2] followed by the relative mobility between brackets as given by Schmidt et al. [3]: A1 (0.60), A 2 (0.66), Aa (0.72) etc.; B~ (0.52), B2 (0.60), B3 (0.65) etc. The enzyme chymosin (EC 3.4.23.4) preferentially splits the Phe (105)-Met (106) bond in K-casein giving an N-terminal fragment ca]led para-r-casein, which contains the two cysteines, and a C-terminal fragment of 64 residues called the macropeptide containing all the carbohydrate as well as the genetic substitutions [1]. The macropeptide will be further specified by reference to the parent K-casein molecule e.g. K-casein macropeptide A~ etc. The name "K-casein" will be used for the mixture of carbohydrate-containing and carbohydrate free r-casein fractions which is obtained in a purification procedure such as the method of McKenzie and Wake [10], i.e. a procedure where it is intended to separate the K-casein from the other caseins. MATERIALS AND METHODS r-Casein was prepared by the method of McKenzie and Wake [10] starting from milk from cows homozygous for one of the r-casein variants. The solution obtained after the last ethanol precipitation step was dialyzed three times for at least eight hours each against a 20-fold volume of a 0.002 M EDTA buffer pH 7 and finally three times against distilled water adjusted to pH 7 with ammonia. Fractionation of K-casein was carried out by column chromatography. About 2.5 g of K-casein was fractionated on a DEAE-cellulose (Whatman DE22) column (25 × 3 cm) using a pH-gradient. A flow rate of 66 ml/h of starting buffer was maintained until the non-adsorbed material had been eluted. Hereafter the gradient was started (2 × 2 l) and the flow rate increased to 108 ml/h. The starting buffer contained 5 M urea, 0.01 M piperazine, 0.001 M glycylglycine (to act as a cyanate "scavenger", see ref. 1 l) and 0.003 M mercaptoethanol; the pH was adjusted to 6.5 with HC1. The gradient buffer contained the same components as the starting buffer and in addition 0.05 M NaC1 and HCl. As much HC1 was added to the gradient buffer as was needed to obtain a solution of pH 5.2 when two parts of starting buffer were mixed with one part of gradient buffer. The urea solution was deionized, before making the buffers, on an Amberlite MB-1 mixed bed ion exchange resin column to remove cyanate. Sedimentation experiments were carried out in a Spinco E analytical centrifuge: K-casein A1 (0.60) or B1 (0.52) was dissolved in a buffer containing 0.01 M EDTA, 0.001 M dithiothreitol or 0.003 M mercaptoethanol, 0.15 M NaCl, pH 6.6 and dialyzed against a similar buffer before the experiment. The correction to standard conditions (s20.w) involved the correction for the solvent viscosity to water at the temperature of measurement (usually 20-22 °C) and the viscosity correction for water at temperature of measurement to 20 °C. Boundaries between the protein solution and its diffusate were made in a double sector synthetic boundary cell.

95

Starch gel electrophoresis was carried out at pH 8.6, 5 M urea, 0.03 M mercaptoethanol and a 0.076 M Tris/citrate buffer [12]. The extinction coefficient was measured in a Cary-14 spectrophotometer. For the determination at low and high pH, the protein was dissolved in 0.01 M HC1 and centrifuged in a table top centrifuge. To a part of this solution 0.5 M N a O H was added to make it 0.01 M in NaOH. Another part was used for the concentration determination. This was done by evaporating the dilute HC1 containing solvent on a waterbath followed by drying of the residue for one hour in an oven at 105 °C. As an alternative and check the solution was also freeze-dried followed by drying in the oven. Since the protein is in the chloride form, its chloride content was determined so as to be able to calculate the extinction coefficient for the isoelectric protein. For the determination at neutral pH the protein was dissolved in a 0.1 M Tris.HC1 buffer, pH 7, and centrifuged. The concentration was determined by dialyzing a known amount of the solution against distilled water brought to pH 7 with ammonia. This solution was transferred quantitatively to the freeze drying jar, freeze-dried and oven-dried as described above. Absorbances were corrected for light scattering [13]. N-acetyl-neuraminic acid was determined by the thiobarbituric acid method of Warren [14]. The amide-nitrogen content was determined according to the procedure of Hamilton [15]. Nitrogen was determined by a micro-Kjeldahl method. Phosphorus content was determined by the method given by Griswold et al. [16]. RESULTS

Isolation A typical chromatographic fractionation by means of a pH gradient of Kcasein B is shown in Fig. 1. The fractions indicated in this figure were analysed by starch gel electrophoresis as is shown in Fig. 2. An average 70 % of the protein applied to the column was recovered. The recovery of the B-variant fractions expressed in grams per 100 g of initially applied r-casein is collected in Table I. The fractions Ba (0.65) and B4 (0.67) were formerly designated as one zone with relative mobility 0.66 "loT 280 nm 40 50 6C 70 80 90 B

o

~

D

C

~

5

~

~

F G

E

~

-~

t(h)

H

I

$

K

J

$

lb

1'~

12

Fig. 1. D E A E - c h r o m a t o g r a p h y o f r-casein B. T h e h a t c h e d fractions were isolated a n d a sample o f each was r u n on a urea starch gel (cf. Fig. 2).

96 TABLE I RECOVERY OF K-CASEIN B FRACTIONS Fraction

Recovery (g per 100 g K-casein *)

N o n adsorbed material (para-K-casein etc.)

11

± 3

r-B1 (0.52) K-B2 (0.60) x-B3 (0.65) K-B4 (0,67) K-Bs (0,74) K-B6 (0.77) K-B7 (0.83)

18.1 2,5 6.4 6.0 7.7 6.6 7.8

± 4.9

Remainder (a~t-casein etc.)

8

± ± ± ± ±

1.6 1.1 0.6 1,9 2.1

± 3

* Average values and standard deviations of ten different x-casein preparations; fraction K-B~ (0.60) is the average of three preparations.

Fig, 2, Starch gel electrophoresis of the fractions obtained by chromatography as indicated in Fig. 1. The initial K-casein B was run on track A and L for reference.

97 by Schmidt [3]. The McKenzie and Wake x-casein (sample A, Fig. 2) is usually contaminated by some y-casein and, depending on the success of the ethanol fractionation, more or less a- and fl-casein. Furthermore, if proteolysis has occurred in the milk, para-r-casein is also found in the preparation. From the data above it appears that these contaminations together amount to 20 ~ of the r-casein preparation on average.

Identification and chemical analysis of the r-casein fractions The minor component r-Bz (0.60) has the same relative mobility as r-A1 (0.60) and has been considered to be an artifact formed by the conversion of lysine into homocitrulline [8]. According to Hill and Wake [17], who claimed that this minor rcasein fraction is not an artifact, it is the precursor of the para-r-casein minor band. In an attempt to confirm this last statement we subjected the fractions r-B1 (0.52), K-Bz (0.60) and x-A1 (0.60) to the action of chymosin to produce para-r-casein. From the electrophoretic pattern shown in Fig. 3 it can be seen that the main para-r-casein

Fig. 3. Chymosin treatment of r-casein bands: A, K-casein B; B, K-Bz (0.60); C, r-Bt (0.52) ÷ chymosin; D, x-B2 (0.60) -~- chymosin; E, r-A~ (0.60) + chymosin; F, K-casein B + chymosin.

98 band derived from K-B~ (0.60) has the same mobility as the para-K-caseins from the major fractions K-B1 (0.52) and K-A1 (0.60), which is contrary to the work of Hill and Wake [17]. Since the difference in mobility of the x-B1 (0.52) zone and the K-B2 (0.60) zone is not due to a difference of charge in the para-K part of the molecule it may be concluded that a charge difference exists in the macropeptide part. To try to determine the origin of the charge difference, the AcNeu content was determined of the fractions K-Bz (0.60), K-B3 (0.65), K-B4 (0.67), K-Bs (0.74) and K-A2 (0.66) as well as the phosphate content. The data are collected in Table II. The minor fraction contains only half an AcNeu group per mole E-casein. The reason for this is probably that fraction x-Bz (0.60) also contained material from the K-B t (0.52) and the K-B4 (0.67) zone both devoid of carbohydrate. In the same way fraction K-As (0.66) contained K-A1 (0.60) and K-A4 (0.74) zone material. Although the minor fraction x-B2 (0.60) was more heavily contaminated with the faster moving K-B4 (0.67) and the minor fraction K-A2 (0.66) with the slower moving K-A1 (0.60), their individual AcNeu contents did not differ appreciably. Therefore the data for both fractions are pooled in Table II. The fraction K-B3 (0.65) contains two AcNeu groups per mol and the fraction K-B4 (0.67) has no AcNeu but has one extra phosphate group, i.e. both fractions have two negative charges extra at p H 8.6. TABLE II PHOSPHATE AND AcNeu CONTENT OF SOME K-CASEIN FRACTIONS Fraction

~ AcNeu

AcNeu groups per mole K-casein*

%P

Mol P per mol K-casein

Number of observations (n)'*

0.06 ± 0.05

0.04

0.155 ± 0.012

0.95

6

r-A2 (0.66)j

0.71 ± 0.03

0.44

n.d.

n.d.

4

r-B3 (0.65)

r-B4 (0.67)

2.88 ± 0.19 0.18 ± 0.05

1.90 0.11

0.163 ± 0.008 0.295 ± 0.013

1.00 1.81

8 6

r-Bs (0.74)

5.72 ± 0.81

4.09

0.159 i 0.013

0.98

8

(052) K-A1 (0.60)J

r-B~ (o.6o)~

-

* Assuming each AcNeu group (M = 309) adds 726 (a trisaccharide unit) to the peptide molecular weight of 19 030. ** Each preparation of the fraction was analysed in duplicate: n/2 is the number of different preparations. Standard deviations are given.

Experiments with the main K.casein fraction Ultracentrifugation. In sedimentation velocity experiments of the K-B1 (0.52) fraction in the SH-form, two peaks were observed in the synthetic boundary cell: a slowly moving one with a sedimentation coefficient of 1.4 S and a fast moving peak of about 11 S (cf. Fig. 4). The sedimentation coefficient of the fast peak exhibited the same poor reproducibility when different preparations were compared as was reported by Talbot and Waugh [7], i.e. a spread of three S-units. Measurements on the fast peak were done in experiments where a single sector aluminium centerpiece was used at 60 000 rev./min. Under these circumstances the slow peak does not detach

99

Fig. 4. Sedimentation of K-B1(0.52) in a double sector synthetic boundary cell, 44 000 rev./min : A, 7.5 mg/ml; B, 1 mg/ml total concentration. itself from the meniscus. Sedimentation and area determinations of the slow peak were done in experiments where the double sector synthetic boundary cell was used at 42 000 rev./min. In this case the fast peak moves away from the boundary in a relatively short time, leaving behind the slow peak (cf. Fig. 4). The area of the slow peak did not depend on the total concentration in the concentration range from I to 6 mg/ml. The concentration of the slow component calculated from the area was 0.35 mg/ml. The s20,w of the polymer peak of fraction r-B1 (0.52) at a concentration of 4 mg/ml and at 4 °C was 12.8 S, a little less than that found for the same solution at 20 °C: 14.5 S. It shows that in this respect fractionated x-casein is similar to the unfractionated protein [5]. Chromatography. The following behaviour was observed when r-B1 (0.52) was chromatographed on a Sephadex G150 column: a void volume peak containing the polymer was followed by a monomer peak (cf. Fig. 5). The area of the latter did not depend on the area found under the void volume peak. Rechromatography of the void volume material produced an identical pattern with the same surface area of the monomer peak as before. The sedimentation pattern of the void volume peak was identical to that shown in Fig. 4. Physical and chemical analysis. Spectroscopic and chemical data of the carbohydrate free r-casein fraction are collected in Table III. The results for the A- and the B-variant were pooled since no significant differences could be detected between the variants. The extinction coefficients are calculated for the isoelectric protein. The chloride content found was 3.28 ± 0.21 ~ (n = 6), close to the theoretical value of 3.07 ~ calculated from the maximum number of positive charges. It was found that 0.6 ~ H20 remained firmly bound to the protein after drying for 1 h at 105 °C and that the sample weight did not decrease further when heating was continued for longer than one hour. The extinction coefficient determined at p H 7 varied considerably from one preparation to another as indicated by the standard deviation given in Table III. The same can be said for the nitrogen determination where the average

100

I:L 2O 3o 4o E c 50 o eel 60 70 80 90 IO0

i

i

0

1

01

2

3

4

5 6 7

~t(h)

Fig. 5. K-A1 (0.60) on Sephadex GI50, 0.01 M EDTA-F 0.2 NaCl, pH 6.6; column 20 × 1.5 cm, flow rate 10 ml'h-L A part of the void volume peak (left curve) was diluted and rerun on the same column (right curve). value 15.6 % is lower than the theoretical value for r-B1 (0.52): 16.3 % N. It was not possible to control this variability. N o correlation could be f o u n d between the nitrogen content and the extinction coefficient at p H 7 or between the nitrogen content and the refractive index increment. The amide content remained ambiguous. The a m o u n t o f NH4C1 produced during 2, 4 and 6 h refluxing of the protein in 2 M HC1, extrapolated to zero time resulted in 1.52 % (s = 0.08) amide N, corresponding to 21 amide groups per K-casein molecule (theoretical 1.54 % N). The correlation coefficient o f the linear regression of 14 values was 0.50, which means that the observed time dependence in this regression analysis has a chance of more than 5 % to occur completely fortuitously. In this case one can disregard the time dependence and one can take the average o f all values. The result is 1.66 % N (s = 0.13), corresponding to 22 amide groups per molecule (theoretical 1.62% N). The n u m b e r o f amide groups given by Mercier et al. [4] is 21, which corresponds with the extrapolated value given above. TABLE III PHYSICAL AND CHEMICAL PARAMETERS OF K-A~ (0.60) AND K-B1 (0.52) /7

E]~m(276 nm) in 0.01 M HCI

9.23 ± 0.08"

E~°~,m(291 nm) in 0.01 M NaOH

13.11 ± 0.06

El°~m(280 nm) pH 7

10.0

dn/dc (546 nm) ml/g Nitrogen %

15.6

* Standard deviations are given.

± 0.3

0.184 ± 0.003 ± 0.4

4 4 10 10 22

!01 DISCUSSION

The identification of r-casein fractions The zones r-B2 (0.60) and K-A2 (0.66) on mercaptoethanol/urea/starch gel electropherograms contain K-casein fractions which have one more negative charge than the main fractions r-B1 (0.52) and r-A1 (0.60), respectively. It is in these zones that the K-casein fraction with one AcNeu group must be located. It follows from our results that, for some unknown reason, the relative amount of that fraction is small. This causes the apparent complexity of the minor zones, for if only a relatively small part, e.g. 5 or 10~, of the main fraction is modified by carbamylation or deamidation, the amount in the minor zone will be doubled. The properties of such a mixed fraction may then reflect more the properties of the contaminant than those of the originally occurring component. Therefore we support Hill and Wake's hypothesis [17] that fractions r-B2 (0.60) on r-A2 (0.66) are not artifacts produced during preparation and electrophoresis of K-casein. The next fraction r-B3 (0.65) contains two AcNeu groups per molecule, Table II, i.e. two negative charges more than r-B1 (0.52). A slightly higher mobility, no AcNeu and one extra phosphate group was found for the next fraction r-B4 (0.67). Although this fraction has the same net charge as fraction r-B3 (0.65) at pH 8.6, and therefore should have the same mobility, the AcNeu groups in the latter fraction probably occur at the end of a trisaccharide unit (or are part of a tetrasaccharide unit, see ref. 17), giving a branched molecule. The resulting increase in friction with the gel matrix causes the lower mobility of zone r-Ba (0.65). The fact that both fractions r-B3 (0.65) and r-B4 (0.67) occur in approximately equal amounts explains why formerly about one AcNeu group per molecule was found by Schmidt et al. [3] in their fraction B I I I , which consisted of both the above mentioned fractions. The use of a pH gradient results in a better separation between these fractions than was formerly obtained with a NaC1 gradient. The existence of K-casein fractions with one extra phosphate group also explains why apparently not all the AcNeu can be removed by treatment with neuraminidase [3] and why fraction r-B4 (0.67) is eluted from the column before fraction r-Ba (0.65) at pH 5.5. The last fraction analysed, r-Bs (0.74), Table II, contains four AcNeu groups, a fraction with three AcNeu groups is apparently missing. Taking into account the relative proportions of the fractions one can assume that attachment of AcNeu groups occurs predominantly in multiples of two. This would agree with the average carbohydrate composition of K-casein from bovine milk determined with gas-liquid chromatography by Fournet et ai. [18] (their Table I) i.e. 1 galactose: 1 N-acetylgalactosamine: 2 AcNeu and would mean a predominance of their proposed carbohydrate chain containing two AcNeu groups. Only two threonines are now needed to explain K-casein heterogeneity: the Thr (131) and Thr (133), which have been indicated by Joll6s et al. [19]. A carbohydrate chain containing two AcNeu groups linked to each accounts for three fractions. This number is doubled by attaching a second phosphate group to the peptide chain. Four of the six combinations are accounted for in Table II. Because r-B6 (0.77) is eluted from the DEAE-column before x-B5 (0.74), Fig. 2, fraction r-B6 (0.77) probably contains two phosphate groups and one carbohydrate chain with two AcNeu groups; compare with the behaviour of fractions r-B3 (0.65) and K-B4 (0.67), Fig. 2 and Table II. The last fraction, r-B7 (0.83), may contain either two phosphate groups and two carbohy-

102 drate chains or one phosphate group and three carbohydrate chains. According to the "code sequence" hypothesis [20] that the Ser in Ser-X-Glu (X = any amino acid) should be phosphorylated, Ser (127), which is at present one of the exceptions to this rule, is the most likely residue for linkage with a second phosphate group.

Association behaviour of the main r-casein fraction The observations using the ultracentrifuge and the experiments with the Sephadex column show that r-casein in the SH form associates according to a monomer-polymer mechanism strongly reminiscent of the behaviour of fl-casein and of micelle formation in soap solutions. In contrast to fl-casein, r-casein does not depolymerise at 4°C which suggests that hydrophobic interactions are not the primary driving force for K-casein polymerisation. The sedimentation coefficients show a tendency to decrease somewhat at concentrations lower than 1 mg/ml indicating a reduction in polymer size as the total concentration approaches the monomer concentration. The fact that the protein concentration between monomer and void volume peak (cf. Fig. 5) is close to or equal to zero is an indication for a system which attains an equilibrium slowly. Equilibrium is attained, however, within a few hours as rechromatography of the void volume peak gives the same amount of monomer (cf. Fig. 5) and because the period between successive synthetic boundary experiments, usually half an hour, did not appear to be critical for the observed pattern. For the interpretation of the sedimentation experiments it is probably not essential to know whether equilibration is slow or fast. I f it is slow the area under the slow peak corresponds to the monomer concentration in the initial solution. If equilibration is fast the Gilbert theory predicts [21] that in the limit of a large degree of polymerisation the area under the slow peak is a good approximation of the monomer concentration in the plateau region. In both cases the area of the slow peak corresponds to the monomer concentration and should be independent of total concentration: this behaviour was found for r-casein. If the degree of polymerisation of about 30 (see ref. 22) may be equated to "a very large one", it is possible to calculate the standard free energy difference per mole (monomer) between the polymeric and the monomeric state. The equilibrium constant can be calculated if the degree of polymerisation is known. To draw the parallel between the association phenomena in soap solutions and in Kcasein solutions further, the standard free energy difference will be calculated from tzo _ / ~ o = RTIn [cmc] (see e.g. Tanford (23)) where #o is the standard chemical potential and [cmc] ( = critical micelle concentration) in mol fraction units. The same equation follows of course from the Gilbert theory [19]. The result for r-casein fraction K-B~ (0.52) at p H 7 and ionic strength 0.2 is --36 kJ.mo1-1. REFERENCES 1 Mackinlay, A. G. and Wake, R. G. (1971) in Milk Proteins (McKenzie, H. A., ed.), Vol. II. Chapt. 12, Academic Press, New York 2 Pujolle, J., Ribadeau-Dumas, B., Garnier, J. and Pion, R. (1966) Biochem. Biophys. Res. Commun. 25, 285-290 3 Schmidt, D. G., Both, P. and de Koning, P. J. (1966) J. Dairy Sci. 49, 776-782 4 Mercier, J. C., Brignon, G. and Ribadeau-Dumas, B. (1973) Eur. J. Biochem. 35, 222-235 5 Waugh, D. F. and yon Hippel, P. H. (1956) J. Am. Chem. Soc. 78, 4576-4582 6 Swaisgood, H. E., Brunner, J. R. and Lillevik, H. A. (1964) Biochemistry 3, 1616-1623 7 Talbot, B. and Waugh, D. F. (1970) Biochemistry 9, 2807-2813

103 8 Rose, D., Brunner, J. R., Kalan, E. B., Larson, B. L. and Melnychyn, P. (1970) J. Dairy Sci. 53, 1-17 9 Gamier, J., Mocquot, G., Ribadeau-Dumas, B. and Maubois, J. L. (1968) Ann. Nutr. Aliment. 22, B495-B552 10 McKenzie, H. A. and Wake, R. G. (1961) Biochim. Biophys. Acta 47, 240-242 11 Stark, R. G. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, p. 592, Academic Press 12 Schmidt, D. G. (1964) Biochim. Biophys. Acta 90, 411-414 13 Wetlaufer, D. B. (1962) in Advances in Protein Chemistry (Anfinsen, Jr., C. B., Anson, M. L., Baily, K. and Edsall, J. T., eds.), Vol. 17, p. 309, Academic Press, New York 14 Warren, L. (1959) J. Biol. Chem. 234, 1971-1975 15 Hamilton, L. (1960) in A Laboratory Manual of Analytical Methods of Protein Chemistry (Alexander, P. and Block, R. J., eds.), Vol. 2, pp. 59-100, Pergamon Press, Oxford 16 Griswold, B. L., Humoller, F. L. and Mclntyre, A. R. (1951) Anal. Chem. 23, 192-194 17 Hill, R. J. and Wake, R. G. (1969) Biochim. Biophys. Acta 175, 419-426 18 Fournet, B., Fiat, A. M., Montreuil, J. and Joll/~s, P. (1975) Biochimie 57, 161-165 19 Joll6s, J., Fiat, A. M., Alais, C. and Joll6s, P. (1973) FEBS Lett. 30, 173-176 20 Mercier, J. C., Grosclaude, F. and Ribadeau-Dumas, B. (1971) Eur. J. Biochem. 23, 41-51 21 Cann, J. R. and Goad, W. B. (1970) Interacting Macromolecules, pp. 107, 161, Academic Press, New York 22 Slattery, C. W. and Evard, R. (1973) Biochim. Biophys. Acta 317, 529-538 23 Tanford, C. (1973) The Hydrophobic Effect, pp. 45-49, Wiley