Glycoprotein II The Isolation and Characterization of a Major Antigenic and Non-Haemagglutinating Glycoprotein from Phaseolus Vulgaris

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

35587

GLYCOPROTEIN II THE ISOLATION AND CHARACTERIZATION OF A MAJOR ANTIGENIC AND NON-HAEMAGGLUTINATING GLYCOPROTEIN FROM PHASEOLUS VULGARIS

A. PUSZTAI

AND

W. B. WATT

The Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB (Great Britain) (Received January 8th, r97o)

SUMMARY

r. The isolation of Glycoprotein II from the seeds of kidney bean is described. The purification was achieved by extraction of the pH 5 insoluble proteins of the seeds at pH 8.3 followed by high-voltage electrophoresis and chromatography on Sephadex G-200 and DEAE-cellulose columns of the soluble proteins. 2. Glycoprotein II was found to be in the form of a monomer and essentially homogeneous at neutral and slightly alkaline pH values by disc gel electrophoresis, immuno-diffusion and -electrophoresis, velocity and equilibrium ultracentrifugation. Its monomer molecular weight was 140 ooo ± 2000. Its hydrodynamic properties indicated a voluminous and asymmetric ((3 = 2.56 · ro- 6 ; Ve = 5. 72 · ro-18 ml) particle with large amounts of entrained solvent. Between pH 3-4 and 6.6 it was mainly in the form of a tetramer (molecular weight of about 560 ooo) with smaller amounts of monomer and higher oligomers also present. These oligomers were found not to be in a rapid chemical equilibrium and their conversion rates were infinitely slow at these pH values. Between pH 2.2 and 3-4 the glycoprotein was almost exclusively in the form of the monomer. 3· Glycoprotein II was also shown to be homogeneous by chromatography and velocity and equilibrium ultracentrifugation in dissociating media. It was however dissociated to about one quarter (35 ooo-43 ooo) of the monomer molecular weight in these solvents. 4· The carbohydrate part was mainly composed of D-mannose and D-glucosamine. No uronic acids were present. The amino acid analyses revealed a definite deficiency in the sulphur-containing amino acids, especially in cyst(e)ine. Phosphoruscontaining compounds were shown to be absent. 5. Glycoprotein II was found to be a strongly antigenic protein. It had however no haemagglutinating activity for rabbit erythrocytes.

Biochim. Biophys. Acta, 207 (r970) 4r3-43r

A. PUSZTAI, W.

B.

WATT

INTRODUCTION Glycoproteins are ubiquitous in the Plant Kingdom1- 3. They include such potentially interesting substances as haemagglutinins 4- 9 with mitogenic activity 6·10 ·11 carried possibly by the same glycoprotein molecule, toxins 12 , plastocyanins 13 and enzymes14 - 17 . Some of the glycoproteins are also presumed to contribute to the poor nutritive value of unheated legumes 18 . Glycoproteins containing hydroxyproline have been reported to be associated with structural elements of walls of cultured plant cells19 .20 and of normally grown plant tissues 21 . Several of these glycoproteins containing hydroxyproline have recently been purified 22 . Other highly purified glycoproteins from higher plants have also been described 23 - 25 whose biological function is unknown at present. There has also been some progress made towards the elucidation of the nature of the carbohydrate-peptide linkage in some of these glycoproteins26-28. A preliminary report of the isolation of Glycoprotein II, one of the largest single protein constituents of the seeds of kidney bean, has already appeared 29 . In the present paper a full account of the isolation and properties of this glycoprotein is given. MATERIALS AND METHODS Kidney bean was a variety called 'haricot' and sold locally by W. Smith and Sons (Aberdeen). The results of the chemical analyses for these seeds have been given before30 . Phytohaemagglutinin P was obtained from Difco Laboratories, Detroit, Mich. U.S.A. Guanidinium · HCl (B. D. H. Poole, Dorset, Great Britain) was purified as described before31 . 5 M solutions of this reagent had extinction values ofless than 0.030 against a water reference at 280 nm. The following proteins with their molecular weight and origin in parentheses were employed for the approximate calibration of the Sephadex G-200 column equilibrated with 5 M guanidinium · HCl: bovine serum albumin (69 ooo, Armour); ovalbumin (45 ooo, Sigma); {$-lactoglobulin-A (36 ooo, Koch-Light); pepsin (34 500, Armour); chymotrypsinogen-A (25 ooo, Sigma); myoglobin (I7 Soo, Koch-Light) and cytochrome-c {I2 400, Sigma). Dry matter of all materials was determined by drying over P 20 5 to constant weight in a vacuum desiccator. Nitrogen and phosphorus were determined as described before32 . Uronic acids were determined according to BITTER AND MuiRs 3 . Neutral sugars were estimated by the phenol-sulphuric acid method34 . Carbohydrate, hexosamine and amino acid analyses were carried out as described before 22 ·24 . Conditions for hydrolysis to liberate amino acids were: 5.6 M HCl at I04°. Samples were hydrolysed in sealed tubes under nitrogen for 24, 48 and 72 h with an approximate ratio of material to volume of acid of I mg to I mL For the estimation of individual sugars' samples were hydrolysed and de-ionised as described by CHESHIRE AND MuNDIE 35 . The resulting free sugars were reduced with NaBH 4 , acetylated and determined by gas chromatography36. Tyrosine and tryptophan were determined by the spectrophotometric method Biochim. Biophys. Acta, 207 (1970) 413-431

GLYCOPROTEIN Il FROM P. vulgaris of BENCZE AND ScHMID 37 • The measurements were made on the protein samples with and without added tryptophan. Rabbit antisera against crude extracts of kidney bean and against purified glycoproteins were prepared as described before30 . Immuno-electrophoresis and -diffusion were carried out as before30 . Haemagglutination tests: Rabbit blood was collected into a polypropylene tube (previously rinsed with a heparin solution) which contained a o.g% (w/v) NaCl solution to give an approximately ro-fold dilution of the erythrocytes. All materials tested for haemagglutinating activity were also dissolved in the saline solution to give a concentration of o.r% (wfv). These were then serially diluted and mixed with an equal volume of diluted erythrocytes (final vol. 0-4 ml). The tubes were left to stand at room temperature for 2 h. The degree of clumping was determined under a microscope. Under these conditions Phytohaemagglutinin P. included in each series of testing as a control gave definite clumping at r [lg level. Buffer solutions: Two buffer solutions were generally used: (A) o.os M Tris0.025 M acetic acid, pH 8.3 and (B) 0.04 M sodium acetate-acetic acid, pH 4.g6. Both contained o.r M NaCl. Molecular sieve and ion-exchange chromatography and high-voltage electrophoresis were carried out as described before 24 . Acrylamide gel electrophoresis: This was carried out by the gel rod technique in Poulik's buffer system as described by TOMBS AND AKROYD 38 . The progress of migration was visually checked by including trace amounts of bromophenol blue in the sample. The resulting gel rods were stained by immersing them in a r% (w/v) solution of naphthalene black dissolved in 7% (v/v) acetic acid for r h and then electrolytically de-stained in 7% (v/v) acetic acid. Chromatography on Sephadex G-200 in 5 M guanidinium · HCl: The gel was equilibrated with a 5 M solution of guanidinium · HCl for a month before pouring the column (roo em X 2.2 em). Under these conditions a flow rate of about 8-ro ml/h was obtained. The void volume of the column was determined with Blue dextran. The effluents were monitored at 280 nm with a Beckman DB recording spectrophotometer equipped with a circular 24-h attachment. The elution volume of the reference proteins was determined. Chromatography on Bio Gel P roo in phenol-acetic acid-water (r :r :r, wfvfv): Experimental conditions and the calibration of the column were as before 22 • Sedimentation velocity experiments were performed in a Spinco Model E analytical ultracentrifuge at 59 780 rev.jmin and 20° in a standard single sector 12-mm cell or at so 740 rev.fmin in a double sector cell made of epoxy resin filled with powdered aluminium. The concentration of the glycoprotein varied between o.r and r.o gjroo ml. More dilute solutions, down to 0.03 gfroo ml were examined in a 30-mm single sector cell. The schlieren optical system was used throughout. For the evaluation of the sedimentation coefficients a 'Sevel' IBM-Fortran computer program was used. The sedimentation coefficients from the schlieren patterns were estimated at zero time with residual mean squares of unconstrained regression and with regression through the estimated zero time position. The program included adjustment of the slopes for temperature, viscosity and density of solutions. A close leakage tolerance of 0.0025 em was automatically used. The estimation of the combined concentration correction factors incorporating radial dilution and angular factors for preselected exposures were also included in the program. Biochim. Biophys. Acta, 207 (1970) 413-431

A. PUSZTAI, W. B. WATT

Diffusion coefficients were obtained from the results of low speed (ro 589 rev.fmin) centrifugation in a capillary-type synthetic boundary-forming cell. The results were calculated by the height-area method. Approximate diffusion coefficients were also estimated from the results of immunodiffusion experiments39 • The optimum concentration of the glycoprotein against the antiserum was previously determined by serial dilution. High speed sedimentation equilibrium experiments were performed at 20° as described by YPHANTIS40 . The cell used in all runs had a six-channel epoxy centerpiece and sapphire windows. The solvent channels were filled with o.or ml of FC 43 fluorocarbon oil (Beckman, Glenrothes, Great Britain) and 0.12 ml of buffer. The sample channels had 0.02 ml of oil and o.r ml of sample. Protein concentrations were between o.ors and o.oso gjroo ml. Rayleigh interference optics were used. The experiments were generally carried out with an An-D rotor but for speeds below r6 ooo rev.fmin an An- J rotor was used. Speeds were checked from odometer readings with a stopwatch. The interference patterns were photographed on either Kodak II-G or on Ilford R 30 Trichrome plates. Vertical fringe displacement was read on a Shadomaster Model CRP Mk II projection microscope with coordinate measuring stage (Watson Manasty and Co., Great Britain). Usually three fringes were read and these after correcting for blank readings were averaged. Calculation of the various molecular parameters were performed as suggested by YPHANTIS 40 . Partial specific volume: This was determined in a pycnometer of a size of about so ml. Concentration of the glycoprotein varied from o.so to r.so gfroo ml and the measurements were carried out in both Buffer A and B solutions. The results were calculated from the relation

Vapp =

roo

roo-c

dptoteln

dsolveut

-----Cprotein

where dprotein and dsolvent are the densities of the protein and the solvent at 20.00° and Cprotein is the glycoprotein concentration (gfroo ml) in solution. Vapp was found to be similar in the two solvents and was independent of the protein concentration in the range measured. All results were therefore combined to give o.7r8 ± o.oo6. In multicomponent systems an operationally defined apparent quantity l/J' (refs. 41, 42) is measured by first dissolving the protein in the buffer solution and then dialysing it to osmotic equilibrium against a large volume of the same salt solution. In the calculations instead of dsolvent, dctiffusate is substituted in the above equation. Values for l/J' in Buffer A and B solutions were found to be very similar to Vapp values. A mean combined value of 0.716 ± 0.004 was calculated from the results. In similar measurements a mean l/J' value of 0.740 ± 0.004 was obtained for Glycoprotein II at four protein concentrations in 5 M guanidinium · HCl solutions containing o.r M z-mercaptoethanol. Viscosimetry: The measurements were only made with Buffer A. Ostwald viscometers with an outflow time for water of about 215 sec were used. All measurements were carried out at zo.ooo ± O.OOJ The concentration of the glycoprotein varied between 0.37 and I. I I gfroo ml. All values were corrected for the density of the solvent and solutions. 0

Biochim. Biophys. Acta, 207 (1970) 413-431

•

GLYCOPROTEIN

II

FROM

p. vulgaris

RESULTS

Purification of Glycoprotein I I

The initial stages of the purification were the same as those used for the preparation of Glycoprotein I (ref. 24). The precipitate obtained on dialysis of the original kidney bean extract against the 0.033 M sodium acetate-acetic acid buffer (pH s.o), served as starting material in the present investigation. A typical experiment is described in detail. All steps were carried out in a cold room (0-4°). The pH 5 precipitate which contained 3·43 g or 6g.7°/cl of total seed nitrogen extracted 24 was extracted with about 200 ml of o.r M Tris-o.os M acetic acid buffer (pH 8.3). The resulting slurry was centrifuged at 5300 X {',in an MSE Major refrigerated centriguge for r h. The sediment was stirred up in roo ml of the same buffer and spun again. The sediment was discarded. The combined slightly cloudy supernatants contained 2-4 g or 48.8% of total seed nitrogen extracted and were further purified by high-voltage electrophoresis (Fig. r). The contents of Tubes r6-24 were pooled. This fraction contained o.g8 g or rg.g% of the total seed nitrogen extracted and was composed mainly of an approximately 7-S glycoprotein. It also contained small amounts of haemagglutinins and several high molecular weight protein contaminants. The solution was then saturated with solid (NH 4 ) 2S0 4 (760 g/1) and the resulting precipitate was collected by centrifugation. It was then dissolved in and dialysed against Buffer A. The ammonia-free solution (about roo ml) was concentrated to about so ml by ultrafiltration (Sartorius SS membrane filter, CB., porosity< 5 nm; Schleicher and Schull, West Germany) and chromatographed on a Sephadex G-200 column (Fig. z). The contents of Tubes 75-90 were pooled and found to contain 0.52 g or ro.6% of the total seed nitrogen extracted. This fraction when examined in the ultracentrifuge in Buffer A was made up almost exclusively of molecules with an approximate sedimentation coefficient of 7 S. The solution was then applied on to a DEAE-cellulose column equilibrated with a o.os M Tris-0.025 M acetic acid buffer (pH 8.3) containing

+

1.0

50

60

70

80

90

FRACTION

100

No

110 120 130

Fig. I. Separation of the proteins of kidney bean by high-voltage electrophoresis. Conditions: Hannig apparatus; 0.1 M Tris-0.05 M acetic acid buffer (pH 8.3); 30 Vfcm; 190 rnA; rate of buffer flow in the cuvette. 50 ml/h; sample introduction rate. approx. 2 ml/h; sample introduction point indicated by arrow. The extinction values of the contents of the 48 collection tubes were read at 280 nm in 1-cm cells. Fig. z. Separation of proteins of kidney bean obtained by high-voltage electrophoresis on a Sephadex G-200 column (1oo em x 4 em) equilibrated with Buffer A. The effluents were continuously monitored at 280 nm to locate the position of the proteins.

Biochim. Biophys. Acta. 207 (1970) 413-431

A. PUSZTAI, W. B. WATT

+

a

2

3

4

b

c

10

20

30

40 50 GO 70 FRAC fiON No

80

90

100

110

Fig. 3· Chromatography of Glycoprotein II on a DEAE-cellulose column (34 em X 4 em). The column was equilibrated with o.os M Tris-0.025 M acetic acid buffer (pH 8.3) containing o.os M NaC!. The chromatogram was developed with a gradient of NaCl in the Tris-acetic acid buffer. The concentration of NaCl in the eluted fractions (0-0) was determined by titration with standard I M AgN0 3 solution with K 2Cr0 4 as indicator. The effluents from the column were continuously monitored at 28o nm to locate the position of the proteins (e-e). Fig. 4· Tests for purity of Glycoprotein II. a. The patterns of proteins obtained by disc gel electrophoresis: I, starting material; 2, Fractions 75--90 from Sephadex G-200 chromatography; 3, Fractions 55-70 from Sephadex G-2oo chromatography; 4. Fractions 63-75 from DEAE-cellulose chromatography. To demonstrate the presence of small amounts of contaminating proteins the amount of glycoproteins in the various preparations applied to the gel rods was more than so 11-g. b. The patterns of proteins obtained by immunoelectrophoresis: I, original extract of the seeds of kidney bean; 2, the purified glycoprotein from Fractions 63-75 from DEAE-cellulose chromatography. The precipitin lines were obtained by using an antiserum produced against an original extract (pH S.o) of the seeds of kidney bean. c. The pattern of immunodiffusion of Glycoprotein II against antisera as above.

0.05 M NaCl and developed with an NaCl gradient (Fig. 3). Contents of Tubes 63-75 were combined. Yield: 0-45 g or g.r% of total nitrogen extracted. Biochim. Biophys. Acta, 207 (I97o) 4I3-43I

GLYCOPROTEIN

Il

FROM

p. vulgaris

Properties of and tests for the purity of Glycoprotl!in II The various stages of purification of the glycoprotein were followed by disc gel electrophoresis (Fig. 4a). The final product gave one main band with only traces of slower moving components. The glycoprotein when injected into rabbits elicited the formation of precipitating antibodies. Fig. 4b shows the results of immunoelectrophoresis of the purified glycoprotein and of the starting material against antisera produced against crude extracts of the seeds of kidney bean. In the early stages of its purification the various preparations of this glycoprotein showed a definite haemagglutinating activity. This activity, however, was not present in the final product when tested at a concentration of I mg/ml. All the original haemagglutinating activity was recovered from the high molecular weight protein fractions obtained by chromatography on Sephadex G-200 (Fig. 2). These had activities comparable to that of Phytohaemagglutinin P. Chemical composition An aliquot of the combined material obtained from the DEAE-cellulose column was exhaustively dialysed against water. The glycoprotein precipitated out during dialysis. For this reason no de-ionisation by mixed-bed ion exchangers could be carried out. The dialysed solution with the precipitated glycoprotein was freeze-dried and then dried to constant weight. All chemical analyses were carried out on this material. An elementary analysis (Dr. F. Pascher, Mikroanalytisches Laboratorium; 53 Bonn, Buschstrasse 54, West Germany) gave the following results expressed as % (w/w), C, 49.04; H, 7.og; 0, 27.79; N (Dumas), I5.64; S (total), 0.24; S (inorganic), nil; ash, 0.37. There were only traces of phosphorus found. Kjeldahl N estimations in our laboratory gave I5.54% in good agreement with the results of the Dumas N estimations. Hydrolysis with I M HCl at I00° for various lengths of time up to 24 h released a hexosamine component which was indicated by chromatography on ionexchange resin columns and on paper to be glucosamine. A maximum amount of r.Io% was released between 6- and 8-h hydrolysis under these conditions. The neutral sugar content expressed as mannose 34 was found to be 4-46%. No uronic acids could be demonstrated. Amino acid analyses on the 24-, 48- and 72-h hydrolysates were carried out in triplicate. These results together with the sugar composition and other data are given in Table I. Physical properties As the glycoprotein was insoluble in water in the absence of salts and could not therefore be de-ionised, its approximate iso-electric point was determined from the pH of exhaustively dialysed solutions containing precipitated proteins. Different initial pH and buffer composition (citrate-phosphate buffers, pH 2.2-S.o) had no appreciable effect on the final pH of 5-42 of the exhaustively dialysed solutions. This is taken as an indication that pH 5-4 approximates to the true isoelectric point of Glycoprotein II. The glycoprotein when examined by sedimentation velocity analysis in Buffer A gave a major symmetrical sedimenting component with a minor higher molecular weight component, usually less than s% of the total area, which was just discernible at the beginning of the run. The sedimentation coefficient of the major component Biochim. Biophys. Acta, 207 (1970) 413-431

A. PUSZTAI, W. B. WATT

420

TABLE I ANALYTICAL VALUES FOR GLYCOPROTEIN

II

Details of purification and methods of analyses are described in the text. The mean of the results of the analyses are expressed as g anhydrous amino acid or anhydro sugar residues per roo g dry weight.

Amino acid

gfroo g dry wt.

Sugar

gfwo g dry wt.

Asp Thr* Ser* Glu Pro Gly Ala Cys** Val*** Met** lie*** Leu Tyr Phe NH 3 Lys His Arg Tyrt Trpt Glucosamine

12-41 3·41 6.69 15·14 2.88 2.68 2.99 0.28 5.16 o.69 5.6! 9.10 3-49 6.64 !.83 5·56 2.64 5·04 3.21 o.8o 0.99

Mannose Galactose Arabinose Xylose Neutral sugars at

3.20 Traces Traces 0.35 4·46

Total

94·03

% ofN

accounted for

98·7

* Obtained by 72-h hydrolysates. ** Results were *** Results were t Results were

a linear extrapolation to o h of the results of analyses on the 24-, 48- and obtained after performic acid oxidation. calculated from analyses on the 72-h hydrolysates only. obtained by the spectrophotometric method of BENCZE AND ScHMm 37 •

3:

o'

Ji'7.0

- -·-

(}1

0-2

9f

0·3 0·4 0·5 0·6 0·7 0·8 0·9 100ml

1~

Fig. 5· The dependence of s 20 ,w values of Glycoprotein II monomer on protein concentration. All experiments were performed in Buffer A. The points in the plot represent the mean values of separate experiments and of separate readings of these experiments; the vertical bars indicate the magnitude of the standard deviation from the mean values.

Biochim. Biophys. Acta, 207 (1970) 413-431

GLYCOPROTEIN

II

FROM

P. Vttlgaris

421

showed a negative concentration dependence (Fig. 5). The relationship between s (or rjs) and c was not linear. A reasonable agreement with the experimental results however could be obtained by dividing the concentration range into two sections within which the relationship between s and c was close to linear. The lower concentration range from 0.04 to 0-40 gjroo ml could then be approximately fitted with the following equation: s 20 ,w = 7·59 (r - 0.171 c) S. The amount of the minor high molecular weight material was not dependent on the ionic concentration of the solution at this pH value in the NaCl concentration range of 0.004-I.O M. Moreover, the sedimentation coefficient measured at a protein concentration of o.6r gjroo ml of the major component was also fairly constant (s 20 ,w = 6.94 ± o.ro S) and only below 0.05 M NaCl concentration showed a decrease to S2o,w = 5·89 sat 0.004 M. This was probably due to the lack of enough supporting electrolytes and consequent primary and secondary charge effects and, possibly, to molecular expansion. When, however, Glycoprotein II was examined near its iso-electric point in Buffer B at pH 4·94 it was found to contain at least four components, one major with an approximate sedimentation coefficient of r8 S (about 84% of the total area) and three minor. One of these last was a component with an approximate s value of 7 (about 5% of total); a second one with an s value of about 27 (about ro% of the total). Finally, the third minor component (about r% of the total) had an s value in excess of 33 S. In the concentration range studied (o.o4-1.0 gjroo ml) no changes in the proportion of the individual sedimenting boundaries were observed. In addition, all boundaries were clearly separated. These indicate that the various components are not in a rapid equilibrium of association-dissociation 43 . The sedimentation coefficients of the various molecular species showed some slight concentration dependence (Fig. 6). The limiting values estimated from these plots were as follows: 7.2; r8.9 and 28.3 S. No accurate estimate of the S 20 ,w value for the minor (approx. 33 S) component could be obtained. In view of these findings a thorough study of the effects of pH on the sedimentation velocity patterns of Glycoprotein II was undertaken. For these studies aliquots of the glycoprotein were exhaustively dialysed against a 0.02 M citrate0

TABLE II THE RELATIVE CONCENTRATION OF THE INDIVIDUAL SEDIMENTING COMPONENTS OF GLYCOPROTEIN II AS A FUNCTION OF THE

pH

OF THE MEDIUM

The results were obtained from sedimentation velocity runs and expressed as per cent of the total area in each individual run. The total area in all runs was constant within + 3 to -4% of a common mean value. The absolute concentration of the glycoprotein in these experiments was o.6r gfroo mi. The values refer to the pictures in Fig. 7·

Relative concentration

S2o,w

of component

pH of the medium: 7.48

7·2 IO approx. 18.9 28.3 33

97·5 2.5 Nil Nil Nil

6.96

6.42

,5.89

98-4 3-4 Nil Nil Nil

16.8 Nil 78.0 5·2 Nil

5-4 Nil 84.8 ro.5 ~il

,5.JI

4·79

].85

3.42

3.04

2.77

2.25

7.24* 5 .4I

4·3

2.7 Nil 87·7 9.6 Nil

2-4 Nil 9L8 5·9 Nil

33·3 Nil 6 5 .6 I. I Nil

97·6 2.4 Nil Nil Nil

98.2 I.8 Nil Nil Nil

96.6 3-4 Nil Nil Nil

96.o 4·0 Nil Nil Nil

~il

82.3 II.4 I.9

* These solutions contained o.o5 M EDTA.

Biochim. Biophys. Acta, 207 (1970) 413-431

5·0 Nil 89·3 5·8 Nil

A. PUSZTAI, W. B. WATT

pH 7.48 50• 88min

pH 6.96 60° 16min

pH 6.42 55° 20 min

-direction of centrifugol field

__l_j__l

29 28

pH5.89 55° 16 min

27

20 ~· 19 cf>

18

pH 4.39 65° 12min

_j_ _1 _j_

26

~

pH 5.31 60° 16min

1

...___._-;---..-----.c!____

pH 3.85 65° 16min

JL 0·1

02

g3

gf

04 05 100ml ('/,)

06

pH 2.25 45° 44min

pH 3.42 55° 20min

~ pH 7.24EDTA 55° 88min

pH 2.77 5Q•

80min

L

pH 5.41 EDTA 55° 32 min

Fig. 6. The dependence of s 20 ,w values of the three components of Glycoprotein II obtained at pH 4.96 on protein concentration. The concentration on the abscissa is the sum of all the components present. All experiments were performed in Buffer B. The experimental points represent the mean values obtained in a number of separate experiments. The results have not been corrected for the Johnston-Ogston effect. Fig. 7· Sedimentation velocity patterns of Glycoprotein II obtained at pH values from 2.2 to S.o with and without the presence of EDTA.

phosphate buffer44 containing o.r M NaCl in the pH range from about 2.2 to 8.o. The results are given in Fig. 7 and Table II. From these it is clear that above pH 6.6 and below 3-4 the glycoprotein existed almost exclusively in the form of the 7-S component which is probably the monomeric form of the glycoprotein. In between these pH values the r8.9-S component predominated, probably a tetramer, with smaller amounts of monomer and higher oligomers also present. Some of these solutions were then re-examined under similar conditions in the same buffer but with the addition of EDTA. No changes in the sedimentation patterns or coefficients were found however (Fig. 7). Diffusion coefficients. Diffusion coefficients were determined at pH 8.3. There was an appreciable dependence of D 20 ,w values on protein concentration (Fig. 8). The following equation gave a reasonable fit to the experimental data: D 20 ,w = 5·53 (r- 0.175 c) ·ro- 7 cm 2 ·sec-1 where cis the concentration of the glycoprotein in gjroo ml. Very similar results were obtained from immunodiffusion experiments (Fig. 4c). A mean value of 5.5 · ro- 7 cm 2 • sec- 1 was obtained consistently with several Biochim. Biophys. Acta, 207 (rg7o) 413-431

GLYCOPROTEIN

Il

FROM

P. vulgaris

1·7

H ~

r 70

r--

0

1·5 1·4

1·3

6 0 5·0

~

~

0

1·1

4·0 02 04 0·6 08 gjlOOml

10

Fig. 8. The dependence of D 20 ,w values of Glycoprotein II monomer on the protein concentration. The results were calculated from the patterns of low speed centrifugation (ro 589 rev.fmin). All experiments were performed in Buffer A. The points of the plot represent mean values of separate experiments and of separate readings of these experiments. The vertical bars indicate the magnitude of the standard deviation from these mean values. Fig. g. The dependence of the relative viscosity values of Glycoprotein II monomer on the protein concentration. All experiments were performed in Buffer A. The line to fit the experimental values was obtained by a least squares treatment of the data and corresponds to a limiting value of 0.0695 dlfg.

5.01'----L-..J...__.___.__......___J_

_.L_..L---l

200 400 600 800 1000 1200 1400 1600 1800 V(r) -

Yo

7·0

G so 20

[ . _ _ J __

43-0

_L__..J...__

_j___

43·6 43·9 43-3 , 2 ( c m 2)

__J

44·2

50

c

o

e

. o

6·60

e

A. 6·62

6·64

(r) em

Fig. ro. The results of high speed equilibrium centrifugation experiments on Glycoprotein II monomer. The experiments were performed in Buffer A at pH 8.3. a. A plot of log vertical fringe displacement vs. the square of distance in the cell. b. The dependence of point weight average (e), of point number average (0) effective reduced molecular weights and zan-aw values (D) on protein concentration as expressed by vertical fringe displacement. c. The extrapolation ofO'n and aw values plotted against distance to the base of column. Conditions of the experiments: o.o2o gfroo ml protein concentration; Buffer A; 20.0°; 17 g8o rev.fmin; 48 h.

Biochim. Biophys. Acta, 207 (rg7o) 413-431

A. PUSZTAI, W.

B.

WATT

different antisera produced against the pure glycoprotein or against crude kidney bean extracts. Viscosity. Following ScHACHMAN's recommendation 45 'f)rel values vs. glycoprotein concentration were plotted (Fig. g). A limiting value of o.o6g5 ± o.ooso dljg was obtained from a least squares treatment of the data. High speed sedimentation equilibrium experiments. These were performed at pH 8.3 in Buffer A and at 20°. They indicated, in agreement with the results of sedimentation velocity runs, that Glycoprotein II contained a small amount of higher molecular weight material. The amount of this minor component, however, was not dependent on the original protein concentration in the range studied. Plots of log vertical fringe displacement vs. distance were linear except close to the bottom meniscus (Fig. roa). Point weight and point number average molecular weights were computed for each experimental point (displacements greater than 50 fl) as suggested by YPHANTIS 40 and these were plotted against fringe displacement (Fig. rob). As sedimentation velocity runs indicated the presence of no component smaller than the major component an extrapolation of these values to zero concentration gave 140 ooo as the molecular weight of the monomer, the smallest molecular species present at pH 8.3. This value was found to be independent of the original protein concentration in the range of o.ors-o.oso gjroo ml. The weight average molecular weight was obtained by extrapolating the point number average molecular weight values plotted against the distance to the bottom meniscus to give a value of M w = rso ooo and the z average, Mz = r6r 700, from the similarly extrapolated weight average values. These values corresponded to 7 and g% dimer, respectively, assuming that all the

3·2

a

3·0 2· 8

!

0

>

2 ·6

)~

~

4·8

N I

E 4·6

u

\..:l4 ·4

2 -~

4-2

!:"' 2·2

4·0

>

2 0 1 8 1· 6

/

50 /

-

~

8

~~

4·

6

N

430

43·5 r2 (c m2)

44·0

<.!> ~ ~ ~

-L._j

200 400 600 800 1000 1200 YrrJ - Yo

c

s s ~ ~ e

• !

'~

r

2

~-o

base-

6 58

6·6

6 62

6 {;~

6 66

( r) em

Fig. r r. The results of high-speed equilibrium centrifugation experiments on Glycoprotein II at pH 4.96. a. A plot of log vertical fringe displacement vs. the square of distance in the cell. For this run a protein preparation obtained from the DEAE-cellulose column was used without any further separation. band c. Similar plots to those described in Fig. ro but obtained on the glycoprotein tetramer purified by chromatography on Sepharose 4B columns. Conditions of the experiments were the same for both runs and were as follows: o.or 6 gfroo rnl protein concentration; Buffer B; 20.o 0 ; 7928 rev.fmin; 48 h.

Biochim. Biophys. Acta, 207 (1970) 413-431

GLYCOPROTEIN

II

FROM

P. vulgaris

deviation was due to the presence of a dimer in the sample. A similar approach at pH 4.96 revealed considerably more pronounced polydispersity (Fig. rra). The approximate weight- and z-average molecular weights were as follows: M w = 602 ooo and M z = 654 ooo. The purified tetramer obtained by chromatography on a Sepharose 4B column gave a less complicated picture although a close inspection of log fringe displacement vs. distance plots at the bottom meniscus revealed some slight deviation from linearity. The molecular weight for the smallest (main) species present, 561 ooo, however was very close to Mw = 567 ooo and Mz = 577 ooo (Figs. rrb and rrc). In addition, the molecular weight of the tetramer was found to be independent of the initial protein concentr3.tion in the range of o.or5-0.050 gjroo ml. Studies in dissociating media. Sedimentation velocity experiments were performed on the protein dissolved in and dialysed against solutions of 5 M guanidinium · HCl containing o.r M 2-mercaptoethanol. The results were converted to s 20 ,w values. Experimentally determined values of I.I2I2 for density and r.4r8 for the relative viscosity of the diffusate and a
A. PUSZTAI, W. B. WATT

3 0

0

2·0

>-

a

1·8 c:S

N


2 5

I

>-

"' 2 0

1·6

3;

b

~

1 ·4 I 5

1. 0

'---'--J...._-l..._.J...__J

0·2 0·4 0·6 0·8 gf lOOg

1·0

41 5

4l0

( r 2 ) em 2

42·0

Fig. 12. a. The dependence of s 20 ,w values of Glycoprotein II on protein concentration in 5 M guanidinium · HCl containing o. r M 2-mercaptoethanol. b. The results of high speed equilibrium ultracentrifugation in the above solvent. Log of vertical fringe displacements were plotted against the sqare of distance in the cell. Conditions of the experiment: 0.030 gfroo ml protein concentration; 20.0°; 44 700 rev.fmin; 48 h.

a

0

~2

"'

I }'"I

,...____.. >-----<

~~

3 0

I v\

0

j

1 0

ro

~

~

~

ro

oo oo

FRAC TlON

No

~

b

2 0

m w m I· 2

30

40

50

40

50

60

GO

70

80 90

70

80

90

FRACTION

No

100

110 120

c

I 0

~ 0 8 N

"'

0 6 0·4 0 ·2

30

FRACTION

100 110

120

No.

Fig. 13. a. The separation of the tetrameric form of Glycoprotein II from the monomer at pH 6.56 by chromatography on a Sepharose 4B column. The range of collection for the monomer and for the tetramer are indicated by horizontal bars in the diagram. b. Re-chromatography of the monomer ( 0) and tetramer (e) obtained from the first Sepharose chromatogram. c. The separated components were dialysed first against a citrate-phosphate buffer of a pH value higher than 7, then against the original pH 6.56 buffer and re-chromatographed on the same column. The effluents from the column were continuously monitored at z8o nm to locate proteins.

Biochim. Biophys. Acta, 207 (1970) 413-431

GLYCOPROTEIN

II

FROM

P. vulgaris

427

components into each other. When examined in the ultracentrifuge both behaved as homogeneous components with a characteristics value of 7 S and rg S, respectively. The initial distribution pattern (about 6o% tetramer and 40% monomer) could however be re-established from each of the separated components by first dialysing them against a buffer solution of above pH 7 and then against the original buffer of pH 6.56 (Fig. 13c). Both dialysis steps had to be performed for at least 4-5 days to complete this process. Failure to do so resulted in incomplete conversion. The process of monomer to tetramer conversion could however be speeded up by exposure to pH values lower than pH 6.56 and, conversely, tetramer to monomer conversion was speeded up by pH values higher than 7·

DISCUSSIOK

The present paper described the isolation of one of the largest single glycoprotein constituents from the seeds of kidney bean accounting for at least ro% of the total nitrogen of the seed. The carbohydrate part of Glycoprotein II resembled that of a number of other well-characterized soluble plant glycoproteins 23 -- 27 . Its characteristic feature was the presence of glucosamine and mannose as the main carbohydrate constituents. The presence of the small amounts of xylose (Table I) is more open to doubt. In contrast to glycoproteins obtained from black kidney beans8 other sugars such as glucose, galactose or rhamnose were found only in trace amounts. Uronic acids were also found to be absent. The amino acid composition (Table I) showed a definite deficiency in sulphur-containing amino acids, especially, in cyst(e)ine. The absence of phosphorus indicated that the final preparation of Glycoprotein II was free from nucleic acids or from phytic acid which are known and common contaminants of seed proteins. By all criteria-chemical, physical or immunochemical-Glycoprotein II was found to be free from other proteins or other macromolecular contaminants. At neutral or at slightly alkaline pH values it was composed of almost exclusively the 140 ooo molecular weight component. As this species was the lowest molecular weight component found in dilute aqueous buffers it is regarded as the monomeric form of the glycoprotein. Similarly, the glycoprotein also existed mainly as a monomer below pH 3-4 but, interestingly, no further dissociation into subunits occurred even at pH z.z (Fig. 7). These results obtained by velocity and equilibrium ultracentrifugation were also supported by the evidence of the high degree of homogeneity obtained in the various electrophoretic examinations such as disc gel or immunoelectrophoresis (Figs. 4a and 4b). Between pH 3-4 and 6.6 the tetramer was the main molecular form with a most likely molecular weight value of 560 ooo. There were, however, appreciable amounts of monomer (about S%) and higher oligomers (about ro%) also present. All evidence obtained was consistent with the idea that these oligomers were not in a rapid equilibrium of association-dissociation 43 at these pH values. Accordingly, the various forms once formed at one pH value could readily be separated and isolated by chromatography at the same pH value (Figs. 13a and 13b). Their interconversion could be brought about only by appropriate changes in the pH of the medium (Fig. 13c). This pH-controlled association-dissociation reaction was rather slow. Metal cations, however, were apparently not involved in this reaction Biochim. Biophys. Acta, 207 (1970) 413-431

A. PUSZTAI, W. B. WATT

since the presence of EDTA in the buffer solutions of various pH values caused no changes in the sedimentation patterns of Glycoprotein II (Fig. 7). It is to be noticed that the molecular weight value calculated for the monomeric form from S 20 ,w and D 20 ,w values (M 8 ,n) was just over rzo ooo. This value was much lower than that obtained from equilibrium centrifugation. It was thought that in spite of the uncertainty in arriving at the correct value for S 0 20 ,w (Fig. 5) this discrepancy was outside the experimental error, i.e. to bring M 8 ,n into line with those obtained from equilibrium runs it would require an so 20 ,w value of nearly 9 S. This seems rather unlikely, especially, as it has been noticed before that if a curvature exists in a plot of s vs. c, this curvature actually decreases as the concentration approaches zero 46 •47 . In comparison, the precision of the techniques used in the present work for measuring diffusion coefficients was not high. Moreover, the error of these measurements should increase with decreasing protein concentration. If, for example, all values below 0.5 gjroo ml protein concentration (Fig. 8) were neglected and no concentration dependence of D 20 ,w values was assumed, a mean value of 4.8 for D 20 ,w would be obtained. With this value in the Svedberg equation an Ms,D value of over 137 ooo is calculated. This approaches the values obtained by equilibrium centrifugation. In 5 M guanidinium · HCl the monomeric form of Glycoprotein II underwent further dissociation into subunits (protomers). The results of velocity and equilibrium sedimentation gave no evidence of inhomogeneity. The macromolecular component in this case was at thermodynamic equilibrium with respect to interaction with the diffusible components present 41 •42 • An apparent subunit weight of 43 ooo ± z6oo was calculated from equilibrium runs with the aid of the experimentally measured quantity
0

Biochim. Biophys. Acta, 207 (r970) 4r3-43r

GLYCOPROTEIN Il FROM P. vulgaris were obtained for the monomer at pH 8.3 where the glycoprotein had a net charge. The high ionic concentration of the medium, however, tended to suppress the magnitude of the charge effects on the parameters measured. The intrinsic viscosity value of 0.0695 dljg was higher than could be expected for a compact globular protein59 • It was, however, too low for a random coil. The moderately high value for [?]] might then indicate asymmetry or expansion or both. Attempts to evaluate these effects separately from the combined viscosity and sedimentation data along the lines proposed by CREETH AND KNIGHT 60 ·61 were inconclusive. The value of 2-46 obtained for K, 1[1 ] of Glycoprotein II was higher than r.6 predicted from theory for rigid spherical particles or random coil polymers in good solvents. The significance of the higher than theoretical value is not clear as asymmetry produces values lower than r.6. It is to be noticed that CREETH AND KNIGHT also tabulated a number of other proteins with values for Ks/[ 1 J greater than r.6 (ref. 6o). On the other hand, the calculation of the coefficient ~ of ScHERAGA AND MANDELKERN 62 gave a value of 2.56 · ro- 6 ± o.os · ro- 6. This corresponded to a relatively high frictional ratio of about 1.84, an axial ratio of about r6 and a viscosity increment of 28.3 if as a model a prolate ellipsoid of revolution was chosen. The volume of the effective hydrodynamic ellipsoid was calculated from: roo·M·[I)]

Ve=~~~

N·v

where N is Avogadro's number, vis the viscosity increment, M is the monomer molecular weight and [17 J is the intrinsic viscosity (dljg). Ve was found to be 5. 72 · ro-18 ml. The molecular volume calculated from 17 = 0.718 was on the other hand only about o.r8 · ro-18 ml. The ratio, Ve/17 mol, was over 30 and this high value indicated a voluminous particle with large amounts of solvent entrained63 . The theoretical basis for relating these two molecular volumes quantitatively, however, is not clear. For this reason it was felt that calculation of the major axes of a hydrodynamic ellipsoid would be purely hypothetical. Most of the above considerations applied to the monomer at pH 8.3. Near the isoelectric point, however, the glycoprotein was mainly in the form of a tetramer. Although this form was studied less extensively than the monomer some of their parameters can be compared. Thus, a similar value for 17 of 0.718 was obtained at both pH values. The ratio of four for the molecular weight of the tetramer near the isoelectric point and the monomer at pH 8.3 obtained from high speed sedimentation equilibrium experiments is supported by the relation, (s 0 20 ,w tetramerjs 0 20 ,w monomer)3i2 = 3.93, obtained from sedimentation velocity runs. This also indicated a similar frictional ratio for the two molecular forms. It is to be noticed, however, that none of the oligomers at pH 4.96 showed any anomalies in their s vs. c relationship (Fig. 6) similar to that exhibited by the monomer at pH 8.3 (Fig. 5). Moreover, the concentration dependence of the s values was less pronounced for the tetramer than for the monomer and obeyed the relation: s 20 ,w = r8.9 (r - 0.037 c) S. This compared with a K 8 value of o.r7r for the monomer. According to some preliminary data the tetramer, in mixtures containing small amounts of other oligomers, had viscosity values somewhat lower than the monomer at pH 8.3. Thus, the above results were not entirely consistent with either the absence or presence of conformational changes occurring in the monomer to tetramer conversion. Biochim. Biophys. Acta, 207 (1970) 413-431

430

A. PUSZTAI, W. B. WATT

The pH-controlled limited association-dissociation reaction observed for Glycoprotein II was similar to that exhibited by several other plant proteins and is probably a fairly general property of a number of seed proteins 64 •65 • The general occurrence and the high content of glycoproteins in the seeds of higher plants1- 3 when compared to the relatively small amounts found in the green parts66 may indicate some special role for these compounds in the life cycle of the plant. Although, at present, no special function can be ascribed to Glycoprotein II, its relative abundance and the characteristic patterns of its carbohydrate make-up and markedly strong antigenicity suggest it could be useful as a marker in plant physiological studies. ACKNOWLEDGEMENTS

The authors are grateful to Professor P. Johnson and to Drs. R. H. Smith and E. I. MacDougall for reading the manuscript and for advice; to Drs. J. S.D. Bacon and M. V. Cheshire and Mr. C. M. Mundie at the Macaulay Institute for Soil Research, Aberdeen for the determination of sugars by gas chromatography; to Mr. I. McDonald for writing a 'Sevel' program fL•r the IBM II30 computer for the evaluation of sedimentation coefficients; to Miss I. Duncan and Mr. J. C. Stewart for skilful technical assistance. REFERENCES I 2 3 4 5 6 7 8 9 ro

II

12 13 14 15 I6 17 r8 19 20 21 22 23 24 25 26 27 28 29

A. PUSZTAI, Nature, 20! (1964) 1328. A. PuszTAI, Abstr. Commun. 6th Intern. Congr. Biochem., New York, I964, II p. 153. A. PuszTAI, Biochem. ]., 94 (r965) 6o4. S. WADA, M. J. PALLANSCH AND I. E. LIENER, j. Biol. Chem., 233 (1958) 395· T. TAKAHASHI, P. RAMACHANDRAMURTHY AND I. E. LIENER, Biochim. Biophys. Acta, 133 (1967) 123. D. A. RIGAS AND E. A. JoHNSON, Ann. N.Y. Acad. Sci., II3 (1964) Soo. W. G. JAFFE, Acta Cient. Venezolana, 13 (1962) roo. W. G. JAFFE AND K. HANNIG, Arch. Biochem. Biophys., ro9 (r965) So. H. LIS, C. FRIDMAN, N. SHARON AND E. KATCHALSKI, Arch. Biochem. Biophys., II7 (I966) 301. P. C. NowELL, Cancer Res., 20 (r96o) 462. D. A. RIGAS, E. A. JoHNSON, R. T. JoNES, J.D. McDERMED AND V. TISDALE, in G. PARISSAKIS, Chromatographie et Methodes de Separation Immediate, I965, Vol. 2, National Technical University, Athen, 1966, p. 151. w. G. JAFFE, F. WAGNER, P. MARCANO AND R. HERNANDEZ, Acta Cient. Venezolana, rs (r964) 29. S. KATOH, l. SHIRATONI AND A. TAKAYIMA, j. Biochem., 51 (1962) 32. T. AKAZAWA, K. SAIO AND N. SuGIYAMA, Biochem. Biophys. Res. Commun., 20 (I965) rr4. L. M. SHANNON, E. KAY AND J. Y. LEw,]. Biol. Chem., 241 (1966) 2166. T. MURACHI, A. SUZUKI AND N. TAKAHASHI, Biochemistry, 6 (1967) 3730. E. ToRo-GoYcO, A. MARETZKI AND M. L. MATos, Arch. Biochem. Biophys., r26 (r968) 91. I. E. LIENER, Am.]. Clin. Nutr., II (1962) 28r. D. K. DoUGALL AND K. SHIMBAYASHI, Plant Physiol., 35 (1960) 396. D. T. A. LAMPORT AND D. H. NORTHCOTE, Nature, r88 (1960) 665. N.J. KING AND S. T. BAYLEY,]. Exptl. Botan., 16 (1965) 294. A. PuszTAI AND W. B. WATT, European]. Biochem., 10 (1969) 523. A. PuszTAI, Biochem. ]., 95 (I965) 3C. A. PUSZTAI, Biochem. j., IOI (Ig66) 379. I. KoSHIYAMA, Agr. Biol. Chem., 30 (I966) 646. H. Lis, N. SHARON AND E. KATCHALSKI,]. Biol. Chem., 24I (I966) 684. T. TAKAHASHI AND I. E. LIENER, Biochim. Biophys. Acta, I54 (I968) 560. D. T. A. LAMPORT, Nature, 2I6 (1967) I322. A. PuszTAI, Biochem. ]., 99 (I966) 93·

Biochim. Biophys. Acta, 207 (I970) 4I ~-43I

GLYCOPROTEIN 30 3I 32 33 34 35 36 37 38 39 40 4I 42 43 44 45 46 47 48 49 50 5I 52 53 54 55 56 57 58 59 6o 6I 62 63 64

II

~ROMP.

vulgaris

431

A. PUSZTAI, Biochem. j., 94 (I965) 6II. A. PuszTAI, Arch. Biochem. Biophys., I26 (rg68) 28g. A. C. }E>~NINGS AND W. B. WATT,]. Sci. Food Agr., r8 (I967) 527. T. BITTER AND H. M. MuiR, Anal. Biochem., 4 (rg62) 330. J\1. DuBors, K. A. GILLES, J. K. HAMILToN, P. A. REBERS AND F. SMITH, Anal. Chem., z8 (1956) 35°· M. V. CHESHIRE AND C. M. MUNDIE,]. Soil Sci., 17 (Ig66) 372. E. D. CROWELL A:--ID B. B. BURNETT, Anal. Chem., 39 (rg67) I2I. W. L. BENCZE AND K. SCHMID, Anal. Chem., 29 (1957) II93· M. P. ToMBS AND P. AKROYD, Shandon Instr. Appl., r8 (rg67) r. A. C. ALLISON AND J. H. HUMPHREY, Immunology, 3 (rg6o) 95· D. A. YPHANTIS, Biochemistry, 3 (1964) 297· E. F. CASASSA A:--ID H. EISENBERG, Advan. Protein Chem., Ig (rg64) 287. E. REISLER AND H. EISENBERG, Biochemistry, 8 (1969) 4572. G. A. GILBERT, Proc. Roy. Soc. London, Ser. A, 250 (1959) 377· T. C. :VIclLVAINE,]. Biol. Chem., 49 (Igzi) r83. H. K. ScHACHMAN, in S. P. CoLOWICK A>~"D N. 0. KAPLAN, Methods of Enzymology, Vol. 4, Academic Press, New York, 1955, p. IOI. R. CECIL AND A. G. 0GSTON, Biochem. ]., 44 (I949) 33· R. L. BALDWIN, Biochem. j., 65 (I957) 503. H. K. ScHACHMAN AND S. j. EDELSTEIN, Biochemistry, 5 (rg66) 268r. E. P. K. HADE AND C. TANFORD,]. Am. Chem. Soc., 8g (rg67) 5034. W. W. KIELLEY AND W. F. HARRI:--IGTON, Biochim. Biophys. Acta, 41 (rg6o) 401. M. E. NoELKEN AND s. N. TIMASHEFF, }. Bioi. Chem., 242 (rg67) soSo. L. THELANDER, European}. Biochem., 4 (rg68) 407. S. KATZ, Biochim. Biophys. Acta, 154 (Ig6H) 468. F. j. CASTELLINO A:--ID R. l3ARKER, Biochemistry, 7 (1968) 2207. A. ULLMA>~"N, M. E. GoLDBERG, P. PERRIN AND J. MoNOD, Biochemistry, 7 (rg68) 26r. T. WIELAND, P. DUESBERG AND H. DETERMAN, Biochem. Z., 337 (rg63) 303. H. OLESEN AND P. 0. PEDERSEN, Acta Chem. Scand., 22 (rg68) 1386. P. F. DAVISON, Science, r6r (rg68) go6. J. T. YANG, Advan. Protein Chem., r6 (rg6I) 323. J. :VL CREETH A>~D C. G. KNIGHT, Biochim. Biophys. Acta, roz (I965) 549· J. M. CREETH AND C. G. KNIGHT, Chem. Soc. London Spec. Pub!., 23 (rg68) 303. H. A. SCHERAGA AND L. MANDELKERN, j. Am. Chem. Soc., 75 (1953) I79· H. K. ScHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, New York and London, 1959, p. 236. J. J. RACKIS, A. K. SMITH, G. E. BABCOCK AND H. A. SASAME,]. Am. Chem. Soc., 79 (1957)

4655· 65 P. JoHNSON AND E. M. SHOOTER, Biochim. Biophys. Acta, 5 (rg5o) 36r. 66 A. C. jENNINGs, A. PuszTAI, R. L. :VI. SYNGE AND W. B. WATT,}. Sci. Food A gr., 19 (rg68) 203.

Biochim. Biophys. Acta, 207 (1970) 413-431