Biochimica et Biophysica Acta, 379 (1975) 317-328 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 36945 N A J A M E L . 4 N O L E U C A (FOREST COBRA) VENOM P U R I F I C A T I O N A N D SOME P R O P E R T I E S OF PHOSPHOLIPASES A

FRANCOIS J. JOUBERT and STEPHANUS J. VAN DER WALT National Chemical Research Laboratory, Councilfor Scientific and Industrial Research, P.O. Box 395, Pretoria (South Africa) (Received August 15th, 1974)

SUMMARY Three phospholipases A (Fractions DE-I, DE-II and DE-III) were purified from Naja melanoleuca (Forest cobra) venom by a combination of gel filtration on Sephadex G-50 and chromatography on DEAE-cellulose. The purified phospholipases A were homogeneous by various physicochemical criteria. Whereas Fraction DE-I contains 118 amino acid residues, Fractions DE-II and DE-III comprise 119 residues. The three enzymes are cross-linked by seven disulphide bridges, have asparagine as N-terminal amino acid and the C-terminal is glutamic acid or glutamine. The molecular weights of the three phospholipases A from sedimentation analysis at p H 2.1, also by the sodium dodecylsulphate-gel method and calculated from the amino acid composition, were close to 13 000. Studies of circular dichroism in the spectral region between 195 to 305 nm showed that the three phospholipases A contain similar helical contents but revealed conformational differences between their side-chain chromophores. INTRODUCTION Phospholipase A (EC 3.1.1.4) has been purified from several sources, viz. porcine pancreas [1], bee venom [2, 3] and also various snake venoms [4--17]. Kocholaty et al. [18] found that Ndja melanoleuca snake venom was particularly rich in phospholipase A. It was the highest in phospholipase A acivtity of all Crotalidae, Elapidae and Viperidae species tested. Whereas the purification and the primary structure of a number of neurotoxins and cytotoxin from Naja melanoleuca venom were reported [19-22], little is known about the phospholipase A from this snake venom. The present study is concerned with the purification and some properties of three phospholipases A from N. melanoleuca venom. EXPERIMENTAL PROCEDURE Materials Desiccated Forest cobra (N. melanoleuca) venom was obtained from D. Muller,

318 Professional Snake Catcher (Pty), 215 Barkston Drive, Blairgowrie, Johannesburg. DEAE-cellulose was a microgranular preparation (DE-52) from Whatman. Sephadex G-50 was obtained from Pharmacia. Dipalmitoyl-L-a-lecithin was supplied by Fluka.

Physicochemical methods Sephadex G-50 and DEAE-cellulose columns were prepared as recommended by the manufacturers and the eluates monitored at 280 nm with a Beckman spectrochrom analyzer. Moving boundary electrophoresis was carried out at 1 °C in a Beckman Model H electrophoresis-diffusion apparatus. A barbiturate-NaCl buffer of ionic strength (1) 0.1 and pH 8.6 was employed. Disc electrophoresis at pH 9.4 using a 7.5 ~o gel was performed according to the method of Ornstein and Davis [23]. Sodium dodecylsulphate-gel electrophoresis at pH 7.2 using a 10 ~o gel was carried out as described by Shapiro et al. [24]. The ultracentrifuge data were obtained at 20 °C with a Beckman Model E analytical ultracentrifuge. The measurements of sedimentation coefficients were carried out at 60 000 rev./min. Molecular weights were estimated by the rapid sedimentation equilibrium method of Yphantis [25]. An eight-channel cell with 0.8-mm column heights and ultracentrifuge speed of 16 000-22 000 rev./min were employed. Partial specific volumes were calculated from the amino acid compositions [26]. Diffusion coefficients were determined in the ultracentrifuge at 5600 rev./min using a synthetic boundary cell for boundary formation. Density and viscosity values for guanidinium and urea solutions were taken from Kawahara and Tanford [27], but were measured for the other solutions. Circular dichroism measurements were made with a Jasco J-20 spectropolarimeter. The percentage a-helix was calculated from the equation developed by Greenfield and Fasman [29]: a helix =

n m - - 4000 33 000 -- 4000

[0]208

The units of [0] are degrees.cm 2. dmole-1. Helix and/%structure content were also estimated as described by Bannister et al. [29] (their Eqn 2), using the reference spectra of Chen et al. [30].

Determination of phospholipase A For monitoring of column eluates the turbidimetric method of Marinetti [31] was employed. The effect of pH on phospholipase A was determined with dipalmitoylL-a-lecithin as substrate according to the method of Dennis [32]. For the determination of the positional specificity of phospholipase A the method described by Bores and Viljoen [16] was used.

Reduction and S-carboxymethylation The phospholipases A were reduced with dithiothreitol and S-carboxymethylated with iodoacetate as described [33].

Amino acid analyses Amino acid analyses were performed with an automatic Beckman amino acid

319 analyzer. Samples were hydrolyzed with 6 M HC1 at 110 °C for 24, 48 and 72 h in sealed evacuated tubes; phenol was added to prevent destruction of tyrosine [34]. For the determination of tryptophan the samples were hydrolyzed with 3 M p-toluenesulphonic acid as described by Liu and Chang [35]. Free sulphydryl groups, in 6 M guanidinium chloride, were assayed in intact phospholipase A samples according to Ellman [36].

N-terminal and C-terminal analyses The N-terminal amino acids of reduced and S-carboxymethylated phospholipases A were carried out manually by the Edman procedure as described by Peterson et al. [37]. The C-terminal amino acids of reduced and S-carboxymethylated samples were determined by the tritium labelling technique of Matsuo et al. [38] as modified by Haylett et al. [39]. RESULTS

Purification of phospholipase A Gel filtration of forest cobra venom through Sephadex G-50 in 0.2 M NH4HCO3 solution yielded the elution pattern shown in Fig. 1. Six fractions were evident of which the $3 and $4 fractions exhibited phospholipase A activity. The major Fraction /

I

0.4 E c

o 0.2

o,

,

,

,

:

I

60

:

Fraction No.

t

120

;

Fig. 1. Gel filtration of 2 g crude Naja melanoleuca venom on Sephadex G-50 (3.8 cm × 450 cm) in 50 ml 0.2 M NH4HCO3 (pH 7.8) solution. The flow rate was 100 ml/h, the column temperature 5 °C and the eluate was monitored at 280 nm. The asterisks indicate phospholipase A activity. $3 was directly lyophilized. For further fractionation Fraction $3 was dissolved in 20 ml 0.05 M Tris-HCl buffer (pH 8) and applied to a DEAE-cellulose column. The column was developed with a linear gradient of increasing concentration of NaCI (0-0.2 M) in 0.05 M Tris-HC1 buffer of pH 8. The elution pattern of Fig. 2 reveals two major and two minor fractions which all possess phospholipase A activity. The different phospholipase A fractions, after dialysis against distilled water, were lyophilized. Final purification of the four fractions was achieved by rechromatography on DEAEcellulose columns as above. The elution diagrams each showed a major and one or two minor fractions. The major fractions were recovered and after dialysis against distilled

320

I~-I 0.8

r

~

-

/

( l-"

DE-E

1

DE-IT

~OA

I1

!

\D;-E

J

i

O0

40

80 Fraction No

120

Fig. 2. Chromatography of Fraction $3 on DEAE-ce]lu]ose column (].9 cm × 50 cm). A linear gradient of 2 ] from 0 to 0.2 M NaC] in 0.05 M Tris-HCl buffer (pH 8) was used to elute the column. The flow rate was ]00 m]/h, the column was kept at 5 °C, and the eluate was monitored at 280 nm. The asterisks indicate phospholipase A activity.

water, they were lyophilized. This gave four phospholipase Fractions DE-l, DE-II, DE-Ill and DE-IV, and the yields of the fractions were, respectively, 0.9, 1.1, 3.2 and 0.6 ~.

Purity of the phospholipase A fractions The purity of the four phospholipase A fractions was ascertained by several methods. In the ultracentrifuge a single symmetrical sedimentation peak was obtained for the four fractions. Moreover each fraction gave a single band by sodium dodecylsulphate-gel electrophoresis. Moving boundary electrophoresis of Fractions DE-I, DE-II, and DE-III in barbiturate buffer (I -- 0.1, pH 8.6) revealed well-defined single peaks for both descending and ascending boundaries but for Fraction DE-IV two distinct peaks were observed. Disc electrophoreiss at pH 9.5 showed a single band for Fractions DE-I, DE-II, and DE-Ill but two bands for Fraction DE-IV. No further purification of Fraction DE-IV was attempted. Some properties of phospholipase A Fractions DE-l, DE-H and DE-Ill The amino acid composition of the three phospholipases A are given in Table I. Whereas Fraction DE-I contains 118 amino acid residues, Fraction DE-II and DE-Ill both comprise 119 amino acid residues. They have asparagine as N-terminal amino acid. The tritium labelling method gave glutamic acid as C-terminal amino acid for the three phospholipases A. No free sulphydryl could be demonstrated in the three fractions. The pH dependence of the sedimentation coefficients of phospholipase A Fraction (DE-III) at a fixed concentration (1 70) is shown in Fig. 3. In each experiment a single peak was found for the enzyme during sedimentation runs. The sedimentation coefficients decreased significantly at low pH and also at high pH but they were reasonably constant from pH 4.5 to 8. The change in sedimentation coefficients was influenced by the ionic strength of the buffers.

321 TABLE I AMINO ACID COMPOSITION OF PHOSPHOLIPASE A FRACTION DE-I, DE-II, AND DE-III Samples were hydrolyzed for 24, 48 and 72 h. Values are given as moles of residue per mole. Amino acid

Moles of residue per mole

Lys His Arg Asp Thr* Ser* Glu Pro Gly Ala (~ys**" Val*** Met Ile*** Leu Tyr Phe Trp Free sulphydryl groups Total N-terminal C-terminal

Fraction DE-III

Fraction DE-II

Fraction DE-I

4.0 (4) 2.9 (3) 5.9 (6) 19.5 (20) 5.5 (6) 5.6 (6) 6.1 (6) 3.7 (4) 8.9 (9) 10.7 (11) 13.9 (14) 3.9 (4) 0.9 (1) 5.5 (6) 3.0 (3) 8.7 (9) 3.9 (4) 2.3 (3) 0 119 Asp Glu

5.1 (5) 2.0 (2) 6.0 (6) 19.7 (20) 6.9 (7) 5.9 (6) 8.1 (8) 3.1 (3) 9.0 (9) 10.1 (10) 14.2 (14) 4.0 (4) 0.9 (1) 5.1 (5) 3.0 (3) 8.7 (9) 3.9 (4) 3.1 (3) 0 119 Asp Glu

8.0 (8) 2.9 (3) 6.0 (6) 18.5 (19) 6.6 (7) 3.1 (3) 6.9 (7) 4.2 (4) 8.9 (9) 8.6 (9) 13.7 (14) 3.9 (4) 0.8 (1) 4.9 (5) 3.2 (3) 8.5 (9) 3.8 (4) 3.1 (3) 0 118 Asp Glu

* Extrapolated to zero hydrolysis time. ** Determined as S-carboxymethylcysteine. *** After 72-h hydrolysis.

g

[

l

I

I

I

I

I

I

I

i

l

I

I

I

I

I

I

2.6 2.4

~> ~2.0 to

2

4

I

I

I

6

OH

8

10

Fig. 3. Sedimentation coefficients of Naja melanoleuca phospholipase A Fraction DE-III in various buffers at a fixed concentration (1 g per 100ml). Buffer: (a) phosphate--citric acid, I = 0.I; (b) phosphate-citric acid, I = 0.2; (c) formic acid-NaOH, I = 0.1 ; (d) Tris-HCl, I = 0.1 ; (e) glycineNaOH, I = 0.1. S o m e p h y s i c o c h e m i c a l p r o p e r t i e s o f p h o s p h o l i p a s e A, F r a c t i o n D E - I , D E - I I a n d D E - I I I are s u m m a r i z e d i n T a b l e II. W h i l s t n e g a t i v e v a l u e s were o b t a i n e d for t h e e l e c t r o p h o r e t i c m o b i l i t i e s o f F r a c t i o n D E - I I a n d D E - I I I , a positive v a l u e was a p p a r e n t for F r a c t i o n D E - I . T h e s e d i m e n t a t i o n coefficients a n d t h e m o l e c u l a r weights ( r a p i d

322

c5c5o

-

0

0

g

g

g~

"~" ¢,~

0", ~-

0o ~ t'q oh

Z


~EE

323 TABLE III CONCENTRATION DEPENDENCE OF THE SEDIMENTATION COEFFICIENTS ($20.~) AND MOLECULAR WEIGHTS (RAPID SEDIMENTATION EQUILIBRIUM) OF PHOSPHOLIPASE A (FRACTION DE-III) Concn (g/100 ml)

s~0,w (S) (I = 0.3, pH 7.2)

Mol. wt I = 0.3, pH 7.2

-I = 0.1, pH 2.1

1.00 0.78 0.50 0.39

2.78 2.79 2.78 2.81

25 900 25 900 26 500 27 200

14 900 14 700 14 800 14 300

sedimentation equilibrium) of the three phospholipases A in the various buffers were slightly dependent on concentration. Table III shows typical values. The values reported in Table II were obtained by extrapolation to zero concentration. It was quite obvious that for Fraction D E - I l l the sedimentation coefficients and diffusion coefficients were greatly influenced by the pH. The molecular weight of Fraction D E - I I I calculated from sedimentation analysis was of the order of 26 000 in the p H range 7.0-7.6. In the presence of 6 M guanidinium chloride, 2 M urea and 0.005 M CaCI2, however, the molecular weight was diminished. At p H 2.1 a much smaller molecular weight for Fraction D E - I l l was found. This value which was in the order of 14 000 was in accord with the molecular weights obtained from the sodium dodecylsulphate-gel method and calculated from the amino acid composition. For phospholipase A Fraction DE-I, and DE-II, sedimentation coefficients and diffusion coefficients were determined at p H 2.1; the values obtained were in agreement with values for Fraction DE-Ill. The molecular weights calculated for Fraction DE-I and DE-1I by several methods were also in the vicinity of 13 000.

Specificity of the phospholipases A The specificity of the phospholipase A Fractions DE-I, D E - I I and D E - I l l was established by investigation of their action on egg lecithin. It was apparent that predominantly unsaturated fatty acids were released by all three fractions. The three enzymes are, therefore, of the phospholipase A2 type.

Effect of pH The effect of p H on the phospholipase A activity was examined using dipalmitoyl-L-a-lecithin as substrate. As can be seen in Fig. 4 phospholipase A Fraction D E - I I and D E - I l l exhibited a broad p H optimum with optimum activity being found at p H 8.5 to 9. As illustrated in Fig. 4, the activity of Fraction DE-I was not markedly affected by p H and a very broad optimum was found at p H 7.5-8.5. It was, however, quite apparent that in the p H range 6.5-9.0 the specific activity of phospholipase A Fraction DE-I was significantly lower than of Fractions D E - I I or DE-Ill.

Circular dichroism spectra As shown in Fig. 5 the circular dichroism spectra from 200 to 245 nm of phospholipase A Fractions DE-l, D E - I I and D E - I l l are very similar. The spectra showed

324 E

g .c_ E 5.0

t~

_o

o

(a)

2.5

J

to

--~ 0

:~

t

t

6

I

7

8

pH

9

Fig. 4. The effect o f p H on the activity o f Naja melanoleuca phospholipase A (a) Fraction DE-I, (b) Fraction D E - I I and (c) Fraction DE-III. The assay enzyme system contained 1 m M dipalmitoyl-L-alecithin, 2 m M Triton X-100, 20 m M CaC12 1 m M E D T A and 0.02fig phospholipase A in a volume 4 ml and the temperature was 40 °C.

two negative maxima in the region of 210 and 223 nm. The [0] values at the maxima for the three phospholipases A are given in Table IV. The [0] values at 208 nm and the helical content computed from the [0120s ,m values are also listed in Table IV. The three phospholipases A have similar helical contents. The amount of a-helix calculated by the method of Bannister et al. [29] was somewhat higher, viz. 28, 27 and 22 ~o, respectively. The percentage of fl-structure found was --8, --2.5 and 4 ~ . The negative values found, show that the reference

18

12

6

0

I

I

2~)0

J

I

I

I

-6

-12

2,20

I Anm

2~.0

Fig. 5. Circular dichroism spectra o f phospholipases A: (a) Fraction DE-I at p H 6, (b) Fraction D E II at p H 6 and (c) Fraction DE-III at p H 5 from 195 to 245 nm.

325 TABLE IV THE RESULTS OF THE CIRCULAR DICHROISM ON PHOSPHOLIPASE A FRACTION DE-I, DE-II AND DE-III [0] values are given in degree.cm2.dmole -1. Phospholipase A Fraction DE-I at pH 6 Fraction DE-II at pH 6 Fraction DE-III at pH 5

Maximum ~, (nm)

[0] value

Maximum ~ (nm)

[0] value

[0]208.m value

a-helix

211 210 208

--9 400 --10 000 --9 000

221 222 222

10 900 10 600 9 300

8600 9600 9000

15.9 19.3 17.2

%

spectra do not form a true basis for the phospholipase A. However, they do indicate that there is little or no fl-structure present. The spectra of the three enzymes in the region of 250 to 300 nm, however, differed markedly as shown in Fig. 6. In this region the spectra exhibited strong negative bands with quite different [0] values. These results suggest that the three phospholipases A differ in the conformation of their side-chain chromophores, probably owing to the tyrosyl and tryptophyl residues and the disulphide cross-links. i

I

I

I

I

I

i

"--"- (c)

50 Ce]

too

150 I

I

3. n m

2&o

I

Fig. 6. Circular dichroism spectra of phospholipases A: (a) Fraction DE-I at pH 6; (b) Fraction DEII at pH 6; (c) Fraction DE-III at pH 5 from 250 to 290 nm. DISCUSSION The isolation procedure for three phospholipases A from N. melanoleuca was relatively simple. Utilizing a two-step purification four phospholipase A fractions were prepared. On the basis of their monodisperse behaviour during sedimentation velocity, free boundary electrophoresis, polyacrylamide-gel electrophoresis and sodium

326 dodecylsulphate-gel electrophoresis three out of the four fractions (DE-l, DE-II and DE-III) were regarded as being homogeneous. Furthermore, the near integral values for amino acid residues of the three enzymes also indicate homogeneity. The three phospholipases A are particularly rich in aspartic acid and the corresponding amides, and also in alanine, tyrosine and glycine. In contrast they all contain a single methionine residue. The three fractions comprise 14 half-cystine residues and since no free sulphydryl groups could be detected, they are cross-linked by seven disulphide bridges. Furthermore, the number of arginine, glycine, leucine, tyrosine, phenylalanine and tryptophan residues are invariant for the three phospholipases A. A high proportion of the amino acids for the three enzymes differ by not more than one or two residues. On the basis of their similar amino acid compositions and the fact that the three phospholipases A each have the same N-terminal and the same Cterminal amino acid, they can be regarded as homologous enzymes. However, the electrophoretic mobilities of the fractions were different, and revealed that Fractions DE-II and DE-Ill are negatively charged at pH 8.6 whilst Fraction DE-I is positively charged at this pH. It is thus apparent that the isoelectric points of Fractions DE-1I and DE-III are below pH 8.6 and that of Fraction I is above pH 8.6. This contrast suggests a distinct difference in their amino acid compositions especially of acidic and basic amino acids. The amino acid compositions of phospholipase A, Fractions DE-I, DE-11 and DE-Ill from N. melanoleuca venom were compared to that ofphospholipase A purified from several sources, i.e. the venom from Hemachatus haemachatus [40], Naja nigricollis [10], A. halys blomhofJ7i [12], Bitis gabonica [16], Bitis arietans [17], Vipera palestinae [15], and Laticauda semifasciata [9] and porcine pancreas [1]. The comparison was limited to phospholipases A which contain 108-140 amino acid residues. High contents of aspartic acid, half-cystine, glycine and tyrosine are common features of all phospholipases A. The phospholipases A contain 12-14 half-cystine residues and consequently their cross-linking must be very similar. The enzymes are deficient in histidine, methionine and tryptophan. The number of basic amino acid residues reveals an interesting trend whereas the number of histidine residues ranged from 1 to 3 and the arginine residues from 3 to 6, the number of lysines of various phospholipases A is quite variable and ranged from 4 to 17. Molecular weights were reported in the literature for phospholipases A isolated from porcine pancreas [1], two bee venoms [2, 3] and various snake venoms [4-17, 41]. One striking feature is the range of the molecular weights which have been reported. For various phospholipases A the gel filtration and sodium dodecylsulphate-gel methods revealed predominantly a smaller molecular weight than obtained by sedimentation analysis. Wells and Hanahan [7] purified two phospholipases Az from Crotalus adamenteus venom, both having identical amino acid composition and molecular weight by sedimentation analysis of 30 000. Later, Wells [41] showed that these phospholipases A 2 are dimers of identical subunits of molecular weight, 15 000 by gel filtration in 6 M guanidinium chloride containing 0.1 M mercaptoethanol. The molecular weights of the three phospholipases A from N. melanoleuca were calculated from sedimentation and diffusion data, the rapid sedimentation equilibrium method, sodium dodecylsulphate-gel method and the amino acid composition. The molecular weights ranged from 12 800 to 27 600. Ultracentrifugal studies at pH 7.0-7.6 revealed the higher molecular weight. This value is almost double the molec-

327 ular weight estimated by s e d i m e n t a t i o n analysis at p H 2.1, sodium d o d e c y l s u l p h a t e gel m e t h o d or calculated f r o m the a m i n o acid c o m p o s i t i o n . It would appear that the lower molecular weight of 13 400 represents the m o n o m e r of the p h o s p h o l i p a s e molecule a n d the higher value of 26 800 represents the dimer. I n the presence of, for example, urea, g u a n i d i n i u m chloride or Ca 2÷ molecular weights between these extremes were found. A d y n a m i c e q u i l i b r i u m seems to exist between m o n o m e r a n d dimer which can be shifted towards either of the forms d e p e n d i n g o n c o n d i t i o n s such as ionic strength a n d pH. ACKNOWLEDGEMENTS The authors are greatly indebted to D r D. P. Botes for valuable discussions, to M r J. N. Taljaard for his skilful assistance with experimental work, a n d to M r L. M. d u Plessis a n d Mrs S. Vladar for fatty acid analyses.

REFERENCES 1 De Haas, G. H., Postema, N. M., Nieuwenhuizen, W. and Van Deenen, L. L. M. (1968) Biochim. Biophys. Acta 159, 103-117 2 Munjal, D. and Elliott, W. B. (1971) Toxicon 9, 403-409 3 Shipolini, R. A., Callewaert, G. L., Gottrell, R. C., Doonan, S., Vernon, C. A. and Banks, B. E. C. (1971) Eur. J. Biochem. 20, 459-468 4 Currie, B. T., Oakley, D. E. and Broomfield, C. A. (1968) Nature 220, 371 5 Wu, T-W. and Tinker, D. O. (1969) Biochemistry 8, 1558-1568 6 Hachimori, Y., Wells, M. A. and Hanahan, D. J. (1971) Biochemistry 10, 4084-4089 7 Wells, M. A. and Hanahan, D. J. (1969) Biochemistry 8, 414-424 8 Augustyn, J. M. and Elliot, W. B. (1970) Biochim. Biophys. Acta 206, 98-108 9 Tu, A. T., Passey, R. B. and Toom, P. M. (1970) Arch. Biochem. Biophys. 140, 96-106 10 Wahlstr6m, A. (1971) Toxicon 9, 45-56 11 Salach, J. I., Turini, P., Seng, R., Hauber, J. and Singer, P. (1971) J. Biol. Chem., 246, 331-339 12 Kawauchi, S., Iwanaga, S., Samejima, Y. and Suzuki, T. (1971) Biochim. Biophys. Acta 236, 142160 13 Lo, T-B., Chang, W-C. and Chang, C-S. (1972) T'ai-Wan I Hsueh Hui Tsa Chih 71,318-322 14 Vidal, J. C., Cattaneo, P. and Stoppani, A. O. M. (1972) Arch. Biochem. Biophys. 151, 168-179 15 Shiloah, J., Klibansky, C., De Vries, A. and Berger, A. (1973) J. Lipid Res. 14, 267-278 16 Botes, D. P. and Viljoen, C. C. (1974) Toxicon, in the press 17 Howard, N. L. (1974) Toxicon, in the press 18 Kocholaty, W. F., Lepford, E. B., Daly, J. G. and Billings, T. A. (1971) Toxicon 9, 131-138 19 Botes, D. P. (1972) J. Biol. Chem. 247, 2866-2871 20 Carlsson, F. H. H. and Joubert, F. J. (1974) Biochim. Biophys. Acta 336, 473-469 21 Shipolini, R. A., Bailey, S. G. and Banks, B. R. E. (1974) Eur. J. Biochem. 42, 203-211 22 Poilleux, G. and Boquet, P. (1972) C.R. Hebd. Sc. Acad. Sci. Paris, Ser. D 274, 1953-1956 23 Ornstein, L. and Davis, B. J. (1962) Disc Electrophoresis, preprinted by Distillation Products Industries, Rochester, New York 24 Shapiro, A. L. Vinuela, E. and Maizel, J. V. (1967) Biochem. Biophys. Res. Commun. 28, 815-820 25 Yphantis, D. A. (1960) Ann. N.Y. Acad. Sci. 88, 586-601 26 Cohn, E. J. and Edsall, J. T. (1943) Proteins Amino acids and Peptides, p. 370, Rheinhold Publishing Corp., New York 27 Kawahara, K. and Tanford, C. (1966) J. Biol. Chem. 241, 3228-3232 28 Greenfield, N. and Fasman, G. D. (1969) Biochem. 8, 4108-4116 29 Bannister, W. H., Bannister, J. V., Camilleri, P. and Ganado, A. L. (1973) Int. J. Biochem. 4, 365-371 30 Chen, Y-H., Yang, J. T. and Martinez, H. (1972) Biochemistry 11, 4120-4131

328 31 32 33 34 35 36 37 38 39 40 41

Marinetti, G. V. (1965) Biochim. Biophys. Acta 98, 554-565 Dennis, E. A. (1973) J. Lipid Res. 14, 152-159 O'Donnell, I. J., Frangione, B. and Porter, K. R. (1970) Biochem. J. 116, 261-268 Sanger, F. and Thompson, E. O. P. (1963) Biochim. Biophys. Acta 71, 468-471 Liu, T. Y. and Chang, Y. H. (1971) J. Biol. Chem. 246, 2842-2848 Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 Peterson, D. P., Nehrlich, S., Oyer, P. E. and Steiner, D. F. 0972) J. Biol. Chem. 247, 4866-4871 Matsuo, H., Fujimoto, Y. and Tatsumo, T. (1966) Biochem. Biophys. Res. Commun. 22, 69-74 Haylett, T., Swart, L. S. and Parris, D. (1971) Biochem. J. 123, 191-200 Joubert, F. J. (1975) Eur. J. Biochem, in the press Wells, M. A. (1971) Biochemistry 10, 4074-4078