Further purification of sheep plasma erythropoietin

BIOCHIMICA ET BIOPHYSICA ACTA BBA

487

3753

FURTHER

PURIFICATION

OF SHEEP

PLASMA E R Y T H R O P O I E T I N

E. G O L D W A S S E R , W. F. W H I T E * AND I~. B. T A Y L O R * *

Argonne Cancer Research Hospital***, and the Department of Biochemistry, the University of Chicago, and Research Department, Armour and Co., Chicago, Ill. (U.S.A.) (Received April 25th, 1962)

SUMMARY

A method of partial purification of sheep plasma erythropoietin by ion-exchange chromatography, ammonium sulfate precipitation and batch adsorption is described. The most active fraction obtained has an activity of 450 units/mg protein and represents a purification factor of about 64000 over the starting plasma. It has the highest activity yet reported for any preparation. Antiserum to a less purified erythropoietin preparation (3 units/mg protein) does not precipitate or inactivate the hormone but does react with impurities. Some chemical properties of the most active fraction are described.

INTRODUCTION

In the past few years there has been a considerable revival of interest in erythropoietin, the hormone which stimulates the formation of red blood cells. The introduction of a relatively short assay method 1 has made it feasible to attempt purification of this hormone, which in a purified state might be used for the treatment of some anemias, and will be of great value in studies of the early stages of red cell differentiation. Although much evidence points to the kidney as the site of formation of erythropoietin 2, 3, kidney extracts have only feeble erythropoietic activity, and plasma from anemic animals has been our source of the hormone. Recently there have been some studies on urinary erythropoietin, especially of human origin 4,5, but as yet there is no body of evidence relating the properties of plasma and urinary hormones. The partial purification of rabbit plasma erythropoietin, which in general seems to resemble the sheep hormone, has been described6, 7. In previous communications from these laboratories we have reported on the partial purification of sheep plasma erythropoietin and on some properties of these preparationsS, 9. This report is concerned with further progress in the purification of this hormone. A preliminary account of this work has been reported 1°. * P r e s e n t a d d r e s s : A b b o t t Laboratories, N o r t h Chicago, Ill. (U.S.A.). ** P r e s e n t a d d r e s s : Radcliffe I n f i r m a r y , Oxford U n i v e r s i t y , . Oxford (Great Britain). *** O p e r a t e d b y t h e U n i v e r s i t y of Chicago for t h e U n i t e d States A t o m i c E n e r g y C o m m i s s i o n .

Biochim. Biophys. Acta, 64 (1962) 487-496

488

E. GOLDWASSER, W F. WHITE, K. B. TAYLOR MATERIALS AND METHODS

The production of plasma from phenylhydrazine-treated sheep and several stages of its purification using ion-exchange methods have already been described s. The four-day assay method, based on measurement of incorporation of 5"Fe into newly formed red cells in the fasted rat was used as previously described1, s with one modification. Sprague-Dawley male rats weighing between 165 and 195 g before the start of the fasting period were selected for assay purposes. One unit of erythropoietic activity has been defined as that amount of material which, when injected into rats under the standardized conditions of assay, will give rise to an increment of iron incorporation (over control values) equal to that given by 5 tzmoles of CoClz administered under the same conditions s, n. We have chosen to retain this definition of a unit of activity rather than adopt the one proposed recently 12 which is based on a fixed percentage of incorporation, since we feel that the latter fails to allow for the variable sensitivity from assay to assay. Each assay included a standardized preparation or cobaltous chloride at 2 or more dose levels in order to evaluate activity in units and interpolation was done from a plot of increment of per cent incorporation of 59Fe vs. log of dose. DEAE-cellulose was obtained from the Brown Paper Co., XE-97 resin from Rohm and Haas, Bio-Rex 7° from Bio-Rad Laboratories, 5"FeCI3 from Abbott Laboratories and complete Freund adjuvant from Difco Laboratories. Hydroxylapatite was prepared according to the method of TISELIUS13 and calcium phosphate gel by the method of TsuBoI AND HUDSON14. The methods of both LOWRY1~ and WESTLEY AND LAMBETH1~ were used for protein determinations, crystalline bovine serum albumin (Armour) serving as a standard. Sialic acid determinations were done by using MAUZERALLAND GRANICK'S modification 17 of the direct Ehrlich's reaction ts. This method was first standardized using a sample of human orosomncoid (II % sialic acid) kindly supplied by Dr. R. WINZLER of the University of Illinois School of Medicine, after which a crude sample of sheep orosomucoid (8.7 % sialic acid) was used as a standard. Total carbohydrate was determined with the carbazole reagent 19 using an equimolar mixture of glucose, fructose, ribose, galactose and mannose as a standard. Hexosamine was determined by the method ot BLIXz°. Gel electrophoresis was essentially as described by SMITHIES*~ but on a smaller scale and using polyacrylamide22 as the supporting medium. 1.2 ml of IO % dimethylaminoproprionitrile and 1.2 ml of IO % ammonium persulfate were added to ioo ml of filtered 5 % cyanogum 41. in o.o81 M Tris, o.oo3 M EDTA and o.o12 M boric acid at pH 8. 7. The solution was mixed and poured into plastic troughs 88 mm × 15 mm × 4-5 mm, covered with a strip of Parafilm and allowed to stand until the gel was firm (i h). About 2o ~g of the sample in the same buffer was applied to a piece of Whatman No. 40 filter paper (about 2 × 6 mm), held on a piece of razor blade and inserted into a slit about 3o mm from one end of the gel. The bottom surface of the trough was immersed in an ice bath throughout this procedure and the subsequent electrophoresis. After the sample was in place, about 21 mm of gel were cut off each end leaving a 46-mm strip of gel and two wells in the * C y a n o g u m 41 is a m i x t u r e of a c r y l a m i d e a n d N,N'-methylene-bis a c r y l a m i d e m a d e b y A m e r i c a n C y a n a m i d Co. a n d sold b y E. C. Corporation. t

Biochirn. Biophys. Acta, 64 (I96z) 487-496

SHEEP PLASMA ERYTHROPOIETIN

489

trough. The wells were filled with pH 9.2 buffer IO times as concentrated as that used to prepare the gel, platinum wire electrodes were clipped in place at the ends, and 25 mA were applied to the gel (at constant current) for 15 rain. After this time, the gel strip was immersed in 1% Buffalo black in acetic acid - methanol - water (I : 5 : 5, v/v/v) for 2 rain. The gel was then washed free of stain with several changes of the same solvent mixture. The somewhat dehydrated gel was next immersed in 7.5 % acetic acid-3o % glycerol until it had regained its original transparency at which time photographs were made. Rabbits were immunized against partially purified erythropoietin (Step Ill) as follows: 50 mg of the erythropoietin concentrate was dissolved in 2.5 ml of o.15 M NaC1; 7.5 ml of complete Freund adjuvant was added and the mixture emulsified for 5 rain by ejection from a Io-ml syringe through a wide-bore cannula. The emulsified antigens were given in doses of 5 ° mg per rabbit by multiple simultaneous intramuscular and subcutaneous injections in the dorsal region between the scapulae. Each rabbit received 2 sets of injections 12 days apart. The antisera were collected 30 days after the second injection. A similar procedure (but eliminating the second set of injections) was used with guinea pigs for the preparation of anti-sheep al-glycoprotein. Immuno-diffusion experiments were done on microscope slides using 1% Ionagar (Consolidated Laboratories) containing 1% sodium azide. After allowing time for diffusion, the agar gels were placed into o.15 M NaC1 in the cold overnight to remove the unprecipitated proteins. The washed gels were then stained with the same dye used for the polyacrylamide gels, washed free of dye with methanol-acetic a c i d water (5:1:5, v/v/v) and allowed to dry in air to a very thin, tough film, tightly adhering to the glass slide. The slides were then placed in a photographic enlarger and negative prints were made of the stained precipitin bands.

Preparation of starting material The modified large scale production of erythropoietin concentrates by the pilot plant of the Armour Pharmaceutical Co. is carried out according to the following brief outline. The process differs in some respects from the three-step method previously published 8, and the authors are indebted to Dr. R. KUTZ of the Armour Pharmaceutical Co. for details of these procedures*. 5° 1 of the chilled plasma obtained from phenylhydrazine-treated sheep was brought to pH 4.5 and dialyzed against 5 volumes of cold water for 12 h. 750 g oi DEAE-cellulose, washed to remove fines and equilibrated with 0.0375 M NAC10.025 M NaH2PO , (pH4.5), was added to the plasma, mixed for 9 ° rain, the D E A E cellulose collected on a Biichner funnel, and washed with the same buffer until the A 2s0 of the wash was less than o. 2. Elution was done with o.5 M NaC1-o. I M Na 2HPO4 until the A is0 was less than 0.2; the eluate was dialyzed against water for 48 h and lyophilized. This fraction is called Step I. The potency of Step I is usually about 0. 5 to 1.o unit/mg of protein and the recovery of activity about 60-65 %. The Step I fraction was dissolved in IO volumes of 0.02 M NaH2PO4-o.I8 M NaC1 buffer (pH 6.0) and added to a 15 × 60 cm column of washed XE-97 resin equilibrated at pH 6.0. The input to the column was followed by the same buffer until the effluent * The large scale production of erythropoietin concentrates was done under Contract H-5393-C1 between the U.S. Public Health Service and the University of Chicago.

Biochim. Biophys. Acta, 64 (I962) 487-496

490

E. G ( ) L I ) W A S S E I G \¥. F. W H I T E ,

K. B. TAYIA)R

A 2s0 was less than o.2. The total effluent, usually 0--9 1, contains the erythropoietin fraction termed Step II. This fraction is no longer accumulated as such and therefl)re no characterization of the pilot plant material is available. This procedure used to obtain Step II, as indicated in a previous paper s, serves to remove much of the pyrogenic material contaminating Step I. Step I I effluent was immediately brought to p H 5.o and run through a smaller (7.5 x 45 cm) column of XE-97 equilibrated at p H 5.o. At this stage the active hormone is adsorbed b y the resin. Elution is carried out as a batch process by raising the p H to 6.o5 with 5 N NaOH using an autotitrator over about a I-h period. This eluate is dialyzed, lyophilized and is termed Step I I I . Step I I I potency is usually in the range 2-4 units/mg protein. A comparison of Step I and Step I I I fractions by gel electrophoresis is seen in Fig. I. Although in moving boundary electrophoresis Step I I I erythropoietin appears to be homogeneous at p H 8.o as shown in earlier work s, the higher resolving power of the gel method makes clear the heterogeneity of this fraction. Typical results of the combined ion-exchange purification methods are presented in Table I which shows an overall purification of about 4oo-fold by these methods, with a recovery of about 35 % of the starting activity. RESULTS AND DISCUSSION

When used to immunize rabbits, the Step I I I erythropoietin fraction proved to be antigenic, giving rise to easily detectable precipitins (see Fig. 2). Neither precipitating nor neutralizing antibodies toward the active erythropoietin were demonstrated, however. When 1.5 ml of antiserum and IO units of the Step I n fraction were mixed under conditions allowing precipitation of the a n t i g e n - a n t i b o d y complex, the full activity was recovered in the supernatant fraction (Table II). The table includes a control experiment done with normal serltm since a small precipitate was observed TABLE

I

RESULTS OF TYPICAL LARGE SCALE PURIFICATION OF 5 ° 1 OF ANEMIC-SHEEP PLASMA Fraction

Original plasma Step I Step III

Yield (g) 4000 22. 5 3.3

Units]rag protein

Recovery (%)

0.007 o.8 3 .0

TABLE

ioo 64 35

Purification

-112 × 43 ° ×

II

TEST OF RABBIT ANTI-STEP UI SERUM P r e c i p i t a t e s w e r e s u s p e n d e d in o.15 M NaC1. All i n j e c t i o n s f o r a s s a y w e r e d o n e s u b c u t a n e o u s l y .

Precipitate Supernatant

A nti-serum (units)

Normal serum (units)

o.5 6.5

o.o 7.o

B i o c h i m . B i o p h y s . A c t a , 64 (1962) 4 8 7 - 4 9 ()

SHEEP PLASMA ERYTHROPOIETIN

491

when Step I I I and normal serum were mixed. In other experiments with widely differing ratios of antiserum to antigen the activity was always recovered almost completely in the supernatant, even when there was maximal precipitin formed. While these data do not rule out the possibility that a soluble complex did form which was dissociated upon iniection into the assay animals, further studies with the agar-gel immuno-diffusion technique demonstrated a vanishingly small amount of precipitin band formation with more highly purified fractions and the antiserum.

A.

I2ff

B.

111

III

AI

C.

L.

.

.

.

Fig. i. Gel electrophoresis of sheep p l a s m a erythropoietin fractions. Arrows indicate s t a r t i n g positions. The n u m b e r s assigned to the various b a n d s are a r b i t r a r y designations. All b a n d s with the same n u m b e r have been s h o w n to have the s a m e m i g r a t o r y rate.

~2

8

Fig. 2. I m m u n o - d i f f u s i o n of e r y t h r o p o i e t i n fractions. I n each case the a n t i s e r u m is in the central well and test s u b s t a n c e s are in peripheral wells.

The anti-Step I I I serum was used as a qualitative indicator of non-erythropoietin, antigenic materials b y the immuno-diffusion technique. A comparison of such analyses of the various fractions will be discussed below. Preliminary experiments using partially purified antibody as a means of removing impurities have not yet yielded any fraction with increased specific activity. Ammonium sulfate fractionation was carried out in the cold on Step I I I at a concentration of about 2o mg/ml. At o.53 saturation with ammonium sulfate at p H 6.3 the precipitate had low activity; when the ammonium sulfate concentration was brought to o.67 saturated at p H 3.3, a second precipitate formed which contained about one-half the starting activity while the supernatant contained only a small fraction of the activity. The results of such a fractionation are summarized in Table I I I . The o.67 saturated precipitate obtained by this method we have termed Step IV. The overall purification factor at this stage is 43oo with a recovery of about 17 % Biochim. Biophys. Acla, 64 (1962) 487-496

492

E. GOI.I)WASSI~I,:, W. F. WHITE, K. B. "I'AYI.()I,~

of the original activity. Comparisons of Steps I, I I I and IV by gel (qe<'trophoresis and by immuno-diffusion are shown in Figs. I and 2 A and B. When the fractions were examined by gel electrophoresis (Fig. I) we could see that the ammonium sulfate procedure resulted in a lowered amount of component No. i, an increase in No. 2 and the appearance of a component (No. 2a) not previously seen. From data presented below it will be clear that neither component (2 nor 2a) seems to be erythropoietin but that they appear to be only contaminants concentrated by the same procedures as the active hormone. TABLE IIi AMMONIUM

SULFATE

FRACTIONATION

Fraction

Step I i I o.53 s a t u r a t i o n p H 6.3 precipitate o.67 s a t u r a t i o n p H 3.3 precipitate Supernatant

OF STEP

III

ERYTHROPOIETIN

Weight (g)

Units/~ g protein

Recovery of activity (% ;

6.o9

8. 9

IOO

"2,.10

4"4

0.89

3o I Overall recovery

17

49 [.5 8t

Immuno-diffusion revealed several antigenic materials still present in Step IV, despite the three- to four-fold increase in specific activity accompanying the loss of about 95 % of the protein of the Step I I I fraction. in our previous work 8 we had shown that Step I I I erythropoietin when chromatographed on XE-97 according to the method of SCH51ID et al. ~, gave rise to a large amount of an inert el-acidic glycoprotein which seemed to be sheep orosomucoid, and a smaller active fraction which was eluted from the column with the a2-glycoproteins and which we termed fraction 4 B. We had also demonstrated that fraction 4B was free of orosomucoid, but since the large scale method of purification described above is different from the earlier published method, this question was reexamined. The guinea pig anti-sheep ~l-glycoprotein serum was used as an indicator in immunodiffusion and we found no indication of any precipitin band with the Step IV fraction under conditions where there was a band visible with the sheep orosomucoid. Chromatography of Step IV on hydroxylapatite was carried out with the results shown in Fig. 3. Of the 6 peaks obtained only the unadsorbed fraction (A) had erythropoietin at a higher specific activity than the starting material. This fraction usually contained about 5-IO % of the original protein and 3o-5o % of its activity. When the A fraction was divided into two roughly equal parts, the leading half of the peak (AI) showed a significantly higher specific activity (I5o units/rag protein) than did the trailing half (A2) (46 units/mg protein). The reason for this could be clearly seen when the two A fractions were compared with each other and with the B fraction from the same column by immuno-diffusion (Fig. 2c). It is evident that some of the material represented by fraction B is present in A2, while AI seems to be devoid of any antigenic material. Since supplies of the Step I I I and Step IV fractions were severely limited we were forced to use the less active fractions derived from the ammonium sulfate process Biochim. Biophys. Acta, 64 (1962) 487 496

SHEEP

PLASMA

493

ERYTHROPOIETIN

for further work*. In order to arrive at a potency approximately the same as that of Step IV, we subjected the supernatant fraction, which has a potency of about I unit/mg protein, to chromatography on the cation-exchange resin Bio-Rex 7 o, with the results seen in Fig. 4. Contrary to expectations, the active fraction came off the column in the input buffer, somewhat retarded from the main protein peak. When the resin XE-97 (IRe-5 o) (which is very similar to Bio-Rex 7o) was used for chroma~a,o,~ea~,JNca.~.

o, m

ooos

,.*l

10 I I

I

oool~

P~os,~AT~ ,.*0

A,,,,,

t i

c IO

2o

50

40

30

60 70 80 90 FRACTION NUMBER

I00

q

Fig. 3. H y d r o x y l a p a t i t e c h r o m a t o g r a p h y of Step IV. C h r o m a t o g r a p h y of Step I V erythropoietin on h y d r o x y l a p a t i t e . Vertical broken lines indicate buffer changes. Horizontal a r r o w s indicate fractions pooled for assay.

A2so 0900

0.05 M CITRATE pH 5 . 2

B/o-Rex

70 Colum.

2 6 cm • ~ . g cm Dla.

0.800 0700

80 ~

]

0.1 M pHCITRATEs.95

70

0.600

6.0

0500

50

0300

I

I

30

I I

1

I

20

0200

ooo .....

I

o

/ 500

Volume

I000

1500

\

o

2000

o

(ml)

Fig. 4. C h r o m a t o g r a p h y of a m m o n i u m sulfate s u p e r n a t a n t fraction on cation exchanger ]No-Rex 7o. R i g h t - h a n d ordinate refers to a s s a y values of pooled fractions indicated b y b r o k e n line. U n b r o k e n line indicates A ~s0.

* We are grateful to the U.S. Public H e a l t h Service S t u d y Section on H e m a t o l o g y for a s u p p l y of these fractions allocated to one of us (E.G.).

Biochim. Biophys. Acta, 64 (19,52) 487-496

494

E. (;()IA)WASSEI~., ~,V. i.'. W H I T E ,

1,2. B. T A Y L O R

t o g r a p h y of Step I I I (see ref. 8) t h e active fraction came off in tho second buffer. This difference in c h r o m a t o g r a p h i c b e h a v i o r we found to be due to differences between t h e s u p e r n a t a n t fraction a n d Step I I I caused b y the low-pH a m m o n i u m sulfate procedure since u p o n c h r o m a t o g r a p h y of the l a t t e r on B i o - R e x 7o the active fraction was eluted only b y the second buffer as with IRC-5o. Because of the lack of s h a r p resolution on h y d r o x y l a p a t i t e a n d because of other difficulties in the use of this a d s o r b e n t we a t t e m p t e d f u r t h e r f r a c t i o n a t i o n of the e r y t h r o p o i e t i n c o n c e n t r a t e from t h e B i o - R e x 7 ° column using calcium p h o s p h a t e gel in a b a t c h process. IO m l of t h e gel suspension in w a t e r at a d r y weight concent r a t i o n of II.O m g / m l was centrifuged to the b o t t o m of a glass tube. The salt-free* p r o t e i n solution was a d d e d to the p a c k e d gel a n d m i x e d with it for o.5 h in t h e c o l d , a f t e r which t h e gel was r e m o v e d b y centrifugation.The s u p e r n a t a n t from the first gel a d s o r p t i o n was p i p e t t e d on to a second gel pellet a n d the process repeated. Results of such a f r a c t i o n a t i o n are shown in Fig. 5. U n d e r these conditions a b o u t one-half the active m a t e r i a l r e m a i n s in solution while a b o u t 95 % of the 28o-m/x a b s o r b i n g m a t e r i a l is t a k e n out of the solution. A t t e m p t s to elute active m a t e r i a l from the gel h a v e n o t y e t been successful. The purification factor o b t a i n e d b y this m e t h o d is a b o u t I5-fold. W h e n t h e calcium p h o s p h a t e gel s u p e r n a t a n t (Step V) at 45 ° u n i t s / r a g p r o t e i n is c o m p a r e d to the original p l a s m a at o.oo 7 u n i t / m g protein the overall purification factor is 64ooo. U n f o r t u n a t e l y , no good e s t i m a t e of r e c o v e r y of original a c t i v i t y is available since the calcium p h o s p h a t e gel p r o c e d u r e was done on a side fraction of the a m m o n i u m sulfate fractionation. If we could assume similar r e c o v e r y from S t e p IV as from t h e B i o - R e x eluate the overall yield of a c t i v i t y would be 8 - 9 %. The v e r y small a m o u n t of Step V o b t a i n e d in these gel a d s o r p t i o n e x p e r i m e n t s p e r m i t t e d e x a m i n a t i o n of o n l y a few of its properties, I n Fig. I it can be seen t h a t gel electrophoresis shows o n l y one c o m p o n e n t for Step V as a g a i n s t 4 or more for Step IV. W h e n c o m p a r i s o n s w i t h c r u d e r fractions were m a d e we f o u n d t h a t the single e l e c t r o p h o r e t i c c o m p o n e n t was i d e n t i c a l with t h a t b a n d labeled No. i. There is a v e r y faint b a n d p r e s e n t in the immuno-diffusion against a n t i - S t e p I I I serum which u n f o r t u n a t e l y was n o t a p p a r e n t u p o n p h o t o g r a p h i n g the dried film. Some chemical p r o p e r t i e s of the Step V fraction are s u m m a r i z e d in T a b l e IV as TABLE

iV

SOME PROPERTIES OF ERYTHROPOIETIN FRACTIONS

Protein Fraction

).max. (mid)

Az% 280

Step IV

275

5.t

71.5

A i

265 275 270

4.7 IO.O

Step V

Carbohydrate

Hexosamine

67.3

9.3

to.o

7.5

36.8

55.1

12.5

13.8

18.4

80.8

68.1

29.2

17. 5

13.o

LOWR':

(%)

~¥ESTLEY

(%)

(%)

(o~)

Sialic acid (o~/)

* With fractions obtained from columns by the use of citrate buffers it may be necessary to use electrodialysis to reduce the citrate concentration to a level low enough for successful gel adsorption. Biochim. Biophys..4eta, 64 (1962) 487-496

495

SHEEP PLASMA ERYTHROPOIETIN

compared with the Step IV fraction and with the Az fraction previously described. Noteworthy in these preliminary analytical values of Step V are : (a) The large amount of carbohydrate, (b) the relatively low wavelength of the absorption maximum, (c) the high value of the Z °/o absorbancy coefficient. The absorption spectrum of Step V is shown in Fig. 6. The large shift toward the red at high p H indicates a high tyrosine content and a low (if any) tryptophan content for this mucoprotein and serves to explain the large discrepancy between the values for protein obtained by the WESTLE¥ I00 90

i

U n i T * / m ~ p,ol,i

400

80

CALCIUM

1

70

09

_z 60

!

o.

300

O7 <

PHOSPHATE

GEL SUPERNATE

/,

v-o

5O

40

200

i°4 L \\

50

b*d

"\.

../"

"\\

°,

. ,, ....

I00

20

I0

0

I

110

2210

o/

i

230

I

240

rng GEL Fig. 5- Calcium p h o s p h a t e gel f r a c t i o n a t i o n of erythropoietin.

Fig.

K

i

i

250

i

2S0

i

I

270

f

~

280

Wovelength (rn)a)

i

~

290

L

i

300

6. U l t r a v i o l e t a b s o r p t i o n s p e c t r u m Step V e r y t h r o p o i e t i n .

of

AND LAMBETH method and LOWRY method. Since bovine serum albumin was used as a standard it would be expected that proteins with higher tyrosine content would give higher values. The WESTLEY AND LAMBETH method, which does not differentiate proteins on the basis of aromatic amino acids, would be expected to give a truer indication of the protein content except where the non-protein moiety m a y bind copper. Although we recognize that the methods d~scribed here m a y not have led to a pure hormone, we feel that a report at this stage is justified since further purification m a y be very slow owing to the severely limited amounts of crude materials which are available. As more starting material becomes available we will accumulate enough Step V erythropoietin to attempt further fractionation to do more critical tests of homogeneity and to study in greater detail its chemical and physical properties. ACKNOWLEDGEMENT

One of us (K.B.T3 is a Rockefeller Research Fellow. Biochim. t~iophys. Acta, 64 ('1962) 487-496

41)0

E. GOI.I)WASSEI,~,

W. F. WHITE, K. B. TAYL()I{

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