Purification and properties of blood hemoglobin from the fresh-water cladocera, Moina macrocopa and Daphnia magna

BIOCHIMICAET BIOPHYSICAACTA

349

BBA 35716 P U R I F I C A T I O N AND P R O P E R T I E S OF BLOOD H E M O G L O B I N FROM T H E F R E S H - W A T E R CLADOCERA, MOINA MACROCOPA AND D A P H N I A MAGNA

HIROSHI SUGANO AND TAKEO HOSHI Department of Chemistry and Biology, Faculty of Science, Niigata University, Niigata (Japan) (Received August Ilth, I97o)

SUMMARY

I. From extracts of the blood of the pink animals of Moina macrocopa and Daphnia magna, hemoglobins were purified b y sucrose density gradient centrifugation, gel filtration, and DEAE-cellulose column chromatography. 2. Sedimentation and electrophoretic analyses indicated that the hemoglobins were homogeneous. 3. The molecular properties ofMoina laemoglobin were as follows: sedimentation coefficient 17.8 S; diffusion coefficient 2.62. lO -7 cm2/sec; intrinsic viscosity 0.045 dl/g; tool. wt. 6.7O.lO5; frictional ratio 1.31; isoelectric point 5.2; iron content o.317%; and heme content 37-38. 4. The values of Ps0 were 2.1 m m Hg for Moina and 3.5 m m Hg for Daphnia at 20 ° and p H 7.2, and the Bohr effect was hardly observed in both species. 5. Absorption spectra showed a similarity with mammalian hemoglobin with regard to spectral maxima and relative extinction coefficients.

INTRODUCTION Daphnid animals synthesize the blood hemoglobin in their bodies in low 0 2 environments. The animals look pink or even red in extreme conditions when the hemoglobin content is high. The nature of this hemoglobin is detectable, to some extent, within the body in situ. Studies on the properties of hemoglobin in vitro are scant owing to the difficulty of collection of the pink animals, or isolation of the blood and pure hemoglobin. Although several attempts have been made to isolate and purify the daphnid hemoglobin 1-4, they remain unsuccessful. The only literature reference to the molecular weight is that of SVEDBERG AND ERIKSSON-QuENSELI: from sedimentation studies on the blood extract, they estimated the molecular weight of hemoglobin of Daphnia pulex to be approximately 4.2" lO5(16.3 S). Sedimentation or other studies on the purified hemoglobin of Daphnia pulex and other cladoceran species have not yet, as far as we are aware, been carried out (see ref. 5).

Biochim. Biophys. Acta, 229 (1971) 349-358

350

H. ,qU(;ANO, T. HOSHI

The molecular weights of hemoglobins from various animals have been estimated: they range from I - i o 4 - 3 ,io 4 (Paramecium 6, Chironomus 7,s and Petr~> myzon 9) to 2.IO6-3.IO 6 (Arenicola and Lumbricus 7 and Tubifex~°). Therefore Daphnia is one of a few groups in the animal kingdom whose hemoglobin is situated in an intermediary position between the two extreme values of molecular weights. WITTENBERG el aI. n recently reported that the molecular weight of Ascaris hemoglobin is 3,28 "IOs. Moina, the specimen collectable in a large amount in our district, contains a blood hemoglobin similar to that of Daphnia. This paper describes the methods of isolation and purification of the Moina and Daphnia hemoglobins from the blood and some properties of the purified hemoglobins, such as molecular weights, isoelectrie points, absorption spectra, iron content, and Ps0 values. A preliminary report has been published elsewhere p2

MATERIALS AND METHO1)S

Materials The pink animals of Moina macroc@a Straus, whose hemoglobin c,mtent is high, were used. They were collected from the pond of the Suwa Shrine in the Oono district, the body size of the animals being checked by use of the plankton net, mesh of size I m m x I mm. Almost all the animals were adult females with the eggs or embryos in the brood pouch. They were washed in tap water several times and finally in physiological saline solution. The mass of the animals was then put into a suitable bag of silk or cotton gauze, and left hanging in the air to remow" excess water. The last drop of water surrounding the cloth was removed with filter paper. The wet weight was measured. The animals were stored in a refrigerator until use. The pink specimens of Daphnia magna Straus were obtained by the method of low-O., culture la, since they were not naturally available in our district. Analytical methods A Spinco Model L or HitaehiModel4oP ultracentrifuge was used in tile isolation procedure. Sedimentation analyses were performed with a Spinco Model E ultracentrifuge. Viscosity measurements were carried out in an Ostwald Fenske viscometer. Diffusion coefficients were measured with a cell of Neurath type in a Hitachi Model HTB-2 electrophoretic apparatus equipped with Schlieren optics. A thermostatic bath was controlled at 20 ° --~ 0.005 °. Spectral studies were carried out in a Beckman DU spectrophotometer. Moving boundary electrophoreses were performed with a Hitachi ModelHTB-2 apparatus; starch-gel electrophoreses according to the method of SMITHIES14 with the use of partially hydrolyzed starch prepared from Wako potato starch. Protein concentration was determined by the method of FOLIN AND CIOCALTEU 15 or refractometrically by using Schlieren optics. The iron content was measured by the method of CAMERON16. The value of Ps0 was obtained from O o-dissociation curves of hemoglobin determined by the method of RIGOStv with a slight modification 18. DEAE-cellulose was obtained from Serva Entwicklungs-labor; Sephadex G-2oo from Pharmacia Fine Chemicals. Biochim. [¢iophys, Acta, 229 (197I) 349-358

HEMOGLOBINS OV Moina macrocopa AND Daphnia magna

351

Isolation and purification of hemoglobin from blood The blood sample containing hemoglobin was prepared by the freezing and thawing method 4 from the pink animals collected in the bag and subjected to centrifugation at 20 ooo rev./min for 20 min to remove debris and other impurities. The supernatant thus obtained is referred to as "blood extract". A higher tendency to autoxidation of Moina hemoglobin was observed when compared with human hemoglobin. Therefore, except in the experiments on oxygenation, the blood extract was treated with CO in order to protect the hemoglobin from autoxidation. CO was prepared by decomposition of formic acid in hot H2SO 4, followed by washing in a solution of KOH. The experiments were carried out below 5 ° throughout and in the dark if necessary. Sedimentation analysis of the blood extract showed the existence of three components; a major peak at 4-6 S, a peak at about 15 S, and colored I8-S peak. So the sucrose density gradient centrifugation was used as the first isolation procedure. Each 1.5 ml of 20, I5, io, and 5% sucrose solutions in 0.03 M phosphate buffer (pH 7.2) was laid in the centrifuge tube, successively in the order described. After standing overnight, 3 ml of the blood extract were placed on the surface of the sucrose solution. The colored hemoglobin fraction condensed in the lower zone of the tube after centrifugation at lO5 ooo × g for 4-5 h was collected with a syringe and combined. Next, the colored hemoglobin fraction was subjected to gel filtration using Sephadex G-2oo. The gel, equilibrated with 0.03 M phosphate buffer (pH 7.2), was introduced into the column tube and washed several times with the same buffer to form a bed of the column (2 cm x 20 cm). The colored hemoglobin fraction was applied and eluted with the same buffer. Sedimentation analysis of the colored fraction collected suggested that the gel filtration was useful for complete removal of the slowly sedimenting materials and reduction of the content of the uncolored I5-S component to some extent. Finally, DEAE-cellulose column chromatography was used to remove the uncolored I5-S component and to obtain pure hemoglobin. DEAE-cellulose was treated with o.I M NaOH, rinsed with water until the filtrate became free from alkali, suspended in 0.03 M phosphate buffer (pH 7.2), packed into the chromatographic tube (column 2 cm × 5 cm), and washed thoroughly with the same buffer. After adsorption

Fig. I. S e d i m e n t a t i o n p a t t e r n of purified Moina h e m o g l o b i n in o.i M p h o s p h a t e buffer (pH 7.2) a t 5o 74 ° r e v . / m i n a n d 23 °. T h e p h o t o g r a p h w a s t a k e n 24 inin after t h e r o t o r r e a c h e d full speed.

Biochim. Biophys. Acta, 229 (1971) 349-358

352

H. SU(;ANO, T. HOSHI

of the hemoglobin sample, previously dialyzed against the same buffer, washing with the same buffer was continued until the effluent showed almost the same absorbance at 28o nm as the washing buffer. Elution of the hemoglobin was done bv raising the buffer concentration to o.I M. As the elution proceeded, a single colored band gradually moved down. When the sedimentation analysis of the eluted component showed an asymmetrical peak, which was considered to be due to the contamination of I5-S component, a repeat of the same procedure by I)EAE-cellulose colunul chr.matHgraphy was necessary to obtain the purified hemoglobin. About 15 eo mg of the purified hemoglobin were obtained from Ioo g wet weight of pink animals. RESULTS AND I}ISCUBSI()N

Physicochemical properties The sedimentation pattern of the purified Moina hemoglobin is shown in Fig. ~. The peak is single and symmetrical in shape, and a red color characteristic of hemoglobin always moved together with the peak. Therefore isolated and purified hemoglobin from the blood extract of Moina is considered to be homogeneous. As the colored low-molecular-weight component was almost imperceptible in the sedimentation pattern of the blood extract, minor hemoglobin, if it exists, would be in very small amount compared with the main I8-S henmglohin. I;rom the sedimentation measurements of the diluted blood, SVEDBERG AND ERIKSSON-QUENSEL 1 deduced that the Daptmia hemoglobin contains a main component of I6.3 S and a varying amount (30 40°'o at neutral pH) of a low-molecular-weight component of 3 4 S, that the main component is stable from pH 4.5 to IO.5, but that tim percentage of the lower component increases with pH, reaching IooG~ at pH H . 5 where the higher component disappears. Preliminary studies 1~ on the pH dependence of sedimentation properties of Moina hemoglobin showed (a) that the purified hemoglobin is stable from pH 6.3 to I x. 5, (b) that it was without accompanying lower component, but

,g

1`=

30

0

02

0.4 Q6 ConcentrQtion (g/dl)

0.8

10

Fig. 2. Sedimentational comparison of l ) a p h n i a (A) and Moina (B) hemoglobins in o.i M phosp h a t e buffer (pH 7.2) at 5-' ~4 ° rev./min and 22 °. The p h o t o g r a p h w a s taken 2o rain after the r o t o r reached full speed. Fig. 3. S e d i m e n t a t i o n rate of Moina h e m o / l o b i n as a function of protein concentration in phosp h a t e buffer (pH 7.2) at l o.2o.

Biochim. l~iophys. .lcla, 229 (1971) 349-35 ~

HEMOGLOBINS OF Moina macrocopa AND Daphnia magna

353

(c) at p H 11. 5, a trace amount of the lower component was sometimes observed, and (d) that at p H 3.5 two further components appeared, which were probably the aggregating products. This last observation is in contrast with the behavior of Daphnia pulex hemoglobin reported by the above authors 1 who found a decrease of sedimentation coefficient at p H 4.0. The occurrence of turbidity in the p H region from about 4-5 to 5-3 is another property of Moina hemoglobin different from that of Daphnia pulex hemoglobin. These differences in the properties of the two hemoglobins m a y be due to the difference in the species and to the measurements on Daphnia pulex hemoglobin being made with diluted blood. As shown in Fig. 2, the hemoglobin isolated from the blood extract of Daphnia magna by the same procedures exhibited, at p H 7.2, a similar sedimenting rate to that of Moina hemoglobin. The sedimentation coefficient at infinite dilution used to estimate the molecular weight was determined from Fig. 3, in which sedimenting rates are depicted as a function of protein concentration. Diffusion measurements were made on the purified Moina hemoglobin dialyzed against phosphate buffer (pH 7.2, I ~ o.15) at a protein concentration of 4.3 mg/ml. 008

~OD6

J OO~

&

&

d3

&

&

Concentration (g/dl)

Fig. 4- Viscosity m e a s u r e m e n t s on Moina hemoglobin in p h o s p h a t e buffer (pH 7.8) at I = o.15.

Coefficients were computed by the height-area method and corrected to the standard condition, but the effect of protein concentration was not examined here. The results of viscosity measurement are shown in Fig. 4, in which a plot of~sp/C versus c is drawn, and the intrinsic viscosity [~?1was determined by extrapolating it to infinite dilution. The molecular weight was estimated from a combination of S°2o,w and D~0,w using the following equation~°: RTs M-(I -- Vq)D

The partial specific volume was assumed to be equal to that of horse hemoglobin (0.749) 21. The frictional ratio was obtained from the following formula20:

From the frictional ratio thus obtained, the axial ratio of the molecule was determined by means of the contour chart given by ONCLEY*z, assuming a prolate ellipsoid Biochim. Biophys. Acta, 229 (1971) 349-358

:354

H. SU(;ANO, T. HOSHI

h y d r a t e d to the e x t e n t of 30% (ref. 23). A n o t h e r c o m p u t a t i o n to o b t a i n the molecular weight a n d shape was carried out, in which the intrinsic viscosity was c o n v e r t e d to the viscosity i n c r e m e n t c o r r e s p o n d i n g to t h e v o l m n e concentration, a n d then the frictional ratio was d e t e r m i n e d from the c o n t o u r c h a r t given b y OXCLEY 22, assuming again t h a t the molecule was a p r o l a t e ellipsoid with a h y d r a t e d w a t e r of 3oC)i,. B y using a combin a t i o n of s°20,w a n d the frictional r a t i o thus o b t a i n e d , the molecular weight was c o m p u t e d according to the following formula2~): M

102~et)aN'2V(f/Jo)[SJ(I

l'D)a )W~

T h e molecular weight was also e s t i m a t e d from the u l t r a c e n t r i f u g a l m e a s u r e m e n t s according to ARCHIBALD 24. M e a s u r e m e n t s were m a d e at 32oo rev,/min, and the value was o b t a i n e d from r e p e a t e d e x p e r i m e n t s . TABIA£ 1 S U M M A R Y OF P t i * ~ S I C O C t l E M I C A L P R O P E R T I E S

OI; ~ I ( ) I N A H E M O G L O B I N

Property

l "ahw

£'olve nl

s°~o.,,, (~) Dz0,w × io 7 (cm2/sec) Intrinsic viscosity, [~l]'ao,(dl/.<)

17.S 2.~2 o.o45

Phosphate buffer, pH 7.z, l Phosphate buffer, pH 7.8, 1 1)hosphate buffer, pH 7.e, 1

o._,() ().15 ().z()

0.75 O.7o 0.64

Phosphate buffer, ptl 7.z, l

. o.2o

Mol,

wt.

x

io

a

from s and D from s and [~/j from Archibald method fifo, frictional ratio from s and D from E~J] Axial ratio as prolatc from s and D from F~1] Isoelectric point

1.31 1.3(I 3.9 3.7 5.e

The 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 b t a i n e d are s u m m a r i z e d in Table I. As good a g r e e m e n t was o b s e r v e d a m o n g the results d e t e r m i n e d b y the three different methods, the molecular weight for Moina hemoglobin is e s t i m a t e d to be a p p r o x i m a t e l y 6. 7 • I o 5. As for the D a p h n i a hemoglobin, p h y s i c a l m e a s u r e m e n t s for d e t e r m i n i n g the m o l e c u l a r weight were not conducted, but, j u d g i n g from the similar s e d i m e n t i n g rate to t h a t for Moina hemoglobin, tile m o l e c u l a r weight is e x p e c t e d to be close to t h a t of Moina hemoglobin. There is a considerable d i s c r e p a n c y in the molecular weight between this Moina hemoglobin a n d D a p h n i a p u l e x hemoglobin 1 (4.2" IOa). This m a y be p a r t l y due to the use of the non-purified m a t e r i a l s for D a p h n i a pulex a n d to the species difference. Electrophoresis Tile p u r i t y of Moina hemoglobin was e x a m i n e d b y three electrophoretic m e t h o d s : cellulose a c e t a t e m e m b r a n e electrophoresis, starch-gel electrophoresis, a n d m o v i n g b o u n d a r y electrophoresis. As shown in Figs. 5-7, all these m e t h o d s i n d i c a t e d t h a t the purified Moina hemoglobin was homogeneous electrophoretically. The isoelectric point, 5.2, was e s t i m a t e d from t h e p H - m o b i l i t y curve d e p i c t e d b y the moving b o u n d a r y m e t h o d , in which in the p H region between 4-5 a n d 5.3 m e a s u r e m e n t s were Biochim. t~iophys. Acta, 229 (I97 I) 349-358

HEMOGLOBINSOF Moina macrocopa AND Daphnia magna

355

Fig. 5- Cellulose acetate m e m b r a n e electrophoresis o f human(A) and Moina (B) hemoglobins in I mM EDTA, 25 mM boric acid and 45 mM Tris (pH 8.5), d e t e c t e d with a benzidine reagent 2s. Fig. 6. Starch-gel electrophoresis of h u m a n (A) and Moina (]3) hemoglobins in I mM E D T A , 25 mM boric acid and 45 mM Tris (pH 8.5), stained with Amido Black loB.

Fig. 7. Moving b o u n d a r y electrophoresis of Moina hemoglobin in p h o s p h a t e buffer (pH 7.8) at I = o. 15. Duration of t h e e x p e r i m e n t was 12o min at 3.2 V/cm potential gradient.

impossible owing to the turbidity of hemoglobin solution (Fig. 8). The Moina hemoglobin moved faster on cellulose acetate membrane and more slowly on starch gel than did the human hemoglobin. These results can be explained from the lower isoelectric point and the markedly higher molecular weight of Moina hemoglobin compared with human hemoglobin. Daphnia hemoglobin showed a single component on the agar-gel electrophoresis, but it migrated slightly more slowly than Moina hemoglobin. This may be attributed to some differences between amino acid sequences of both hemoglobins.

Absorption spectra Absorption spectra were measured for the oxy-, reduced-, and carboxy-hemoglobins, from Moina and Daphnia, and compared with those of horse hemoglobin ~6 with regard to the wavelengths and relative extinction coefficients. A considerable Biochim. Biophys. Acta, 229 (I971) 349-358

356

H. S I I ( , A N O , T. H O S H I ÷6 4

+2 b

c

7, o

I

I

I

40

,

5.0

m

i

60

70

DH F i g . 8. p H - m o b i l i t y c u r v e o f M o i n a h e m o g l o b i n o b t a i n e d by" t h e m o v i n g b o u n d a r y m e t h o d . A c e t a t e ( p H 4.1 a n d 5.6) a n d p h o s p h a t e ( p H 6 . 0 a n d 6.8) b u f f e r s .

TABLE

eleetrophoretic

11

S P E C T R O S C O P I C D A T A OF T H E H E M O G L O B I N S OF ~'IOINA, D A P H N I A

Hemoglobin

Wavelength (nrn)

AND THE HORSE

Relative extinction coefficient

Daphnia

Horse*

Hb()2

578 542 4:5

576 542 4~4

578 542 4t4

~.o l.O8 9.5 °

i.o 1.03 9.5 o

Hb

559 429

5 ~I 423

556 43 °

I.O 9-5

I.O 9.2

I .o to.7

HbC()

57 ° 539 417

57 o 539 419

57 ° 54 °

I.O 1.16 lO.8

I.O 1.2o 13. 7

l .o :.o2 I3. 7

42o

Moisa

Daphnia

Aloina

Horse* t.o o.95 S.So

* F r o m M. SUZUKI #t al.e%

agreement was found among tile three kinds of hemoglobin (Table [1), suggesting the hemes of Moina and Daphnia hemoglobins to be protohemes like mammalian hemoglobins. The data from pyridine hemochromogen, published elsewhere 18, support this conclusion. Iron content

Table III shows the iron content of Moina hemoglobin. Control analyses were also performed on human hemoglobin prepared by ethanol precipitation from the hemolyzate of human erythroeytes washed repeatedly with physiological saline. The iron content of human hemoglobin (o.336% as a mean value) thus obtained almost agrees with the value (0.346°/'0) estimated from the molecular weight (6.45 "1o4) calculated from the amino acid composition2L Part of the minor discrepancy between the two values m a y be due to a small amount of non-hemoglobin protein in the hemoglobin preparation used here and whose existence was shown by the electrophoretic Biochim. Biophys. Acta, 229 (1971) 3 4 0 - 3 5 8

HEMOGLOBINSOF Moina macrocopa AND Daphnia magna TABLE

III

IRON CONTENTS OF HUMAN AND MOINA

HEMOGLOBINS AND GLOBINS

T h e m o l e c u l a r w e i g h t s , 6 . 4 5 • lO 4 a n d 6. 7 • lO 5, w e r e u s e d t o e s t i m a t e per molecule of human and Moina hemoglobins, respectively.

Sample

Human

357

Fe (%)

Hb

No. i No. 2 No. 3

0.330 0.338 °.341

Moina Hb

B S 84 B S 87 + 8 8 BS 89

o.318 o.321 o.312

Globin

Human Moina

0.027 o.o12

the number

of iron atoms

Number of Ire/ molecule of Hb 3.81 3.90 3-94 38.1 39.4 37.4

analysis. The iron content of the globin fraction, prepared from Moina hemoglobin treated with HCl-acetone, was negligibly low compared with the total iron content of hemoglobin. Since no further examination was performed on the question whether or not the globin fraction used here still contained any heme iron, an approximate value of 37-38 will be appropriate for the number of hemes per molecule of Moina hemoglobin. Based on this number of hemes, the minimal molecular weight of Moina hemoglobin is calculated to be 17 6oo-18 IOO, which is close to that of human hemoglobin (16 IOO). A preliminary experiment TM suggested that the Moina hemoglobin dissociated into the low-molecular-weight component ( < 2 S) in the presence of sodium dodecyl sulfate. Such studies are in progress in our laboratory. TABLE

IV

VALUES OF pso

pH

7.2

6.2 6.8 7.2 8.0

OF M O I N A

Temp.

5°

AND DAPHNIA

HEMOGLOBINS

P6o (ram Hg) Moina

Daphnia

0. 5

--

15 °

I.I

--

2o ° 25 °

2.I 3.5

3.5 --

IO ° (for Moina) 2o ° (for D a p h n i a )

o'5 o.6 o.5 0. 5

3.3 3.4 3.5 3.5

Values of Pso The values of Pso were estimated from the dissociation curves obtained under various conditions. The results are shown in Table IV. Pso of Daphnia hemoglobin obtained here was comparable to that of Daphnia pulex hemoglobin (3.1 mm Hg at 17 °) reported by Fox ~. Ps0 of Moina hemoglobin was a little lower than that of Biochim. Biophys. Acta,

2 2 9 (1971) 3 4 9 - 3 5 8

35 ~

H. SUGANO, T. HOSHI

D a p h n i a h e m o g l o b i n . T h i s m a y b e r e l a t e d w i t h t h e i r h a b i t a t s ; t h e w a t e r in w l f i c h M o i n a d w e l l s is u s u a l l y m o r e f o u l t h a n t h a t i n w h i c h D a p h n i a a r e f o u n d . A s s h o w n in Table

IV, daphnid

r e s u l t is s o m e w h a t

hemoglobins

seem to be almost

different from that reported

lacking in the Bohr

effect. This

by Fox 2 who found a moderate

Bohr

effect in the hemoglobin

o f D a p h n i a p u l e x , b u t a g r e e s w i t h t h a t r e p o r t e d b y Scm':kt';R "s

for Tubifex hemoglobin,

in w h i c h P~0 w a s 2 . 2 m m H g a t 2 0 ° a n d p H 7.o, a n d t h e 1~,o h r

effect was hardly

observed

b e l o w p H 7.5.

ACKNOWLE1)GMF-NTS We wish to thank their cooperation

M i s s i. W a t a n a b e ,

M r . M. K o b a y a s h i

a n d M r . M. H o n m a

fl)r

and skilled technique.

I(EFERENCILS I 2 3 4 5 6 7 8 9 lO II 12 I3 14 15 I6 J7 I8 19 2o 21 22 23 24 25 26 27 28

T. SVEDBERG AND |. [LRIKSSON-(~UENSEL, J. A~I1. Ohen~. Sot:., 5 ~) (1934) ~7oo. t t . M . F o x , J. Exptl. Biol., 2~ (1945) i 6 i . T. HOSHI, Zool, Mag. Tokyo, 7 2 (1963) 32I. T. H o s m ANt) S. NAGUMO, Sei. Rept. Niigata Univ. Ser. D. i (i964) 85. A. ROSSI-FANELLI, E. ANTONINI AND A. CAPUTO, in C. B. A.NFINSEN, Jr., J. T. I".DSALL ANt) F. B. RICHARDS, Advances in Protein Chemistry, Vol. I9, A c a d e m i c Press, New York, i964. p. 75M. H. SMITH, P. GEORGE AN[) J. R. PREER, JR., Arch. Biochem. Biophys., 99 (1962) 313. T. SVEDBERG, J. Biol. Chem., lO 3 (19331 31t. P. THOMPSON, W. BLEECKER AND D. S. ENGLISH, J. Biol. Chem., 243 (I90~) 4403 . N. M. LUMEN AND W. E. LOVE, Arch. Biochem. Biophys., IO 3 (1963) 24 . W. SCHELER AND L. SCHNEIDERAT, Acta Biol. Med. Ger., 3 (1959) 588. B. A. WITTENBERG, T. OKAZAKI AND J. B. WITTENBERG, Biochim. Biophys. Acta. ~ I~ (I9(i5) 496 . T. H o s t t I AND ][{. SUGAN(), .%'ci. Rept. Niigata Univ. Set. D, 2 (19651 t 3. T. Hosl~lI, M. I~.OBAYASHI, M. [-[ONMA AND H. SIJGANO, .S'ei. Rept. Niigata Univ. Nor. 11, (, (19691 155. (). SMITHIES, Biochem..]., 71 (10591 585 . (). FOLIN AND g. CIOCALTEU, J. Biol. Chem., 73 (19291 (127B. F.(-AMt':RON, Anal. Biochem., Ii (E9651 t64. A. RinGs, .]. Gen. Physiol., 35 (195l) 23" T. HOSHI, M. KOBAYASHI ANt) H. ,~UGANO,Sci. ReDt. Niigata Univ. Set. D, 5 (1968) 87. T. HOSHL M. KOBAVASHI AND H. SUGANO, Sci. Repl. Niigata Univ. Set. D, 4 (1967) I. T. SVEI)BERG AND K. (). PEDERSEN, The Ultracentrifuge, C l a r e n d o n Press, t)xford, t94 o, p. 478. G. S. ADAIR AND M. l{. ADAIR, Proc. Roy. Soc. London Net. A, 19 ° 0947) 3~ 1J. g. ()NCLEY, AHn. N . Y . Acad. Sei., 41 (t941) 121. W. L. BRAGG ANt) ~'I. [;. PERUTZ, Proe. Roy. Soc. London Net. A, 2t 3 (tq521 425 . W. J. ARCHIBALD, ,]-. Phys. Colloid Chem., 51 (I947) 12o4. ~. 1{. BOYER, D. C. I"AINER AND M. A. NAUGHTON, Science, 14o (1963) I22~. M. SUZUKI, A. I(AJITA AND C. HANAOKA, J. Biochem. Tokyo, 41 (1954) 4 ol, G. I~RAUNITZER, K. HILSE, V. RUDLOFF AND N. HILSCHMANN, in C. B. ANFINSEN, JR., J. T. J~DSALL AND F. i~. [{ICtIARI)S, Advances in Protein Chemistry, Vol. I9, A c a d e m i c Press, New York, I964, p. 2,. W. SCHELFR, Bioehem. Z., 332 (196o) 300.

Biochim. Biophys. -1 eta, 229 (I97I) 349 358