p. 573 to 578. Pergamon Press. Printed in (

IOCHEMISTRY OF SER1 UMIN-LIKE PROTEIN F

UMIN. kRP,

C Y P R I N U S CARPIO D, KEIICHIROHOSAKA AND RYOITI Sm :y, Nippon Medical School, Sendagi, Bm

~, Japan

(Received 10 December 1973) Abstract--1. The protein component in carp serum which has ha the disc ThJ corresponding to human serum albumin has been characterized. charactc and identified as an albumin-like protein. 2. The purification from carp, Cyprinus carpio, was perform ~erformed using S~ and DEAE cellulose colunm chromatography and isoelectric isoelectri~ focusing. homogenous according to various electrophoreses and an immunologic~ inn 3. The molecular weight was approximately 59,000 and the tl iscelectI with than one sulfhydryl group per molecule was detected by titration titra acid). 4. The fastest moving component of carp serum in cellulose cellulos acetate e with Schiff's reagent. The albumin-like protein in carp serum serur is found fastest-moving component and is one of the proteins which has h~ high soh anic solvent. out with sodium sulfate and by extraction with acidified orgar

INTRODUCTION PHYLOGENETICALLY 6EN~TICALLY, the frog is the lowest animal whose serum albumin has been clearly identified ao et al., 1972, 1973a, Wallace & Wilson, (Nagano 1972). Although a serum protein with the electroic mobility corresponding to human serum phoretic in has been observed in bony fishes (for review albumin t see Engle & Woods, 1960), physicochemical terization has not been reported. Since characte serum albumin has no appropriate enzymatic m and it is difficult activity to facilitate identification ~e overlooked when to define, serum albumin may be present in low concentration. On On the other hand, the electrophoretically fastest-movin 0ving component of some animal sera is considereda not to be serum albumin. F o r example, it has been suggested that the fastest moving component: of lamprey (Rail Hahn, 1962) serum et al., 1961) and salamander (Hahn, tents indicated that is globulin. Since our experiments n protein with the the carp and eel have a serum electrophoretic mobility correslponding to human led this component serum albumin, we have purified ied it as an albuminfrom carp serum and have identified like protein.

retie mobility was purified ) gel filtration I protein was pH 5.2. Less '.-nitrobenzoic gives a color lie site of the ed by salting-

Plasma was w~ carefully taken with a transfer transfer pipette after ation for about 10 rain at 3000 re~,/min. Serum centrifugal was obtail obtained after standing for several h~ hours at room temperature. Ampholine-carrier arnphol! pholytes (40 per cent w/v), Sephadex G-100, crystalline bovine t serum uvant were wq purchased albumin and at Freund's complete adjuvant ma and Difco, from LKB LK1 Produkter, Pharmacia, Sigma respectively. Put Chemicals, respectivel: Ovalbumin, from Wako Pure was purifi et al., ~urified as previously described ONagano (N." acetate sheets (Separax parax) were from 1972). Cellulose C¢ Fuji Photo Film. Protein determination Protein was determined by the methc method of Lowry et al. (1951) using bovine serum albumin as a a standard. In the chromatographic experiments; and those the employing salting-out with sodium sulfate, protein was estimated by the Biuret method or by absorbance at 280 21 nm. Immunological methods Preparation of antisera against carp serum and immunoelectrophoresis were performed by the methods previously reported (Nagano et aL, 1972). Cellulose acetate membrane electrophoresis

ETHODS MATERIALS AND METHODS dly from Nozawaya, Carp were obtained commerciall Tokyo. The blood of carp was withdrawn from the a-heparinized syringe, aorta with a heparinized or a non-he

Electrophoresis on Separax cellulose aacetate sheets et aL, was carried out as previously reported (Nagano (1~ in the 19731)). The detailed conditions are described des~ legend to Fig. 2. Characterization of the Separax paper membrane has been described in our previous pr 573

kGANO, KEIICHIRO HOSAKA AND RYOITI S nsity was traced

sed for disc gel 1 ml) containing to a separating s conducted at el electrophoresis e was performed born(1969),

-out of plasma protein method of Howe (1921) was employed to detersalting-out curve of carp plasma protein. For the ~tion of a saturated solution of sodium sulfate, solid sodium sulfate was added to distilled water lcubation at 37°C was performed overnight. t-tubes warmed at 37°C, 0.2 lift of carp plasma ml of the indicated amount of NazSO4 solution :lded and mixed. After standing for 1 hr at 37°C, 'ecipitated materials were filtered using Toyo aper No. 6. The amount of unprecipitated protein termined by the biuret method and absorbance at L.

in 50 m M sodium et al., 1972, 1973a buffer.

ffer, pH 7.4 (Nagano ed against the same

Electrophoretic pat

,el

F i g u r e 1 shows tl o f h u m a n , bullfro~ (Fig. 1C) h a s a I h u m a n a n d buUfl f r o m carl'p s e r u m is graph n o t sl~ (photogr~ a m o u n t oc f carboh,~ t i o n o f t] o f the a l b u m i determin, ed b y a de oncentratiot m a t e ccon

ctrophoretic patterns el sera. C a r p s e r u m mobility similar to lbumin. This b a n d with Schiff's reagent ring t h a t a detectable :ing. T h e c o n c e n t r a a in c a r p s e r u m was racing. T h e a p p r o x i /ml.

~phoresis o f Electrop~,

Separax sheets

UsualllLy m a m m a fastest-migrating fastest-m T h e electrophoreti elec Separax cellulose a

L is identified as the m e electrophoresis. f carp serum on a s s h o w n in Fig. 2A.

~ion with acidified organic solvent as been reported that only serum albumin is It has ed in the acidified organic solvent (Michael, 1962). extracted F o r thisis extraction, 1 mi of carp serum was mixed with ff 90 9 0 ~ methanol containing 0.05 N trichloroacetic 10ml of acid. The t h e precipitate was removed by centrifugation. To the supernatant, 0.1 N N a O H was added dropwise to give a final p H of about 5-5. The resultant precipitate was dissolved ~solved in 4 mi of water and dialyzed overnight against 0.05 M sodium barbital buffer, pH 8.6.

lsoelectric focusing Eiectrofocusing was performed usm using an electrofocusing column of 110-ml capacity and the carrier ampholyte density gradient with of p H range 4--6. The solution and[ densit in the sucrose were prepared as described in th~ LKB instruction manual. The electrolyte solution for or anode and cathode ~ric acid, respectively. were ethylenediamine and phosphoric solution The sample protein was mixed with the the less-dense le and electrofocused for 22 hr at 400 V. Fractions of 2 ml were collected and pH wass determined with a Beckman expandomatic SS-2 pH meter.

• ..

.i

,.

~

i.

i

.:

i

i

!

.~.~-._iiL..~.. " " - ' " "" ' : - - - ~ - ' - : ' " "; "

~ ~

Titration of sulfhydryl groups

Fig. 2. Separax cellulose acetate electroph ~horesis of carp serum (A) and the albumin-like protein of d carp serum (I3). The amounts of the sample solution solu were as follows: 2 v.l of carp serum and 8'8 v.1 of o albumin-like protein (1"4 mg/ml). The arrow indicate,, cares the position of sample application. Electrophoresis was w carried out at 7 mA/cm for 55 min using 0.05 M sodium so barbital buffer, pH 8.6.

The rate of reaction of SH groupss with 5,5'-dithiobis(25, nitrobenzoic acid) was measuredd by recording the absorbance change at 412 nm usinng a Cary recording spectrophotometer model 14. A molecular extinction coefficient of 13,600 was used (Ellman, nan, 1959). The reacphosphate tion mixture (0"9 ml) contained 45 mnM M sodium sc thiobis(2-nitrobenzoic buffer, pH 7.4, 0.4 mM 5,5'-dithiobis, in-like protein which acid) and 0.27 mg of carp albumin-like had been previously treated with 20 mM mercaptoethanol

T h e fastest m o v i n g c o m p o n e n t o f car•p s e r u m was stained w i t h Schiff's reagent (figure n o t shown). A s n o t e d above, t h e a l b u m i n - l i k e p r o)tein was n o t stained b y Schiff's reagent o n disc gel. gel R e s o l u t i o n o n the cellulose acetate sheet is ppco o r , so it is a m b i g u o u s w h e t h e r the fastest-moving b a n d is comp o s e d o f o n e or m o r e c o m p o n e n t s .

Fig. 1. Acrylamide disc gel electrophoresis. Direction of electrophoresis was from top to bottom (anode). The protein bands were stained with Amido schwarz. The protein band on each gel marked by an arrow indicates an albumin or an albumin-like protein. (A) human, (B) bullfrog, (C) carp and (D) eel serum. Sample solution (0.1 ml) contained 2.7 ~1 of each serum. (E) Purified albumin-like protein of carp serum. About 10 pg of the purified protein was added to 0.1 ml of sample solution containing 12 per cent glycerol. (F) Electrophoretic pattern of carp plasma proteins not precipitated by 70% saturation of sodium sulfate. This protein fraction (4ml as described in Materials and Methods) was dialyzed against distilled water and concentrated to about 1 ml. A sample solution contained 0.04 ml of the concentrated solution and 0.06 ml of 20°k glycerol. (G) Pattern of carp serum extract treated with 90% methanol containing 0.05 N trichloroacetic acid. Sample solution was a mixture of 0.08 ml of the extract obtained as described in Materials and Methods and 0.02 ml of 60% glycerol.

Fig. 4. Tmmunoelectrophoresis of carp serum (upper well) and the albumin-like protein of carp serum (lower well) against anti-carp-serum serum of rabbit. The upper well contained 12 (~1of carp serum and the lower well contained 12 ~1 of 3.9 mg/ml of albumin-like protein. Electrophoresis was performed at 3 mA for 105 min. The anode is left and the cathode is right.

575

Carp serum albumin-like protein Purificationof albumin-likeprotein Purification of the carp serum protein which migrates similar to human serum albumin on disc gel electrophoresis was performed by Sephadex G-100 gel filtration, ion exchange column chromatography using DEAE c&dose and isoelectric focusing. For purification, carp serum or plasma was used. In both cases, similar results were obtained. Carp plasma (4 ml) was applied to a Sephadex G-100 column (25 x 87 cm) equilibrated with 5 mM sodium phosphate buffer, pH 7.0. The same buffer was used for elution. A typical gel filtration profile is presented in Fig. 3A. The 3.0

I

1

pH 5.7, in the reservoir. In the case of the bullfrog, serum albumin is eluted by these conditions (Nagano et al., 1972). The second elution was with 1OOOml of 50mM NaH,P&. A small absorbance peak marked by a bar (peak C) was further purified by rechromatography on a DEAE celhrlose column or by electrofocusing. Pooled fraction C was concentrated with a collodion bag to about 2 ml and electrofocused as described in Materials and Methods. The protein electrofocused at pH 5.2 (Fig. 3C) had the same disc gel electrophoretic mobility as human serum albumin.

,

A

m

a

0

IO

20

30

FRACTION

40

50

NUMBER

C 0

50

100

FRACTION

Fig. 3. Puritication of albumin-like protein. A. Sephadex G-100 gel filtration. Bach fraction was 3 ml. The arrow indicates one bed volume. Peak C marked by a bar was used for the next step. B. DEAE cellulose column chromatography. Each fraction was 1Oml. The arrow indicates the position of elution buffer change. Peak C marked by a bar was used for electrofocusing. C. Elwtrofocusing column chromatography. The arrow indicates the absorbance peak of the albumin-like protein.

150

NUMBER

A

-0

50

100

FRACTION

150

200

NUMBER

An alternative purification of protein in peak C of Fig. 3B involved rechromatography with DEAE cellulose using a linear gradient. The gradient was obtained by using 500ml of 10 mM phosphate buffer, pH 7.1, in the mixing flask and 500 ml of the same buffer containing 0.4 M NaCl in the reservoir. The albumin-like protein was eluted at a NaCl concentration of 0.05-0.1 M (elution profile not shown).

B

combined fraction marked by a bar (peak C) was applied to a DEAE cellulose column (2.3 x 10.7 cm) that had been equilibrated with the above buffer. As seen in Fig. 3B, two sequential methods of elution were used. The first elution was with a gradient using 500 ml of the above buffer in a mixing flask and 500ml of 50 mM sodium phosphate buffer,

Purity The protein isolated by isoelectric focusing or by the rechromatography with DEAE cellulose column was homogeneous according to immunoelectrophoresis, disc gel and cellulose acetate electrophoreses. Figure 1E shows the disc gel electrophoretic pattern of the isolated protein.

576

HIROSHI NAGANO, KEIICHIROH~~AKA AND RYOITI SHUKUYA

The sample migrates as a single band, indicating no polymeric forms are produced although the electrofocused albumin from bullfrog serum (Nagano et al., 1972) polymerizes. As shown in Fig. 2B, the purified protein moves relatively fast to the anode as a symmetrical peak on a Separax sheet. Immunological evidence for homogeneity is demonstrated in Fig. 4. This figure shows the results of immunoelectrophoresis of the purified protein against anti carp-serum serum. A single precipitin line also developed at the cathodic position of the fastest-moving component of carp serum. The fastest-moving component is not serum albumin as shown later. Molecular

weight

Disc gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate was used to estimate the molecular weight of the albumin-like protein of carp. As shown in Fig. 5, a plot of log molecular weight against relative mobility of the marker proteins was linear. The molecular weight of the albumin-like protein polypeptide chain was approximately 59,000. P 0

I BSA

$ iii 9

I

I

I

30-

(TRIMER

1

BSA (DIMER

)

IO BSAlMONOMER

2 -I f20; 2

5

Y

3

g

2

OVALBUMIN

\

0

1

2

4 TIME

6 (MIN

8

IO-

1

Fig. 6. Titration of sulfhydryl groups with 5,5’-dithiobis(Znitrobenzoic acid). Sodium dodecyl sulfate was added to a final concentration of 0.1 per cent at the time marked by the arrow.

The fastest-moving

protein of carp serum

One of the characteristics of serum albumin is the fastest migration to the anode in zone electrophoresis. The fastest-moving component of carp serum, however, is judged not to be serum albumin because of the following reasons. (1) This component is eluted in peak B with Sephadex G-100 gel filtration (Fig. 3B). The molecular weight of this component is estimated to be 150,000-200,000. (2) The fastest-moving component on a cellulose acetate sheet is stained with SchWs reagent. (3) The fastest moving component is salted out at a low concentration of sodium sulfate.

;

I

I

I

I

0.2

0.4

0.6

0.8

Salting-out 1.0

Rf Fig. 5.

0

A plot of log molecular weight vs relative

mobility from disc gel electrophoresis in the presence of sodium dodecyl sulfate. BSA, bovine serum albumin.

polymeric forms of BSA were produced by freezing and thawing of the solution. The molecular weights of the standard proteins were taken as 68,000 for BSA and 43,000 for ovalbumin. Open circle indicates the Dosition of the albumin-like protein of carp serum. Number of suljhydryl groups

Usually less than one thiol group per molecule is titratable using various sources of serum albumin (Fantl, 1972). For this reason the number of thiol groups offers one criterion for the identification of serum albumin. As shown in Fig. 6, titration with 5,5’-dithiobis(2-nitrobenzoic acid) indicates one reactive thiol group per molecule. The addition of sodium dodecyl sulfate (final concentration of of 0.1%) did not reveal additional thiol groups.

curve of carp plasma

One property of serum albumin is that high concentrations of ammonium sulfate or sodium sulfate are required for precipitation. That is, serum albumin has high solubility. In the following experiments, carp plasma was used. The protein fraction precipitated at 70% of saturation with Na,SO, (Fig. 7) has roughly three or four components according to the results of disc gel electrophoresis (Fig. 1F). Apparently four bands are visible in Fig. 1F. The lowest band is the albuminlike protein judging from its mobility. The saltingout experiment indicates that the albumin-like protein is one of the proteins whose solubility is high.

Trichloroacetic

acid-methanol

extraction

Generally, serum albumin is not precipitated by treatment with 90% methanol containing 0.05 N trichloroacetic acid (Michael, 1962). Application of this method for preparation and identification

Carp serum albumin-like protein of carp serum albumin was tried. As shown in Fig. lG, one of the extracted proteins has an electrophoretic mobility similar to human serum albumin.

0

20

40

60

80

100

PERCENT SATURATION

Fig. 7. Salting-out curve of carp plasma with sodium sulfate. o, absorbance at 28Onm; l, absorbance at 540 nm (Biuret reaction). The protein concentration of cam plasma is normalii to 100 per cent. Arrows indicate the positions where new kinds of proteins begin to precipitate. DISCUSSION In this paper we have demonstrated the existence of an albumin-like protein in carp serum by the following criteria: (1) electrophoretic mobility on acrylamide gels is similar to human and bullfrog serum albumin. (2) The molecular weight is about 59,000. This value is similar although somewhat smaller than the reported values of serum albumin from other animals. (3) The isoelectric point is pH 5.2. (4) The protein has no detectable carbohydrates. (5) The number of thiol groups with 5,5’-dithiobis(2-nitroboic acid) is less than one per molecule. (6) The salting-out behavior indicates that this protein has relatively high solubility. (7) The protein is extracted with 90% methanol containing 0.05 N trichloroacetic acid. (8) The protein moves relatively fast to the anode. Properties of the albumin-like protein which differ from the well-characterized serum albumins from other species are as follows: (1) carp albuminlike protein after the isoelectric focusing step does not polymerize as does bullfrog serum albumin (Nagano et al., 1972). (2) The albumin-like protein is not the fastest-moving protein. The fastestmoving component of carp serum is a glycoprotein having a relatively high molecular weight as shown above. (3) The concentration of the albumin-like protein in serum is extremely low (l-2 mg/ml). Since a large amount of albumin-like protein was not available, the 2-(4’-hydroxybenzeneazo)~benzoic acid binding test (Martinek, 1965; Nagano et al., 1973a) and Sevine esterase activity (Casida &

577

Augustinsson, 1959; Nagano et al., 1973a) were not determined. The difficulties of demonstrating serum albumin in fishes are due to the facts that fishes have only a low concentration of serum albumin-like protein and this protein is not the fastest-migrating protein to the anode (Fig. 2). Disc gel electrophoresis seemed to be the most useful method for the demonstration of a low concentration of serum albumin (Komatsu et al., 1970). According to this criterion, eel seemed to have a serum albumin-like protein (Fig. lD> with mobility on disc gels similar to human serum albumin. The concentration of eel serum albumin-like protein is about 0.5 mg/ml. Taking the above results into consideration, bony fishes seem to have an albumin-like protein in serum but its concentration is low. Acknowledgements-We wish to thank Dr. H. Zalkin for reading the manuscript. This work was supported in part by a Grant for scientific research from the Ministry of Education of Japan.

REFERENCES CASIDA J. E. & AUGUSTINSSONK.-B.

(1959) Reaction of plasma albumin with 1-naphthyl N-methylcarbamate and certain other esters. Biochim. biophys. Acta 36, 411-426.

D~~lrsB. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404-427. ELLMAN G. L. (1959) Tissue sulfhydryl groups. Archs Biochem. Biophys. 82, 70-77. ENGLE R. L. & WOODS K. R.

(1960) bio. , Comuarative chemistry and embryology. In The Plasma Protein (Edited by PUTNAMF. W.), Vol. II. pp. 183-265. Academic Press, New York. FANTL P. (1972) Evolutionary trends in plasma mercaptalbumin composition. Comp. Biochem. Physiol. 42B, 403-408. HAHN W. E. (1962) Serum protein and erythrocyte changes during metamorphosis in paedogenic Ambystoma tigrinum mavortium. Comp. Biochem. Physiol. 7, 55-61.

HOWEP. E. (1921) The use of sodium sulfate as the globulin precipitant in the determination of proteins in blood. J. biol. Chem. 49, 93-107. KOMATXJS. K., MILLERH. T., DEVIU~ A. L., OSUGA D. T. & FEENEYR. E. (1970) Blood plasma proteins of cold-adapted Antarctic fishes. Comp. Biochem. Physiol. 32, 519-527. LOWRY 0.

H., ROSEBROUGH N. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with the Folin ohenol reanent. J. biol. Chem. 193.265-275. MAF~TINEKR. G. (1965) Evaluatioh of a dysbindiig method for determination of serum albumin. Clin. r------

--.-

Chem. 11,441-447. MICHAELS. E. (1962) The

isolation of albumin from blood serum or plasma by means of organic solvents.

Biochem. J. 82,212-218. NAGANO H., SHIMADAT.

& SHLJKUYA R. (1972) Purification and properties of serum albumin from Rana catesbeiana. Biochim. biophys. Acta 278, 101-109.

578

HIROSHINAGANO,KEIICHIROHOSAKAAND RYOITISHUKUYA

NAGANOH., SHIMA~AT. & SHUKUYAR. (1973a) Further characterization of bullfrog serum albumin. Biochim. biophys. Actu 310,495499. NAGANO H., SH~MADAT. & SHUKUYA R. (1973b) Purification and characterization of bullfrog serum a,-gfycoprotein. .I. biol. Chem. 248, 5774-5779. RALL D. P., SCHWABP. & ZUBRODC. G. (1961) Alteration of plasma proteins at metamorphosis in the lamprey (Petromyzon marinus dosatus). Science, Wash. 133, 279-280. WALLACED. G. & WILXJN A. C. (1972) Comparison of frog albumins with those of other vertebrates. J. molec. Evol. 2, 72-86.

WEBERK. & OSBORNM. (1969) The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. biol. Chem. 244, 44064412.

Key Word Index-Albumin-like protein; carp serum protein; Cyprinus curpio; serum albumin; serum albuminlike protein; fastest-moving component.