Comp. Biochem. Physiol. Vol. 84B, No. 3, pp. 383-386, 1986 0305-0491/86 $3.00 + 0.00 Printed in Great Britain Pergamon Journals Ltd

CHARACTERIZATION OF THE HEMOGLOBINS AND GLOBINS OF S Y N B R A N C H U S M A R M O R A T U S

BLOCH, 1795 (PISCES, SYNBRANCHIDAE)

WILSON NAKAMOTO*3~ and PAULO EDUARDO DE ABREU MACHADOt *Department of Neurology and Psychiatry, and ~'Department of Internal Medicine, Faculty of Medicine

of Botucatu: Campus of Botucatu, UNESP, Botucatu, Silo Paulo, Brazil

(Received 16 December 1985)

Abstract--1. Total hemolysates of Synbranchus marmoratus Bloch, 1795, captured in Vitoriana, district of Botucatu, State of S~.o Paulo, Brazil, were submitted to agar-starch gel electrophoresis on glass slides using 42 mM-Tris 1.7 mM EDTA~. I mM borate buffer, pH 8.8, for the gel and 10 mM borate-l.7 mM NaOH buffer, pH 8.6, for the cuvette.

2. Three distinct hemoglobin bands were detected, with Hb I being of the cathodic type. 3. Cellulose acetate electrophoresis in 800 mM Tris-2.1 mM EDTA buffer, pH 8.9, containing 6 M urea

and 2.25 mM fl-mercaptoethanol indicated the presence of four globin chains denoted ~ t, t~2, fl and y. 4. It is suggested that the probable tetrameric constitution of the hemoglobin of Synbranchus

marmoratus Bloch, 1795 is Hb I (ct2~,2), Hb II (ct~72) and Hb III (ct~fl2).

INTRODUCTION

Electrophoretic and chromatographic studies of he- molysates of fish from boreal, temperate and tropical regions have shown the presence of multiple hemo- globin bands (Toledo, 1969; Riggs, 1970; Reischl and Tondo, 1974; Fyhn et al., 1978). Fyhn et al. (1978), in a study of hemolysates from Synbranchus mar- moratus captured in the Brazilian Amazon region, revealed the presence of 6-9 hemoglobin bands which characterize 3 different phenotypes, while Wilhelm and Reischl (1981) detected 10 hemoglobin bands in S. marmoratus specimens captured in Rio Grande do Sui, Brazil.

Synbranchus marrnoratus Bloch, 1975, which is found from Northern Argentina to Guatemala and Mexico and throughout almost all of Brazil, has a branchial chamber capable of performing aquatic as well as aerial respiration. S. marmoratus can remain buried in mud for long periods of time like dipnoan fishes (Carter and Beadle, 1931; Johansen, 1966; Rosa, 1978).

The objective of the present study was to in- vestigate the electrophoretic patterns of hemoglobins and polypeptide chains in specimens of Synbranchus marmoratus Bloch, 1795 captured in Vitoriana, Botucatu, State of S~o Paulo, Brazil.

was determined by aga~starch gel electrophoresis on 2.5 x 7.5 cm glass slides. The gel buffer was 42 mM Tris, 1.7mM EDTA, 6.1 mM borate, pH 8.8, and the cuvette buffer was 10 mM borate, 1.7 mM NaOH, pH 8.6 (Hall and Motulsky, 1968). The slides were stained with 0.5% amido black and analysed with a Zeiss densitometer, Model Absorbance Recorder 3.

Globin chains from the total hemolysates and from the chromatographic eluates were separated by electrophoresis on cellulose acetate strips (Chemetron) in 80mM Tris, 2.1 mM EDTA buffer, pH 8.9, containing 6M urea and 2.55 mM fl-mercaptoethanol by the method of Schneider (1974). S. marmoratus hemoglobins were obtained by chro-

MATERIALS AND METHODS

Six specimens of Synbranchus marmoratus Bloch, 1975 were captured in Vitoriana (22°52'S~48°26'W), State of S~,o Paulo, Brazil. Blood (2 ml) was collected from the caudal vein of each specimen with heparinized syringes, and the total hemolysates was obtained by the technique of Drabkin (1946). The number of hemoglobin components

:~Correspondence to be addressed to: Dr Wilson Naka- moto, Faculdade de Medicina de Botucatu, UNESP 18.600, Botucatu, SP, Brazil.

Fig. 1. Agar gel-starch electrophoresis of total Synbranchus marmoratus hemolysate in basic buffer showed three distinct hemoglobin bands. Hb I was in a cathodic position, Hb II in an intermediate position, and Hb III in an anodic

position, O, origin.

383

384 W I L S O N N A K A M O T O a n d P A U L O E D U A R D O D E ABREU M A C H A D O

O.D.

1.0,

0.8,

0.6.

0.4,

0.2,

Et

E3

0 220 TUBES N •

Fig. 2. DEAE-cellulose chromatography of total Synbranchus marmoratus hemolysate on 0.2 M glycine, 0.01% KCN buffer with a pH gradient of 7.16 to 7.52 and a saline gradient of 0.2 M NaCI produced three

eluates, E~, E 2 and E 3.

matography on DEAE-cellulose in 200 mM glycine, 0.01% KCN buffer, with a pH gradient of 7.16 to 7.52 and containing 200 mM NaC1 by the method of Huismann et al. (1974).

Total hemolysates and hemoglobins of S. marmoratus were tested for resistance to alkaline denaturation by the method of Betke et al. (1959).

R E S U L T S

Agar-s tarch gel electrophoresis in basic buffer of total S. marmoratus hemolysates showed the presence of 3 hemoglobin bands (Fig. 1). Under the conditions

employed, hemoglobin I (Hb I) had a cathodic position, hemoglobin II (Hb II), an intermediate position, and hemoglobin III (Hb III), an anodic position. Chromatography on DEAE-cellulose in gly- cine buffer and saline and pH gradients separated 3 eluates (Fig. 2) which, when submitted to agar-starch gel electrophoresis in basic buffer, were shown to correspond to the 3 hemoglobin bands. Thus, eluate 1 (E 0 corresponded to Hb I, eluate 2 (E2) to Hb II, and eluate 3 (E3) to Hb III (Fig. 3).

Electrophoresis of total hemolysates and of the chromatographic eluates El, E 2 and E 3 on cellulose

!ilji!iii i ! i!iii!ii!ji iii!!iii i!! i iii !!! i iiiii ii!!iii!!i ~ ~ ~i:~i i~ ~ ~ ~ ~!: !ii~ i~i ~ ! ~ ~ ~ iii ~i~i ~:~ii~i~:~: ~ ~=~ ~ ~: : : ~ ~ ~ ~i~!~iiiii~iii~ ~II i ~i~ ?~!~i!iii~!i!~! ii ~=~!i ~:~ii~i!

Fig. 3. Agar-starch gel electrophoresis in 42 mM Tris, 1.7 mM EDTA and 6.1 mM borate buffer, pH 8.8 for the gel, and 1.7 mM NaOH buffer for the cuvette, of total hemolysate and of the chromatographic eluates E~, E 2 and E 3 obtained by DEAE-cellulose chromatography of Synbranchus marmoratus total hemolysate in 0.2 M glycine, 0.01% KCN buffer with a pH gradient of 7.16 to 7.52 and a gradient of

0.2 M NaCI. Eluate E~ is contaminated with eluate E 2,

O~ +

i ~I

mnl~,

~y

mm.z~ I ~ , r-"'l

llm ~, r-q

~mp

Hemoglobins and globins of S. marmoratus 385

- - internal acidosis occurring both in fast-water fish (Powers, 1974) and in still-water fish such as Syn- branchus marmoratus, which can live in swamps.

Human foetal hemoglobin (Hb F) is characterized by the fact that it is capable of resisting an alkaline denaturing agent because of the presence of a 7 chain in its tetrametric composition (Perutz, 1974). Since Hb I and II of S. marrnoratus show 100% resistance to alkaline denaturation, their tetrameric structures may have polypeptide chains with structural charac- teristics similar to those of human Hb F. For this reason, the polypeptide chain common to Hb I and Hb II was called type ~, and the 2 different chains were respectively called type ct 2 and ct 1. Hb III showed a polypeptide chain with electrophoretic mobility similar to that of the ct ~ type chain, and the other globin chain was called type/3. Thus, the tetrameric structure of S. marmoratus specimens studied here is assumed to be: Hb I (~2272), Hb II (ct~72) and Hb III

~ l l . t 2 I ~ : l I .j

+

mm~, mm~)s m~m

TH Hbl TH HbK TH Hb~

Fig. 4. (A) Electrophoresis on cellulose acetate strips in basic buffer containing 6 M urea and fl-mercaptoethanol of total Synbranchus marmoratus hemolysates and hemoglobin 1, II and III. (B) Graphic presentation of electrophoretic runs on cellulose acetate strips in basic buffer containing 6 M urea and fl-mercaptoethanol of Synbranchus marmoratus total

hemolysate and hemoglobins I, II and III.

acetate strips in basic buffer containing 6 M urea and fl-mercaptoethanol respectively separated 4 and 2 polypeptide chains for each eluate. The polypeptide chains were called ct~; c~ 2, fl and ~, (Fig. 4).

The test for resistance to alkaline denaturation was positive for the total hemolysate and for hemoglobin I and II, whereas hemoglobin III showed no resistance.

DISCUSSION

The total hemolysates of Synbranchus marmoratus specimens captured in Vitoriana, Botucatu, State of S~.o Paulo, showed 3 distinct hemoglobin bands characterizing a hemoglobin pattern differing from those obtained by Fyhn et al. (1978) and Wilhelm and Reischl (1981). The total hemolysate of S. mar- moratus showed a cathodic hemoglobin (Hb I) similar to the cathodic hemoglobins present in some fast- water fish species such as trout, salmon and Cat- ostomus pantosteus clarkii (Powers and Edmundson, 1972; Bonaventura et al., 1975). The cathodic hemo- globins of these fish species are characterized by the absence of the Bohr effect. The presence of a cathodic hemoglobin may be related to the conditions of

Acknowledgements--We are grateful to Dr Silvio A. Toledo Filho, Inst. de Bloc. de USP, SP, Dr Fausto Foresti, Inst. Basico de Biologla M6dica e Agricola, UNESP, Botucatu; Dr. Arno R. Schwantes, Departamento de Ci~ncias Biol6g- icas, UFSCar, SP and Dr Donn E. Rosen, The American Museum of Natural History, for reading the manuscript.

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386 WILSON NAKAMOTO and PAULO EDUARDO DE ABREU MACHADO

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