Estudo eletroforético de proteínas de músculo esquelético de Micropogonias furnieri (Desmarest, 1823) da costa SE-S do Brasil: 1. Considerações técnicas

Suzuki, Hana; Vazzoler, Anna Emília A. de M; Phan, Van Ngan

doi:10.1590/S0373-55241983000200006

Resumos

No presente trabalho, foram analisados os efeitos de diferentes posições de coleta e de diferentes períodos de preservação das amostras a -15ºC sobre os eletroferogramas de proteínas gerais do músculo esquelético de Micropogonias furnieri, e as condições para extração e a eletroforese das proteínas foram estabelecidas . Foram comparados o poder de extração e de preservação de 4 soluções: água destilada; cloreto de sódio 0,9%; tampão fosfato pH 7,5 µ=0,05 (Connell, 1953); glicerol-EDTA-Tris pH 8,7 (Scopes, 1968). As condições de eletroforese foram investigadas, utilizando-se membranas de acetato de celulose com seis diferentes sistemas de tampões e gel de amido com três diferentes sistemas de tampão. As amostras coletadas de diferentes posições não apresentaram diferenças no número nem na posição relativa das bandas nos eletroferogramas. Foram anotadas alterações nos eletroferogramas conforme o período de preservação das amostras. A solução glicerol-EDTA-Tris pH 8,7 apresentou o maior poder de extração e de preservação. Nos sistemas de tampão testados, o número máximo de 9 bandas foi obtido, tanto em membrana de acetato de celulose como em gel de amido. Melhor separação das proteínas em membrana de acetato de celulose foi obtida com tampão Tris-glicina pH 8,3 (Tris 0,0495M, glicina 0,3836M), e em gel de amido, com tampão do gel constituído por Tris, EDTA e ácido bórico, de pH 8,6 (Huehns, 1968) e tampão da cuba constituído por ácido bórico e hidróxido de sódio (Smithies, 1955).

Eletrofórese; Proteínas; Músculo; Metodologia; Costa sul; Costa sudeste; Cananéia; Micropogonias furwieri; Sciaenidae

Effects of different positions of sampling and different periods of preservation of the samples at -15ºC on the general protein electropherograms of skeletic muscle ofMicropogpnias furnieri, were analysed and the conditions of extraction and electrophoresis of the proteins were established. Extraction and preservation powers of four solutions, distilled water, sodium chloride 0.9%, phosphate buffer pH 7,5 µ=0.05 (Connelly 1953) and glycerol-EDTA-Tris pH 8.7. (Scopes, 1968), were compared. The electrophoresis conditions were investigated using cellulose acetate membranes with six different buffer systems and starch gel with three different buffer systems. Samples of muscle collected from different positions did not show differences either in the number of bands or in their relative migrations in the electropherograms. Alterations in the electropherograms were noted according the period of sample preservation. The greatest extraction and preservation powers were found with the solution glyeerol-EDTA-tris pH 8.7. In the buffer systems used, the maximum number of nine bands was obtained in cellulose acetate membranes as well as in starch gel. The best separation of the proteins in cellulose acetate membranes was found with Tris-glyeine buffer pH 8.3 (Tris 0.0495M, glycine 0.3836M), and in starch gel with gel buffer constituted by Tris, EDTA and boric acid, pE 8.6 (Huehns, 1968) and electrode buffer constituted by boric acid and sodium hydroxid (Smithies, 1955).

Electrophoresis; Proteins; Muscle; Methodology; South coast; Southeast coast; Cananéia; Micropogonias furwieri; Sciaenidae

ARTIGOS

Estudo eletroforético de proteínas de músculo esquelético de Micropogonias furnieri (Desmarest, 1823) da costa SE-S do Brasil. 1. Considerações técnicas^* (* ) Trabalho realizado com apoio financeiro da FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. 74/816, Proc. 75/387 e Proc. 76/262).

An electrophoretic study on proteins of skeleton muscle of Micropogonias furnieri (Desmarest, 1823) from SE-S coast of Brazil. 1. Some technical considerations

Hana Suzuki^** (** ) Aluna do Curso de Pós-Graduação do IOUSP. ; Anna Emília A. de M. Vazzoler^*** (*** ) Atualmente no INPA - Instituto Nacional de Pesquisas da Amazônia. ; Phan Van Ngan

Instituto Oceanográfico da Universidade de São Paulo

RESUMO

No presente trabalho, foram analisados os efeitos de diferentes posições de coleta e de diferentes períodos de preservação das amostras a -15ºC sobre os eletroferogramas de proteínas gerais do músculo esquelético de Micropogonias furnieri, e as condições para extração e a eletroforese das proteínas foram estabelecidas .

Foram comparados o poder de extração e de preservação de 4 soluções: água destilada; cloreto de sódio 0,9%; tampão fosfato pH 7,5 µ=0,05 (Connell, 1953); glicerol-EDTA-Tris pH 8,7 (Scopes, 1968). As condições de eletroforese foram investigadas, utilizando-se membranas de acetato de celulose com seis diferentes sistemas de tampões e gel de amido com três diferentes sistemas de tampão.

As amostras coletadas de diferentes posições não apresentaram diferenças no número nem na posição relativa das bandas nos eletroferogramas. Foram anotadas alterações nos eletroferogramas conforme o período de preservação das amostras. A solução glicerol-EDTA-Tris pH 8,7 apresentou o maior poder de extração e de preservação. Nos sistemas de tampão testados, o número máximo de 9 bandas foi obtido, tanto em membrana de acetato de celulose como em gel de amido. Melhor separação das proteínas em membrana de acetato de celulose foi obtida com tampão Tris-glicina pH 8,3 (Tris 0,0495M, glicina 0,3836M), e em gel de amido, com tampão do gel constituído por Tris, EDTA e ácido bórico, de pH 8,6 (Huehns, 1968) e tampão da cuba constituído por ácido bórico e hidróxido de sódio (Smithies, 1955).

Descritores: Eletrofórese, Proteínas, Músculo, Metodologia, Costa sul - Brasil, Costa sudeste - Brasil, Cananéia - SP, Micropogonias furnieri, Sciaenidae.

SYNOPSIS

Effects of different positions of sampling and different periods of preservation of the samples at -15ºC on the general protein electropherograms of skeletic muscle ofMicropogpnias furnieri, were analysed and the conditions of extraction and electrophoresis of the proteins were established. Extraction and preservation powers of four solutions, distilled water, sodium chloride 0.9%, phosphate buffer pH 7,5 µ=0.05 (Connelly 1953) and glycerol-EDTA-Tris pH 8.7. (Scopes, 1968), were compared. The electrophoresis conditions were investigated using cellulose acetate membranes with six different buffer systems and starch gel with three different buffer systems. Samples of muscle collected from different positions did not show differences either in the number of bands or in their relative migrations in the electropherograms. Alterations in the electropherograms were noted according the period of sample preservation. The greatest extraction and preservation powers were found with the solution glyeerol-EDTA-tris pH 8.7. In the buffer systems used, the maximum number of nine bands was obtained in cellulose acetate membranes as well as in starch gel. The best separation of the proteins in cellulose acetate membranes was found with Tris-glyeine buffer pH 8.3 (Tris 0.0495M, glycine 0.3836M), and in starch gel with gel buffer constituted by Tris, EDTA and boric acid, pE 8.6 (Huehns, 1968) and electrode buffer constituted by boric acid and sodium hydroxid (Smithies, 1955).

Descriptors: Electrophoresis, Proteins, Muscle, Methodology, South coast - Brazil, Southeast coast - Brazil, Cananéia - SP, Micropogonias furnieri, Sciaenidae.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

(Recebido 09-maio-1983; aceito 10-fev-1984)

AMANO, K.; BITO, M. & KAWABATA, T. 1953. Handling effect upon biochemical change in the fish muscle immediately after catch. I. Differences of glycolysis in the frigate mackerel killed by various methods. Bull, japan. Soc. scient. Fish., 19 (4):487-498.
AMLACHER, E. 1961. Rigor-mortis in fish. In: Borgstrom, G., ed. -Fish as food. New York, Academic Press, v.1, p. 385-409.
ANALYTICAL METHODS COMMITTEE 1966. Nitrogen factor for cod fish. Report prepared by the Fish Products Sub-Committee. Analyst, Lond., 91(1085): 540-542.
BECHTEL, M. A. B. & ALVES, J. L. P. 1973. Identificação de espécies de pescado por eletroforese em acetato de celulose. Sér. Tecnol., (3)-GEDIP. Porto Alegre.
BHUSHANA RAO, K. S. P.; FOCANT, B.; GERDAY, Ch. & HAMOIR, G. 1969. Low molecular weight albumins of cod white muscle (Gadus callarias). Comp. Biochem. Physiol., 30:33-48.
BOSCH, M. W. 1951. Quantitative electrophoretic study of rabbit muscle proteins soluble in dilute salt solutions. Biochem. Biophys. Acta, 7:61-86.
CANTAROW, A. & SCHEPARTZ, B. 1962. Biochemistry. Philadelphia, Maruzen Asian Edition, 854p.
CONNELL, J. J. 1953. Studies on the proteins of fish skeletal muscle. 1. Electrophoretic analysis of codling extracts of low ionic strength. Biochem. J., 54:119-126.
______ 1975. The role of formaldehyde as a protein crosslinking agent acting during the frozen storage of cod. J. Sci. Fd Agric, 26:1925-1929.
DAMBERGS, N. 1963. Extractives of fish muscle. 3. Amounts, sectional distributions, and variations of fat, water-solubles, protein and moisture in cod (Gadus Morhua L.) fillets. J. Fish. Res. Bd Can., 20(4):909-918.
de LIGNY, W. 1969. Serological and biochemical studies on fish populations. Oceanogr. mar. Biol., A. Rev., 7:411-513.
FUJINO, K. 1969. Atlantic skipjack tuna genetically distinct from Pacific specimens. Copeia, (3):626-629.
______ 1971. Genetic markers in skipjack tuna from the Pacific and Atlantic Oceans. Rapp. P.-v. Réun. Cons. perm. int. Explor. Mer,161, section 1:15-18.
______ 1976. Subpopulation identification of skipjack tuna specimens from the southwestern Pacific Ocean. Bull. japan. Soc. scient. Fish., 42(11):1229-1235.
______ & RANG, T. 1968. Transferrin groups of tunas. Genetics, 59:79-91.
GORNALL, A. C. ; BARDAWILL, C . S. & DAVID , M. M. 1949. Determination of serum proteins by means of biuret reaction. J. Biol. Chem., 177-751-766.
HAEN, P. J. & O'ROURKE, F. J. 1969. Comparative electrophoretic studies of the water-soluble muscle proteins of some Irish freshwater fishes. Proc. R. Ir, Acad., 68, (B7):101-108.
HAMOIR, G. 1955. Fish proteins. In: Anson, M. L.; Bailey, K. & Edsall, J. T., ed. - Advances in protein chemistry. New York, Academic Press, v. 10, p.227-288.
HOWLET, G. & JAMIESON, A. 1971. A system of muscle esterase in the variants in the sprat (Sprattus sprattus), Rapp. p. -v. Réun. Cons, perm. int. Explor. Mer, 161, section 2:45-47.
HUEHNS, E. R, 1968. Starch gel electrophoresis, section 4 Hemoglobins. In: Smith, I., ed - Chromatographic and electrophoretic techniques. William Heinemann Medical Books, v. 2, p.291-319.
KAWABATA, T. 1953. Studies on trimethylamine oxide-redutase. I. Reduction of trimethylamine oxide in the dark muscle of pelagic migrating fish under aseptic condition. Bull. japan. Soc, scient. Fish., 19(4):505-512.
LOVE, R. M. 1970. The chemical biology of fishes. London, Academic Press, 547p.
MOORE, G. S.; PETERS, H. A. & LEVIN, R. E. 1970. Alterations in the electrophoretic patterns of refrigerated fish. J. Fish. Res. Bd Can., 27:31-38.
MORGAN, R. P. & ULANOWICZ, N. I. 1976. The frequency of muscle protein polymorphism in Menidia menidia(Atherinidae) along the Atlantic coast. Copeia, (2):356-360.
NUMACHI, K. I. 1972, Genetic poly morphism in enzymes and its research applications in fisheries biology. The 2nd International Ocean Developmerit Conference. Preprints (2):1813-1822. Tokyo, October 5-7. Tokyo, Keidanren Kaikan.
SCOPES, R. K. 1968. Methods for starch gel electrophoresis of sarcoplasmic proteins. An investigation of the relative mobilities of the glycolytic enzymes from the muscles of a variety of species. Biochem. J., 107:139-150.
SICK, K. 1965a. Haemoglobin polymorphism of cod in the Baltic and Danish Belt Sea. Hereditas, 54(2): 19-48.
______ 1965b. Haemoglobin polymorphism of cod in the North Sea and North Atlantic Ocean. Hereditas, 54(3):49-73.
SMITH, I. 1968. General principles of zone electrophoresis. In: Smith, I., ed. - Chromatographic and electrophoretic techniques, v. 2, p. 1-16.
SMITHIES, O. 1955. Zone electrophoresis in starch gels: group variations in the serum protein of normal human adults. Biochem. J., 61:629-641.
STECHER, P. G.; WINDHOLZ, M. & LEAHY, D. S. 1968. The Merck index. An encyclopedia of chemicals and drugs. 8th ed. Rahway, N.J., Merck & Co., Inc., 1713p.
TANIGUCHI, N. 1969. Comparative electropherograms of muscle proteins of three species of lizard fishes referable to the genus Saurida Bull. japan. Soc. scient. Fish., 35(9):885-890.
TANIGUCHI, N. & KONISHI, Y. 1971. Muscle protein polymorphism in frigate mackerel collected from the coastal region of Kochi Pref., Japan. Bull. japan. Soc. scient. Fish., 37(7):571-576.
TSAI, H. J. & YANG, R. T. 1975. Electropherograms of muscle extracts of tunas from the waters around Taiwan. Acta oceanogr. taiwan., (5): 131-138.
TSUYUKI, H. 1974. Muscle proteins of fishes. In: Florkin, N. & Scheer, B. T., ed. - Chemical zoology. New York, Academic Press, v. 3, p. 287-305.
THURSTON, C. E. & MacMASTER, P. P. 1960. Variations in chemical composition of different parts of halibut flesh. Fd Res., 25:229-236.
UTTER, F. M.; HODGINS, H. O. & ALLENDORF, F. W. 1974. Biochemical genetic studies of fishes: potentialities and limitations. In: Mallins, D. C. & Sargent, J. R., ed. - Biochemical and biophysical perspectives in marine biology. London, Academic Press, v. 1, p. 213-238.
______ & JOHNSON, A. G. 1972. Biochemical studies of genetics differences among species and stocks of fish. Rep. int. N. Pacif. Fish. Commn, :98-101.

(*

) Trabalho realizado com apoio financeiro da FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. 74/816, Proc. 75/387 e Proc. 76/262).

(**

) Aluna do Curso de Pós-Graduação do IOUSP.

(***

) Atualmente no INPA - Instituto Nacional de Pesquisas da Amazônia.

Datas de Publicação

Publicação nesta coleção
31 Maio 2012
Data do Fascículo
1983

Histórico

Aceito
10 Fev 1984
Recebido
09 Maio 1983

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] AMANO, K.; BITO, M. & KAWABATA, T. 1953. Handling effect upon biochemical change in the fish muscle immediately after catch. I. Differences of glycolysis in the frigate mackerel killed by various methods. Bull, japan. Soc. scient. Fish., 19 (4):487-498.

[2] AMLACHER, E. 1961. Rigor-mortis in fish. In: Borgstrom, G., ed. -Fish as food. New York, Academic Press, v.1, p. 385-409.

[3] ANALYTICAL METHODS COMMITTEE 1966. Nitrogen factor for cod fish. Report prepared by the Fish Products Sub-Committee. Analyst, Lond., 91(1085): 540-542.

[4] BECHTEL, M. A. B. & ALVES, J. L. P. 1973. Identificação de espécies de pescado por eletroforese em acetato de celulose. Sér. Tecnol., (3)-GEDIP. Porto Alegre.

[5] BHUSHANA RAO, K. S. P.; FOCANT, B.; GERDAY, Ch. & HAMOIR, G. 1969. Low molecular weight albumins of cod white muscle (Gadus callarias). Comp. Biochem. Physiol., 30:33-48.

[6] BOSCH, M. W. 1951. Quantitative electrophoretic study of rabbit muscle proteins soluble in dilute salt solutions. Biochem. Biophys. Acta, 7:61-86.

[7] CANTAROW, A. & SCHEPARTZ, B. 1962. Biochemistry. Philadelphia, Maruzen Asian Edition, 854p.

[8] CONNELL, J. J. 1953. Studies on the proteins of fish skeletal muscle. 1. Electrophoretic analysis of codling extracts of low ionic strength. Biochem. J., 54:119-126.

[9] ______ 1975. The role of formaldehyde as a protein crosslinking agent acting during the frozen storage of cod. J. Sci. Fd Agric, 26:1925-1929.

[10] DAMBERGS, N. 1963. Extractives of fish muscle. 3. Amounts, sectional distributions, and variations of fat, water-solubles, protein and moisture in cod (Gadus Morhua L.) fillets. J. Fish. Res. Bd Can., 20(4):909-918.

[11] de LIGNY, W. 1969. Serological and biochemical studies on fish populations. Oceanogr. mar. Biol., A. Rev., 7:411-513.

[12] FUJINO, K. 1969. Atlantic skipjack tuna genetically distinct from Pacific specimens. Copeia, (3):626-629.

[13] ______ 1971. Genetic markers in skipjack tuna from the Pacific and Atlantic Oceans. Rapp. P.-v. Réun. Cons. perm. int. Explor. Mer,161, section 1:15-18.

[14] ______ 1976. Subpopulation identification of skipjack tuna specimens from the southwestern Pacific Ocean. Bull. japan. Soc. scient. Fish., 42(11):1229-1235.

[15] ______ & RANG, T. 1968. Transferrin groups of tunas. Genetics, 59:79-91.

[16] GORNALL, A. C. ; BARDAWILL, C . S. & DAVID , M. M. 1949. Determination of serum proteins by means of biuret reaction. J. Biol. Chem., 177-751-766.

[17] HAEN, P. J. & O'ROURKE, F. J. 1969. Comparative electrophoretic studies of the water-soluble muscle proteins of some Irish freshwater fishes. Proc. R. Ir, Acad., 68, (B7):101-108.

[18] HAMOIR, G. 1955. Fish proteins. In: Anson, M. L.; Bailey, K. & Edsall, J. T., ed. - Advances in protein chemistry. New York, Academic Press, v. 10, p.227-288.

[19] HOWLET, G. & JAMIESON, A. 1971. A system of muscle esterase in the variants in the sprat (Sprattus sprattus), Rapp. p. -v. Réun. Cons, perm. int. Explor. Mer, 161, section 2:45-47.

[20] HUEHNS, E. R, 1968. Starch gel electrophoresis, section 4 Hemoglobins. In: Smith, I., ed - Chromatographic and electrophoretic techniques. William Heinemann Medical Books, v. 2, p.291-319.

[21] KAWABATA, T. 1953. Studies on trimethylamine oxide-redutase. I. Reduction of trimethylamine oxide in the dark muscle of pelagic migrating fish under aseptic condition. Bull. japan. Soc, scient. Fish., 19(4):505-512.

[22] LOVE, R. M. 1970. The chemical biology of fishes. London, Academic Press, 547p.

[23] MOORE, G. S.; PETERS, H. A. & LEVIN, R. E. 1970. Alterations in the electrophoretic patterns of refrigerated fish. J. Fish. Res. Bd Can., 27:31-38.

[24] MORGAN, R. P. & ULANOWICZ, N. I. 1976. The frequency of muscle protein polymorphism in Menidia menidia(Atherinidae) along the Atlantic coast. Copeia, (2):356-360.

[25] NUMACHI, K. I. 1972, Genetic poly morphism in enzymes and its research applications in fisheries biology. The 2nd International Ocean Developmerit Conference. Preprints (2):1813-1822. Tokyo, October 5-7. Tokyo, Keidanren Kaikan.

[26] SCOPES, R. K. 1968. Methods for starch gel electrophoresis of sarcoplasmic proteins. An investigation of the relative mobilities of the glycolytic enzymes from the muscles of a variety of species. Biochem. J., 107:139-150.

[27] SICK, K. 1965a. Haemoglobin polymorphism of cod in the Baltic and Danish Belt Sea. Hereditas, 54(2): 19-48.

[28] ______ 1965b. Haemoglobin polymorphism of cod in the North Sea and North Atlantic Ocean. Hereditas, 54(3):49-73.

[29] SMITH, I. 1968. General principles of zone electrophoresis. In: Smith, I., ed. - Chromatographic and electrophoretic techniques, v. 2, p. 1-16.

[30] SMITHIES, O. 1955. Zone electrophoresis in starch gels: group variations in the serum protein of normal human adults. Biochem. J., 61:629-641.

[31] STECHER, P. G.; WINDHOLZ, M. & LEAHY, D. S. 1968. The Merck index. An encyclopedia of chemicals and drugs. 8th ed. Rahway, N.J., Merck & Co., Inc., 1713p.

[32] TANIGUCHI, N. 1969. Comparative electropherograms of muscle proteins of three species of lizard fishes referable to the genus Saurida Bull. japan. Soc. scient. Fish., 35(9):885-890.

[33] TANIGUCHI, N. & KONISHI, Y. 1971. Muscle protein polymorphism in frigate mackerel collected from the coastal region of Kochi Pref., Japan. Bull. japan. Soc. scient. Fish., 37(7):571-576.

[34] TSAI, H. J. & YANG, R. T. 1975. Electropherograms of muscle extracts of tunas from the waters around Taiwan. Acta oceanogr. taiwan., (5): 131-138.

[35] TSUYUKI, H. 1974. Muscle proteins of fishes. In: Florkin, N. & Scheer, B. T., ed. - Chemical zoology. New York, Academic Press, v. 3, p. 287-305.

[36] THURSTON, C. E. & MacMASTER, P. P. 1960. Variations in chemical composition of different parts of halibut flesh. Fd Res., 25:229-236.

[37] UTTER, F. M.; HODGINS, H. O. & ALLENDORF, F. W. 1974. Biochemical genetic studies of fishes: potentialities and limitations. In: Mallins, D. C. & Sargent, J. R., ed. - Biochemical and biophysical perspectives in marine biology. London, Academic Press, v. 1, p. 213-238.

[38] ______ & JOHNSON, A. G. 1972. Biochemical studies of genetics differences among species and stocks of fish. Rep. int. N. Pacif. Fish. Commn, :98-101.

Brasil