Abstract

Chattrachatchaya Chotichayapong¹, Nison Sattayasai², Nobuyuki Kanzawa³, Toru Tamiya³, Takahide Tsuchiya³ and Saksit Chanthai¹*

DOI : http://dx.doi.org/10.13005/ojc/320119

Myoglobin (Mb) was isolated from ordinary muscle of striped snake-head fish. The crude extract was fractionated by salting out and separated using both Sephadex G-75 and DEAE-cellulose column chromatography and molecular filtration, giving at least two protein bands on SDS-PAGE with molecular mass of 15 kDaas major band and trace of 20 k Dacompared with those of MALDI-TOF-MS: 15,597.68 Da (major peak) and 23,509.42 Da (trace). Thus, this 15 kDa protein would be the fish Mb which was strongly coupled with other proteins of 23.50 kDa. Spectral characteristics of these purified proteins still exhibited maximum absorption at 280 and 410 nm (the Soret peak). Their tryptophan fluorescence appeared at 330 nm also corresponding with horse heart Mb. The partial peptide sequences of 15 kDa protein obtained from LC-MS/MS with in-gel trypsin digestion were homologous with those of some fish species. In addition, the purified Mb was also determined for its partial peptide sequence by digestion with Lys-C endoproteinase and analysis of amino acid sequence using Edman degradation, resulted in only some amino acid residues are identical to Mb from other fish species. It is noted that differences in their amino acid sequences are attributed among fish Mb species of which playing distinct roles of both structural rigidity and functional property.

Myoglobin; Peptide mapping; Edman degradation; Protein purification and characterization

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