Purification, peptide mapping and spectroscopic characterization of myoglobin from striped snake-head fish (ophicephalus striatus)

Abstract

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 kDa as major band and trace of 20 kDa compared with those of MALDI-TOF-MS: 15,597.68 Da (major peak) and 23,509.42 Da (trace). Thus, this 15 kDa protein wouldbe the fish Mb which was strongly coupled withother 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.