Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment α. Acetonitrile gradients at acid and/or neutral pH using μBondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, αCB1 and αCB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on μBondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of αCB2 (95 residues), αCB3 (67 residues) and αCB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment αCB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305–316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120 279–287], the present results enable us to present the complete sequence of fragment α (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alternation of α helices and β structure. The possibility that the non-heme-binding portion of fragment α could constitute a flavin-binding domain is discussed.