Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers.
The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6-A11, A7-B10, and A20-B22. An intermolecular bond of A20-B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6-A11, A7-B10, A20-B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6-A7, A11-B10, A20-B22-cystine]- and [A6-B10, A7-A11, A20-B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.
Maruyama
K
K
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Nagasawa
H
H
Isogai
A
A
Ishizaki
H
H
Suzuki
A
A
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000138:chemical synthesis / Q000737:chemistry*; D007301:Insect Hormones / Q000138:chemical synthesis / Q000737:chemistry*; D019476:Insect Proteins; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D013237:Stereoisomerism; D013820:Thermolysin / Q000737:chemistry
D004220:Disulfides; D007301:Insect Hormones; D019476:Insect Proteins; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D010455:Peptides; C064115:bombyxin IV protein, Bombyx mori; D013820:Thermolysin