Purification and immunochemical detection of a quantitatively major collagen in the dermis of sea cucumber Apostichopus japonicus

• Published in: Fisheries science Vol. 88; no. 1; pp. 173 - 180
• Main Authors: Mizuta, Shoshi, Koizumi, Yuuki, Yokoyama, Yoshihiro, Yoshinaka, Reiji
• Format: Journal Article
• Language: English
• Published: Tokyo Springer Japan 2022; Springer Nature B.V
• Subjects: 2-Mercaptoethanol; Acetic acid; Analytical methods; Apostichopus japonicus; Biomedical and Life Sciences; Collagen; Dermis; Digestion; Disaggregation; Electrophoresis; Ethylenediaminetetraacetic acids; Fibers; Fish & Wildlife Biology & Management; Food Science; Freshwater & Marine Ecology; Gel electrophoresis; Gels; Guanidine hydrochloride; Life Sciences; Marine invertebrates; Original Article; Pepsin; Phosphates; Polyacrylamide; Sodium; Sodium chloride; Sodium dodecyl sulfate; Sodium hydroxide; Sodium lauryl sulfate; Urea; Water purification; Sea cucumber; Body wall; Collagen; Echinoderm; Dermis; Protein; Holothurid
• ISSN: 0919-9268; 1444-2906
• DOI: 10.1007/s12562-021-01564-z

Pepsin-solubilized collagen (PSC) was prepared from the dermis of sea cucumber Apostichopus japonicus (green type) by performing pepsin digestion to collagen fiber pretreated with disaggregating solution (0.1 M Tris–HCl, pH 8.0, containing 0.5 M NaCl, 0.05 M ethylenediaminetetraacetic acid (EDTA), and 0.2 M 2-mercaptoethanol) and 0.1 M NaOH. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the PSC clearly showed two alpha bands under phosphate buffer system in the presence of 3.5 M urea. An antiserum was raised against chromatographically purified major molecular species in the PSC, and immunoblot analyses were performed for the soluble fractions at 4 M guanidine hydrochloride treatment and disaggregation as well as the collagen fiber before and after treatment. These fractions and collagen fibers showed quite similar band patterns to that of the PSC, showing mainly two alpha bands. These combined results suggest that the major molecular species of collagen contains at least two distinct alpha components and that the effect of pepsin digestion is relatively small on the structure of this collagen type.