Immunoblotting identification of jumbo squid (Dosidicus gigas) LOX isoforms and in vitro crosslinking assay over selected collagenous materials

Abstract

Jumbo squid (Dosidicus gigas) muscle hardness has been related to hydroxylysyl-pyridinoline (HP) formation (collagen fibre stabilization) via condensation of oxidized ε-amino groups of lysine and/or hydroxylysine by lysyl oxidase (LOX). Previously published literature has suggested the presence of LOX isoforms in squid muscle. Thus, the objective of the present research was to test the hypothesis that squid LOX isoforms exist in fresh mantle muscle. A semi-pure LOX extract (SPLE) was used to perform an immunoblotting assay using two commercial human-LOX antibodies. Furthermore, SPLE (specific activity of 20 mU/mg of protein) was tested in vitro for possible HP formation using several collagenous materials. Immunoblotting assay confirmed the presence of squid LOX at 32 kDa and its isoforms at 34 kDa and 24 kDa. Although LOX oxidized ε-amino groups on collagenous materials, no HP formation was detected. However, the latter could promote interesting collagen functional modifications.