Extraction and isolation of type I, III and V collagens and their SDS-PAGE analyses

Abstract

Type I, III and V collagens were extracted from bovine dermis and cornea by using pepsin treatment in acetic acid solution, followed by salt precipitation and dialysis, to purify and isolate each type of collagens. The preparation process was analyzed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A reducing agent, 2-mercaptoethanol, was used to remove disulfide bonds and analyze the structure of the bonds involved between α chains in some types of collagens. The use of delayed reducing methods resulted in the difference between α1(III) and α 1(I) chains in a mixture containing type I and III collagens. The structure of disulfide bonds among α chains exists potentially in type V collagen prepared from the pepsin-treatment extraction at 4 °C, which differs from type III collagen in relation to the locations of disulfide bonds. Compared with pepsin-treated collagen at 4 °C, the relative molecular weights of α1(V) and α2(V) chains treated at room temperature decrease by 4.6% and 6.0%, respectively. It is concluded that type I, III and V collagens can be prepared from bovine dermis and cornea by the use of pepsin treatment, salt precipitation and dialysis. The interchain disulfide bonds lie potentially near the edges of termini of type V collagen molecules in extracellular matrix, and a small number of interchain crosslinks exist in type V collagen.