Extraction of Type V Collagen From Bovine Cornea and Its Structural Analysis

摘要

We used bovine cornea as starting material, pepsin treatment in acetic acid solution to extract the mixture of typeⅠandⅤcollagens, and salt precipitation and dialysis to purify and isolate each type of the collagens. The preparation was analyzed using sodium dodecyl sulphate polyacrylamide gel electrophoresis. 2-mercaptoethanol used as reducing agent cut off the disulfide bonds, which was utilized to analyze the structure of disulfide bonds involved betweenαchains in some types of collagens. At the same time, we discovered that the structure of disulfide bonds amongαchains potentially existed in the typeⅤcollagen prepared from the pepsin-treatment extraction at 4℃. Through quantitative analysis, we obtained that, compared with those pepsin-treated at 4℃, the relative molecular weights ofα1(Ⅴ) andα2(Ⅴ) subunits pepsin-treated at room temperature decreased by 4.6% and 6.0%, respectively. It is concluded that typeⅤcollagen can be prepared from bovine cornea by use of pepsin treatment, salt precipitation and dialysis. The interchain and/or intermolecular disulfide bonds potentially lie near the edges of termini of typeⅤcollagen molecules existing in extracellular matrix, and there are few of the intermolecular and/or intramolecular crosslinks formed by lysine or hydroxylysine or histidine residues in typeⅤcollagen.