Analysis of collagen type III by uninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotting: Changes in collagen type III polymorphism in aging rats

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Abstract
A new method of type III collagen analysis by uninterrupted sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) combined with immunoblotting was developed. The electrophoresis was carried out with gels containing 4 M urea. A negatively charged reducing agent, thioglycolic acid, was added to the running buffer of the cathodic reservoir between 15 and 20 min after Bromphenol Blue (BPB) migrated to the top of the separating gel, to reduce interchain disulfide binding of the collagen. The polymorphic type III collagens, i.e., an α‐chain derived from a trimer [α1(III)]3 with interchain disulfide bonds but without covalent cross‐links, α1(III), a β‐chain with covalent cross‐links, β(III), or an α‐chain released from a trimer without reduction of the disulfide bonds, α*1(III), were identified by immunostaining and quantified by densitometry. Using this method, changes in collagen type III polymorphism with aging were examined in the aorta, brachial artery, and skin of rats. The total quantity of collagen type III decreased with aging in all tissues. β(III) was the major component in the aorta and brachial artery, but α1(III) was the major component in the skin. With increasing age from 3 to 60 weeks, the ratio of β(III) to α1(III), which is correlated with the extent of covalent cross‐linking, showed a steep increase in the aorta but only a slight increase in the skin and it remained almost constant in the brachial artery. The ratio of α*1(III) to α1(III), which is correlated with the extent of degradation, increased steeply from 12 weeks to 60 weeks in the aorta; in the brachial artery and the skin, however, it increased up to the 22nd week and was, thereafter, followed by almost constant values. These findings are characteristic of the profile of collagen type III metabolism in each of these tissues with aging.