Quantitation of type I and III collagens using electrophoresis of alpha chains and cyanogen bromide peptides

Abstract

The methods of quantitating the relative amounts of type I and III collagens in samples containing crosslinked collagen chains were evaluated using electrophoresis of alpha chains and cyanogen bromide peptides. The densitometry areas of the αI(I) chains from type I collagen and the αI(III) chains from type III collagen were reduced because of the failure of the crosslinked chains to dissociate. However, the ratios of the unit densitometry areas of these chains (area of chain/μg type I or III collagen loaded) were constant for type I and III collagens prepared from the same samples of tissue. A calibration factor, which was the same for dermis and mitral valve, was derived to convert the densitometry areas of the αI(I) CB8 (type I collagen marker) and the αI(III) CB5 (type III collagen marker) were not reduced by crosslinked collagen chains. A calibration factor was also derived to convert the ratios of the densitometry areas of the marker peptides to weight ratios of type I or III collagens. Almost identical results were obtained when electrophoresis of alpha chains and of cyanogen bromide peptides was used with these calibration factors that quantitate the relative amounts of type I and III collagens in tissue extracts which contained different amounts of crosslinked chains.