Quantitation of collagen fragments and gelatin by deconvolution of polarimetry denaturation curves

A method for quantitating nicked or shortened molecules (fragments) in pepsinized bovine type I collagen preparations using polarimetry thermal denaturation curves is described. The shortened molecules denature about 4 degrees C lower than intact collagen molecules. The analog output of a polarimeter was digitized and stored on a microcomputer disk. A BASIC program was written which retrieves the specific rotation data from the disk, smooths the data with a boxcar average, and plots the derivative of the denaturation curve. The derivative curve was deconvoluted by fitting three Gaussian curves to the derivative curve using published algorithms. The area of the Gaussian centered at 37 degrees C was proportional to the amount of collagen fragments. A good correlation between the amount of fragments determined by polarimetry and by a trypsin sensitivity assay was observed. The overall precision of the method was about 10% RSD, and the method was repeatable by multiple analysts. Application of the method to reconstituted fibrillar collagen samples showed that more fragments are generated when pepsin digestion time is lengthened. By fitting a fourth Gaussian component to the derivative curve, the method can also be used to determine relative amounts of denatured collagen (helix partially unwound but alpha chains not nicked). The detection limit for denatured collagen is about 20%.