title: The chromatographic purification of native types I, II, and III collagens.
abstract: In this paper, a chromatographic method for the purification of native types I, II, and III collagen is described. The method consists of two consecutive gel permeation chromatography steps, followed by anion-exchange chromatography. The two consecutive gel permeation chromatography steps take advantage of the fact that collagens like other asymmetric molecules, elute anomalously late from gel permeation columns, thus allowing one to separate collagens from less asymmetric proteins of comparable molecular weight, notably gelatin, procollagen and higher molecular weight oligomers of collagen. The anion-exchange chromatography separates types I, II, and III collagens from each other with baseline resolution. The collagen products obtained from these procedures are at least 99% pure by a variety of criteria, and in the native state by the traditional criteria of optical rotation, intrinsic viscosity, solubility properties and resistance to non-collagenase proteases. Rat skin type I collagen prepared by this chromatographic method exhibits a higher and sharper thermal transition temperature than an otherwise identical sample of rat skin type I collagen prepared by fractional salt precipitation. In addition, the latter collagen is more susceptible to digestion by trypsin at 37 degrees C. We conclude that salt precipitation of the collagen per se is responsible for a lowering of the Tm values. Our observations indicate that the chromatographic purification of collagen preserves the native structure at a few select sites where high salt concentrations induce irreversible local imperfections of the three-dimensional structure.
mesh_terms: D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000302:isolation & purification*; D006358:Hot Temperature; D008297:Male; D051381:Rats; D012867:Skin / Q000032:analysis; D014357:Trypsin
chemical_list: D003094:Collagen; D014357:Trypsin