Affinity Chromatography of Collagen Glycosyltransferases on Collagen Linked to Agarose

Abstract

Denatured citrate-soluble [rat skin] collagen was coupled to agarose by the CNBr activation technique, and columns prepared from this material were studied for affinity chromatography of collagen glycosyltransferases. Both collagen glycosyltransferases became bound to the column, the degree of binding and the capacity of the column being higher with the glucosyltransferase than with the galactosyltransferase. The addition of Mn2+ enhanced the binding, especially with the glucosyltransferase. The enzymes were eluted from the column with small peptides prepared from collagen and separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase purification of about 5000-fold and a collagen galactosyltransferase purification of about 1000-fold was obtained from chick embryo extract by relatively simple steps.