Use of galactose oxidase in labelling hydroxylysine glycosides of collagen.
Commercial preparations of galactose oxidase were shown to contain a proteolytic component active against collagen and collagen CNBr peptides which could not be inhibited by reagents such as EDTA, PMSF, PCMB and TLCK nor activated by Ca++ or Mn++. 30% of the total galactose oxidase activity could be recovered free of proteolytic activity in a simple one-step combined gel filtration/affinity chromatography procedure. The biological activity of such preparations was tested with reference to collagen hydroxylysine glycosides. It was shown that over 70% of the covalently linked tritium label introduced by KB3H4 reduction after galactose oxidase treatment was not associated with galactose but was present in the same non-specifically labelled components in control experiments. The enzyme could be used in a controlled manner to label hydroxylysine glycosides in collagen by immobilising it on agarose beads. In this form it was easy to store, re-usable and retained 95% of its original activity even after prolonged (18 h) incubation with substrates.
D003094:Collagen; D003488:Cyanogen Bromide; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005692:Galactose Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D006901:Hydroxylysine / Q000031:analogs & derivatives* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011865:Radioisotope Dilution Technique; D014316:Tritium
D004800:Enzymes, Immobilized; D010446:Peptide Fragments; C022819:hydroxylysine glycoside; D014316:Tritium; D006901:Hydroxylysine; D003094:Collagen; D005692:Galactose Oxidase; D003488:Cyanogen Bromide