A chitinase was obtained in milligram amounts from wheat germ by affinity chromatography on chitin followed by chromatography on Sephadex G-50. The purified enzyme was free from wheat germ agglutinin and showed a single peak on sodium dodecyl sulfate-acrylamide gel electrophoresis. Isoelectric focusing on acrylamide gel slabs gave rise to several bands, with isoelectric points ranging between 7.5 and 9.2. The MW of the enzyme, as determined from sodium dodecyl sulfate-gel electrophoresis, was 30,000 and that determined from sedimentation analysis was 33,000; the enzyme exists as a monomer in solution. The enzyme releases oligosaccharides 2-4 units in length from chitin, i.e., it is an endochitinase. When allowed to act on nascent chitin, i.e., chitin that is being synthesized by a preparation of solubilized chitin synthetase, the chitinase shows an enhancement in activity of about 2 orders of magnitude. The products formed under these conditions consist of higher oligosaccharides than those obtained with preformed chitin substrate. Possibly the nascent chitin is more susceptible to the enzyme because its chains have not yet coalesced with others to yield a tighter and more impervious structure. Such a situation may prevail during turnover of structural polysaccharides in vivo; in vitro measurement of polysaccharide breakdown concomitant to synthesis may reflect conditions in the cell better than the static methods usually employed. A method for the separation and measurement of chitin, chitin oligosaccharides and UDP-N-acetylglucosamine by chromatography on DEAE-cellulose paper is also described.
