Studies on collagen. II. Properties of purified collagenase and its inhibition
Journal of Biological Chemistry
In previous papers from these laboratories (1, 2) methods for the purification of the collagenase of Clostridiurm histolyticum were presented, and a calcium ion requirement for the binding of the enzyme to collagen and for the overall enzymatic action was reported. In the present, communication an additional method of purification, which makes possible the large-scale preparation of the enzyme, is described. The sedimentation and diffusion constants and molecular weight of the purified enzyme
... e purified enzyme are also reported. The nature of the end products resulting from the action of collagenase on the citrate-extractable collagens of carp swim bladder and calfskin, as well as on the gelatins derived from these, is considered. We shall also describe the action of the enzyme on collagen in the presence of its derived gelatin; the failure of the enzyme to act on a number of synthetic peptides, amino acid polymers, and esters; and the inhibition of purified collagenase by sulfhydryl-containing substances and by certain metal-sequestering agents. MATERIALS AND METHODS Crude Enzynze-The particular strain of Clostridium histolyticum used in these studies was obtained through the generosity of Dr. Ines Mandl of the College of Physicians and Surgeons, Columbia University. After the organism was plated on anaerobic agar, individual colonies mere selected for culture in the medium suggested by Maclennan, Mandl, and Howes (3). There was some variation in the amount of enzyme produced by subcultures of different colonies, and one was selected which yielded the highest amount of collagenase and relatively little nonspecific proteinase activity. Batches of 10 liters or more of medium were inoculated with this organism, and after a period of growth of approximately 40 hours, the culture was passed through a bacteriological filter. To each liter of filtrate was then added 400 gm. of ammonium sulfate, and the mixture allowed to stand overnight at about 5". The precipitated protein, which had risen to the surface, was scooped off, dissolved in distilled water, and the solution dialyzed for 24 hours against cold running tap water. The dark brown solut,ion was lyophilized. The use of proteose peptone (Difco) in the recommended culture medium has been found to be essential for the masimal yield of collagenase.