ZINC CONTENT AND FUNCTION IN HUMAN FIBROBLAST COLLAGENASE

The zinc contents of samples of human fibroblast collagenase (HFC) pur ified by different procedures and of samples purified by the same proc edure but prepared for analysis by different dialysis protocols have b een determined by atomic absorption spectroscopy. Both the purificatio n method and dialysis conditions affect the zinc stoichiometry. Sample s purified with and without the use of a zinc-chelate chromatography s tep and prepared by dialysis against 1 mM CaCl2 had zinc to enzyme rat ios of 1.46 and 1.22, respectively. When the first sample was prepared by dialysis against 0 and 10 mM CaCl2, the values changed to 0.15 and 1.94, respectively. Thus, the zinc content of HFC is critically depen dent upon the dialysis conditions used to free the enzyme from adventi tious metals. This could account for the disparate reports in the lite rature that give zinc stoichiometries for members of the matrix metall oproteinase (MMP) family of between 1 and 2. The mechanism of inhibiti on of the one zinc form of HFC by 1,10-phenanthroline (OP) and 4-(2-py ridylazo)resorcinol has been studied in detail. Inhibition by both che lating agents is time dependent and biphasic. There is an initial, ins tantaneous inhibition characterized by the involvement of a single inh ibitor molecule that corresponds to the formation of a ternary complex between the zinc atom, enzyme, and chelator. This is followed by a se cond, slower phase involving removal of the zinc atom from the enzyme and its chelation by two molecules of inhibitor. Inhibition of four ot her human MMPs by OP shows similar characteristics and is thought to o ccur by the same mechanism.