Zinc content and function in human fibroblast collagenase.
The zinc contents of samples of human fibroblast collagenase (HFC) purified by different procedures and of samples purified by the same procedure but prepared for analysis by different dialysis protocols have been determined by atomic absorption spectroscopy. Both the purification method and dialysis conditions affect the zinc stoichiometry. Samples purified with and without the use of a zinc-chelate chromatography step and prepared by dialysis against 1 mM CaCl2 had zinc to enzyme ratios 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 dependent upon the dialysis conditions used to free the enzyme from adventitious metals. This could account for the disparate reports in the literature that give zinc stoichiometries for members of the matrix metalloproteinase (MMP) family of between 1 and 2. The mechanism of inhibition of the one zinc form of HFC by 1,10-phenanthroline (OP) and 4-(2-pyridylazo)resorcinol has been studied in detail. Inhibition by both chelating agents is time dependent and biphasic. There is an initial, instantaneous inhibition characterized by the involvement of a single inhibitor molecule that corresponds to the formation of a ternary complex between the zinc atom, enzyme, and chelator. This is followed by a second, slower phase involving removal of the zinc atom from the enzyme and its chelation by two molecules of inhibitor. Inhibition of four other human MMPs by OP shows similar characteristics and is thought to occur by the same mechanism.
D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D020784:Matrix Metalloproteinase 8; D061965:Matrix Metalloproteinase Inhibitors; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012118:Resorcinols / Q000494:pharmacology; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D061965:Matrix Metalloproteinase Inhibitors; D010618:Phenanthrolines; D011480:Protease Inhibitors; D012118:Resorcinols; D017364:Collagenases; D020784:Matrix Metalloproteinase 8; D015032:Zinc; C028398:4-(2-pyridylazo)resorcinol; C025205:1,10-phenanthroline