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Related Experiment Videos

Zinc content and function in human fibroblast collagenase

E B Springman1, H Nagase, H Birkedal-Hansen

  • 1Institute of Molecular Biophysics, Florida State University, Tallahassee 32306, USA.

Biochemistry
|December 5, 1995
PubMed
Summary

The zinc content of human fibroblast collagenase (HFC) is highly sensitive to purification and dialysis methods. Proper dialysis conditions are crucial for accurate zinc stoichiometry in matrix metalloproteinases (MMPs).

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Area of Science:

  • Biochemistry
  • Enzymology
  • Metalloproteinases

Background:

  • Human fibroblast collagenase (HFC) is a matrix metalloproteinase (MMP) involved in tissue remodeling.
  • Previous studies reported varying zinc stoichiometries for MMPs, leading to inconsistencies in understanding their function.
  • The role of zinc in MMP activity and the impact of experimental conditions on its stoichiometry require clarification.

Purpose of the Study:

  • To determine the zinc content of HFC using atomic absorption spectroscopy.
  • To investigate how different purification methods and dialysis protocols affect the zinc stoichiometry of HFC.
  • To elucidate the mechanism of inhibition of HFC by chelating agents like 1,10-phenanthroline (OP).

Main Methods:

  • Atomic absorption spectroscopy was used to quantify zinc content in HFC samples.

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  • HFC was purified using different procedures, including zinc-chelate chromatography.
  • Samples were prepared using various dialysis protocols against different calcium chloride (CaCl2) concentrations.

    • Main Results:

    • Both purification methods and dialysis conditions significantly influenced the zinc-to-enzyme ratio in HFC.
    • Dialysis against 1 mM CaCl2 yielded zinc ratios of 1.46 (with zinc-chelate chromatography) and 1.22 (without).
    • Varying CaCl2 concentrations (0 mM and 10 mM) resulted in zinc ratios of 0.15 and 1.94, respectively, for the same sample.

    Conclusions:

    • The zinc content of HFC is critically dependent on the dialysis conditions used to remove adventitious metals.
    • These findings may explain the disparate zinc stoichiometries reported in the literature for MMPs.
    • Inhibition of HFC by 1,10-phenanthroline and other chelators involves a biphasic mechanism, including ternary complex formation and zinc removal, consistent across other human MMPs.