Purification to homogeneity of latent and active 58-kilodalton forms of human neutrophil collagenase.
Latent and active 58-kDa forms of human neutrophil collagenase (HNC) have been purified to homogeneity. Buffy coats were extracted in the presence and absence of phenylmethanesulfonyl fluoride to generate crude starting preparations that contained latent and active HNC, respectively. The buffers used in preparing these extracts and for all subsequent chromatographic steps contained NaCl at a concentration of 0.5 M or greater, 0.05% Brij-35, concentrations of CaCl2 of 5 mM or greater, and (when feasible) 50 microM ZnSO4 to stabilize the HNC. The collagenase activity in the buffy coat extracts was adsorbed to a Reactive Red 120-agarose column at pH 7.5 in 0.5 M NaCl and was eluted when the NaCl concentration was increased to 1 M. The active and p-(chloromercuri)benzoate-activated latent enzymes were next adsorbed to a Sepharose-CH-Pro-Leu-Gly-NHOH affinity resin in 1 M NaCl at pH 7.5 and desorbed at pH 9 to give a fraction containing only HNC and a small amount of neutrophil gelatinase. The latter enzyme was removed by passage over a gelatin-Sepharose column in 1 M NaCl at pH 7.5. The purified samples of active and latent HNC were obtained with typical cumulative yields of 32 and 82% and specific activities toward soluble rat type I collagen at 30 degrees C of 7200 and 12,000 micrograms min-1 mg-1, respectively. These specific activities are markedly higher than previously reported for HNC. Both active and latent HNC exhibit a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the presence and in the absence of 2-mercaptoethanol. The mobility of latent HNC is consistent with a molecular weight of approximately 58K, with the active form exhibiting a slightly lower (less than 1-2K) molecular weight.
Mookhtiar
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KA
Department of Chemistry, Florida State University, Tallahassee 32306.
Van Wart
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HE
D002469:Cell Separation; D002729:Chloromercuribenzoates; D002846:Chromatography, Affinity; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006801:Humans; D003012:Microbial Collagenase / Q000302:isolation & purification*; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D013379:Substrate Specificity; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; D003012:Microbial Collagenase