title: Eriochrome black T inhibition of human skin collagenase, but not gelatinase, using both protein and synthetic substrates.
abstract: The intracellular degradation of interstitial collagen is accomplished by two neutral metalloproteases, collagenase and gelatinase. Both enzymes are inhibited by metal chelating agents, by certain sulfhydryl reagents, and by similar protein inhibitors. Here, we demonstrate that the dye eriochrome black T (EBT) appears to be unique in its capacity to inhibit collagenase but not gelatinase. Using native reconstituted helical collagen in gel form at 37 degrees C, half-maximal inhibition of collagenase activity by EBT occurs at approximately 45 microM. EBT more effectively inhibits the breakdown of native collagen in solution, with a KI of approximately 8 microM. Using a newly-developed spectrophotometric substrate, AcProLeuGly-S-LeuLeuGly-OC2H5, a KI of 1.4 microM was calculated for EBT on collagenase. Although this same thiopeptolide serves as a substrate for gelatinase with kinetics similar to those of collagenase, no inhibition by EBT was observed. EBT also did not inhibit the gelatinase-mediated breakdown of the natural substrate, gelatin. The data suggest that EBT may have significant potential for allowing the differentiation in biological fluids of two metalloproteases with similar cleavage site specificities.
authors.affiliation: Division of Dermatology, Washington University School of Medicine, St. Louis, MO 63110.
authors.forename: J L
authors.initials: JL
authors.lastname: Seltzer
authors.forename: M L
authors.initials: ML
authors.lastname: Eschbach
authors.forename: J O
authors.initials: JO
authors.lastname: Winberg
authors.forename: E A
authors.initials: EA
authors.lastname: Bauer
authors.forename: A Z
authors.initials: AZ
authors.lastname: Eisen
authors.forename: H
authors.initials: H
authors.lastname: Weingarten
mesh_terms: D001391:Azo Compounds / Q000494:pharmacology*; D005347:Fibroblasts / Q000201:enzymology; D018093:Gelatinases; D006801:Humans; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors*; D010434:Pepsin A / Q000037:antagonists & inhibitors*; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001391:Azo Compounds; D010434:Pepsin A; D018093:Gelatinases; D003012:Microbial Collagenase; C014527:Eriochrome Black T