Novel endogenous inhibitor of sulfur mustard-stimulated protease in cultured human epidermal keratinocytes: Possible application in vesicant intervention

Protease stimulation at the dermal-epidermal junction may be responsible fo r the skin blistering (vesication) action of sulfur mustard (HD), We have p urified a protease to homogeneity from cultured normal human epidermal kera tinocytes (NHEK) exposed to 300 muM HD, In this report, we describe the res ults of our studies on purification and characterization of an endogenous i nhibitor of HD-stimulated protease in NHEK, Purification to homogeneity was accomplished by chromatographic separation of the dialyzed Triton X-100-so lubilized inhibitor using ion-exchange DEAE-cellulose. Analysis of the puri fied inhibitor by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed one polypeptide with an apparent molecular mass of 116 kDa, Activ ity of the inhibitor was screened by incubating different column elute frac tions with protease purified from the same cells. Preliminary results showe d that the purified inhibitor effectively inhibited the protease isolated f rom NHEK, whereas other naturally occurring inhibitors, e.g. soybean trypsi n-chymotrypsin inhibitors, elafin and aprotinin, were ineffective. Although complete characterization and regulation of this inhibitor remain to be re solved, this purification may be a major step towards developing a specific protective measure against HD-induced toxicity.