Abstract
The interaction between subtilisin (Sbt) and Streptomyces subtilisin inhibitor (SSI) was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). When a mixture of Sbt and SSI was incubated together and then mixed with SDS-PAGE sample buffer at room temperature, it had abnormal behavior on SDS-PAGE; a single band corresponding to the complex migrated only to the top of the gel. Sequence analysis of the band electroblotted from the gel showed two major N-terminal sequences corresponding to Sbt and SSI. When the same Sbt/SSI mixture was analyzed by reverse phase-high performance liquid chromatography, protein eluted in positions corresponding to the intact forms of Sbt and SSI. Thus, it was concluded that the Sbt/SSI complex is stable upon exposure to SDS and migrates abnormally as a complex during SDS-PAGE; the abnormal migration stems from the inherent resistance of Sbt to SDS.
The denaturation of the Sbt/SSI complex was examined by heating it at different temperatures, rapid cooling, and SDS-PAGE. The band corresponding to the complex gradually disappeared as the temperature of the complex was increased from 70°C to 85°C; from this, the transition temperature was about 78°C. This temperature is higher than the known transition temperature of the Sbt alone (60°C to 65°C), indicating stabilization of the Sbt by SSI. This transition was accompanied by the appearance of low molecular weight species, none of which corresponded to the intact sizes of Sbt or SSI, indicating that autolysis occurred following denaturation.
