Sodium dodecyl sulfate polyacrylamide gel electrophoresis as a method for studying the stability of subtilisin.
A simple method has been developed to study the stability of subtilisin. Protein incubated at various temperatures in the presence of proteinase inhibitor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and showed a transition from the intact state to the unfolded state between 55 degrees C and 65 degrees C. Additionally, autolysis was also observed above 65 degrees C. In the absence of inhibitor, similar results were obtained below 55 degrees C; however, above 65 degrees C no protein of any size was observed due to extensive autolysis. These results demonstrate that SDS-PAGE can trap subtilisin in the state in which the protein existed prior to the analysis. It can be used to identify the different forms, including autolysis products, of the protein generated by heat denaturation. This method was used to study SDS-induced unfolding of aprA-subtilisin. When the protein was incubated with 0.25% SDS at different NaCl concentrations, a gradual increase in unfolding was observed with increasing NaCl concentration. This change paralleled a decrease in the critical micelle concentration of SDS, indicating that the rate of unfolding of aprA-subtilisin increases with increasing SDS micelle concentration. No detectable unfolding was observed below the critical micelle concentration.
Narhi
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Amgen Inc., Thousand Oaks, CA 91320.
Arakawa
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D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D008823:Micelles; D011480:Protease Inhibitors / Q000378:metabolism; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate; D013381:Subtilisins / Q000032:analysis*; D013696:Temperature
D008823:Micelles; D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate; D013381:Subtilisins