Activity of Subtilisin Carlsberg in macromolecular crowding.
Enzymatic activity of a proteolytic enzyme Subtilisin Carlsberg (SC) in anionic sodium dodecyl sulfate (SDS) micellar medium has been explored and found to be retarded compared to that in bulk buffer. Circular dichroism (CD) study reveals that SDS, which is a potential protein denaturant, has an insignificant denaturation effect on SC. The structural integrity of the protein offers an opportunity to study the functionality of the enzyme SC in a macromolecular crowding of micelles. Dynamic light scattering (DLS) data indicates no sandwich-like micelle-SC complex formation ruling out the possibility of interaction of the enzyme with the hydrophobic core of the micelle. However, steady state and time resolved emission studies on specific and nonspecific fluorescent probes indicate the proximity effect at the surface of the enzyme due to macromolecular crowding of the micelles. The agreement of retarded enzymatic activity in the micellar crowd with a theoretical model ascribed to the facts that substrates are compartmentalized in the micelles and enzyme interacts with the micelle through stern layer.
Shaw
Ajay Kumar
AK
Unit for Nano Science and Technology, Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India.
Pal
Samir Kumar
SK
D002942:Circular Dichroism; D003374:Coumarins / Q000378:metabolism; D005454:Fluorescence Polarization; D007700:Kinetics; D008027:Light; D008823:Micelles; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012967:Sodium Dodecyl Sulfate; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003374:Coumarins; D008823:Micelles; D009842:Oligopeptides; C117513:alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin; D012967:Sodium Dodecyl Sulfate; C037286:benzyloxycarbonylglycyl-glycyl-leucine-4-nitroanilide; D013381:Subtilisins