Role of surfactant on the proteolysis of aqueous bovine serum albumin.
Nonionic and ionic surfactants diminish the initial rate of proteolysis of aqueous bovine serum albumin (BSA) by subtilisin Carlsberg. Surfactants studied include: nonionic tetraethylene glycol monododecyl ether (C12E4); anionic sodium dodecyl sulfate (SDS), anionic sodium dodecylbenzenesulfonate (SDBS), and cationic dodecyltrimethylamonium bromide (DTAB). Kinetic data are obtained using fluorescence emission. Special attention is given to enzyme kinetic specificity determined by fitting initial-rate data to the Michaelis-Menten model. All surfactants reduce the rate of proteolysis, most strongly at concentrations near and above the critical micelle concentration (CMC). Circular dichroism (CD), tryptophan/tyrosine fluorescence spectra, and tryptophan fluorescence thermograms indicate that BSA partially unfolds at ionic surfactant concentrations near and above the CMC. Changes in BSA conformation are less apparent at ionic surfactant concentrations below the CMC and for the nonionic surfactant C12E4. Subtilisin Carlsberg activity against the polypeptide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, decreased due to enzyme-surfactant interaction. At the concentrations and time frames studied, there was no enzyme autolysis. Importantly, aqueous proteolysis rates are significantly reduced at high surfactant concentrations where protein-micellar-surfactant aggregates occur. To explain the negative effect of surfactant on subtilisin Carlsberg proteolytic activity against BSA, we propose that micelle/protein complexes hinder enzyme access.
Porcel
E M Rodriguez
EM
Chemical Engineering Department, University of California, 201 Gilman Hall, Berkeley, California 94720-1462, USA.
Foose
L L
LL
Svitova
T F
TF
Blanch
H W
HW
Prausnitz
J M
JM
Radke
C J
CJ
D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D017510:Protein Folding; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism*; D013501:Surface-Active Agents / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D013501:Surface-Active Agents; D012710:Serum Albumin, Bovine; D013381:Subtilisins