Sodium dodecyl sulfate activation of a plant polyphenoloxidase. Effect of sodium dodecyl sulfate on enzymatic and physical characteristics of purified broad bean polyphenoloxidase.
Latent broad bean polyphenoloxidase was purified and shown to be activated by sodium dodecyl sulfate (SDS). Further characterization of the enzyme was carried out in the presence and absence of SDS. Activation of the enzyme increased in a sigmoidal manner with increasing SDS concentrations up to a maximum of 1.75 mM. The presence of SDS eliminated a low pH optimum induced by acid shocking. Increased thermolability of the enzyme was observed in the presence of SDS as well as an increased binding of [14C]dihydroxy-phenylalanine. Size exclusion chromatography on high performance liquid chromatography showed that the size and apparent molecular mass of the enzyme were slightly altered in the presence (48 kDa) versus absence (47 kDa) of SDS. Although the estimations were larger than those obtained by size exclusion techniques, no large differences in molecular weight were observed after sedimentation equilibrium of the enzyme in the presence (53.9 kDa) and absence (52.3 kDa) of SDS. Relative electrophoretic mobility and intrinsic fluorescence of tyrosine and tryptophan residues increased in a complex fashion as the SDS concentration was increased. Plateau regions in these latter experiments corresponded to concentrations of SDS needed for activation. The ability of SDS to activate the enzyme alters both its enzymatic and physical characteristics and suggests that a limited conformational change, due to binding of small amounts of SDS, may induce or initiate the activation of latent enzyme.
Moore
B M
BM
Chemistry Department, Indiana State University, Terre Haute 47809.
Flurkey
W H
WH
D004156:Catechol Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D004789:Enzyme Activation; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008823:Micelles; D010946:Plants, Medicinal; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014334:Tropolone / Q000494:pharmacology
D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D004295:Dihydroxyphenylalanine; D014334:Tropolone; D004156:Catechol Oxidase