Sodium dodecyl sulfate activation of a plant polyphenoloxidase. Effect of sodium dodecyl sulfate on enzymatic and physical characteristics of purified broad bean polyphenoloxidase.

B M Moore,W H Flurkey

doi:10.1016/s0021-9258(19)34072-4

Abstract

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.

Highlights

Latent broad bean polyphenoloxidase was purified and shown to be activated by sodium dodecyl sulfate (SDS)
Effect of SDS on PPO Actiuity-The purified PPO was activated in a sigmoidal manner with increasing SDS concentrations (Fig. 1)
In contrast to earlier reports which used crude or partially purified enzyme, we have used a highly purified enzyme to examine the enzymatic and physical changes associated with SDS activation

Summary

OF PURIFIED BROAD BEAN POLYPHENOLOXIDASE*

From the Chemistry Department, Indiana State University, Terre Haute, Indiana 47809. Latent broad bean polyphenoloxidase was purified and shown to be activated by sodium dodecyl sulfate (SDS). Activation of the broad bean enzyme was reported by Hutcheson et al [13] who indicated that the membrane-bound and/or soluble enzyme could be activated by SDS, pH, and fatty acids. These findings were observed using purified spinach PPO [10]. Much like plant PPO, activation was more successful and effective using SDS at concentrations below the CMC for SDS They indicated that SDS binding was related to enzyme stabilization and the SDS bound to the enzyme with high affinity at concentrations less than 2 x 10e4 M [17]. Physical characteristics of the latent enzyme, such as size, molecular weight, intrinsic fluorescence, and electrophoretic mobility were examined under similar conditions

PROCEDURES

RESULTS

Purified PPO

DISCUSSION