Triton X-114-aided purification of latent tyrosinase.
Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre- and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.
Núñez-Delicado
E
E
Departamento de Bioquímica y Biología Molecular (A), Facultad de Biología, Universidad de Murcia, Spain.
Bru
R
R
Sánchez-Ferrer
A
A
García-Carmona
F
F
D000364:Agaricus / Q000201:enzymology*; D002021:Buffers; D004156:Catechol Oxidase / Q000737:chemistry / Q000378:metabolism; D002934:Cinnamates / Q000378:metabolism; D003902:Detergents / Q000737:chemistry*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008898:Mimosine / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009183:Mycotoxins / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry*; D011753:Pyrones / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors; D014334:Tropolone / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002021:Buffers; D002934:Cinnamates; D003902:Detergents; D005656:Fungal Proteins; D009183:Mycotoxins; D011753:Pyrones; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D008898:Mimosine; C011890:kojic acid; D014334:Tropolone; D017830:Octoxynol; C010615:Nonidet P-40; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D014357:Trypsin