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Study of the Diphenolase Activity of Mushroom Tyrosinase by Stopped-flow method (CAT#: STEM-AC-0027-WXH)

Introduction

Tyrosinase is a type 3 copper enzyme that catalyses the oxidation by molecular oxygen of phenolic compounds to quinones. The quinone products of this reaction are highly reactive and polymerize non-enzymatically to form melanins. This reaction is the cause of melanogenesis in animals and the enzymatic browning of fruit, vegetables, and fungi.




Principle

Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms.
In its simplest form, a stopped-flow mixes two solutions. Small volumes of solutions are rapidly and continuously driven into a high-efficiency Ball mixer, so mixing is completed in just a few microseconds. This mixing process then initiates an extremely fast reaction.

Applications

Typically used to gain an understanding of reaction mechanisms, including drug-binding processes or following protein structural changes, stopped-flow spectroscopy enables the study of fast reactions in solution over timescales in the range of millisecond to hundreds of seconds.

Procedure

1. In stopped-flow experiments, two, three, or four sample solutions are rapidly mixed and injected into an observation cell.
2. When the flow is stopped, the kinetics are recorded with a detector best suited to the chemical properties of the solutions and the information of interest (e.g. particle size, the environment of the fluorophore, chromophore).

Materials

Stopped-Flows