Polarographic and Spectrophotometric Assay of Diphenol Oxidases Polyphenol Oxidase

The diphenol oxidases (DPOs) includes two major groups of enzymes the ortho-DPOs (also known as catecholases, polyphenol oxidases, and tyrosinases) and the para-DPOs (more usually known as laccases). The names catecholase and laccase are used in this unit. 

Two principal methods are widely used for the assay of DPOs. For enzyme kinetic studies, the most appropriate methods are polarographic and use an 02 electrode (Basic Protocol), which allows the direct measurement of the rate of utilization of oxygen and a true comparison of different phenolic substrates. Minor disadvantages of this method are that it requires more specialized equipment and that assays can only be carried out one at a time. Nevertheless it has proven to be the basis of some excellent undeigraduate biochemical laboratory experiments. 

The other major types of assay are all spectrophotometric (Alternate Protocols 1 and 2) and usually involve measuring the production of the highly colored end-product of the DPO reaction. They are used widely because they are rapid, can be carried out with the simplest of colorimeters/spectrophotometers, and can be adapted to use microtiter plates (Alternate Protocol 3). Thus they are ideal for simple routine comparative or monitoring studies. A major weakness of these colorimetric procedures is that they measure the end-product of a complex sequence of reactions, and different substrates yield different 

Set up an 02 electrode, control box, and data recording system according to the manufacturer s instructions. 

This protocol assumes the use of a strip chart recorder (I or 10 mV input), but other means of continuous data recording could be used. 

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C4.1 Polarographic and Spectrophotometric Assay of Diphenol Oxidases (Polyphenol Oxidase)... 

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