Purification of-galactosidase

Catalytic properties of P-galactosidase BGase follows Michaelis-Menten kinetics (Wallenfels and Weil, 1972) and so that Km = Ks. However, additional enzyme complexes may be distinguished  

Acceptor alcohols (methanol, glycerol, 2-ME, Tris) stimulate the cleavage of o-NPG, but not of other substrates. Heavy metals, orga-no-mercuric compounds, chelating agents (EDTA, citrate), prevent 

For EIA, u-NPG, which is converted to o-nitrophenol (o-NP), is preferred since the relative catalytic rate is considerably higher and less solid-phase interference is expected. Composition of the assay mixture is given in Table 10.8. The coupled reaction is measured by the absorbance of NADH (a = 6.22 cm pmole at 340 nm), whereas in the alternative assay o-NP is measured (a = 18.5 cm / pmole at 405 nm). The change of optical density, which is measured per minute, is used for the calculation of the volume activity in 

The specific activity and the TN can be established after determining the enzyme concentration (Section 9.2.3.4). 

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