By Robert Eisenthal, Michael Danson
In an age of automated and automatic asay structures, it really is attainable to assemble info with no knowing the chemistry or biochemistry concerned. this can be compatible for lab techs who paintings for advertisement businesses, however it isn't a good suggestion for critical scientists. This booklet explains how assays paintings, what can get it wrong and what the constraints are. It additionally stories the kinds of instrumentation customary in assays. it's an exceptional source for laboratory teachers and graduate scholars.
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Additional info for Enzyme Assays: A Practical Approach (The Practical Approach Series, 257)
Under such conditions a sigmoid dependence of initial velocity upon substrate concentration will result if the concentration of one of the substrates is varied at a 24 PRINCIPLES OF ENZYME ASSAY AND KINETIC STUDIES fixed, non-saturating concentration of the other, whereas if the concentration of the other substrate is varied at a fixed, non-saturating concentration of the first, a curve such as that shown in Figure 9 line ( ) will result. Several other mechanisms in which there are alternative (two, or more) ways in which substrates can bind to an enzyme have been shown to give rise to complex steady-state rate equations (60–63).
In this case the magnitude of the burst or lag may depend on the enzyme concentration since this will affect the degree of association. Furthermore, the hysteresis may be dependent on how the reaction is initiated. If the reaction is started by addition of a sample of enzyme from a concentrated stock solution the effects might be different from those observed when the enzyme is diluted into an incomplete assay mixture and allowed to equilibrate before starting the reaction with another component.
However, when enzyme association–dissociation is involved it may not be possible to find an appropriate effector to displace the equilibrium towards the active polymerization state. An alternative approach would be to investigate conditions of pH, buffer composition or ionic strength under which the strength of the intersubunit interactions are such that the polymerization/ depolymerization becomes unimportant over the concentration range used. 2, for example, linearity can be achieved either by inclusion of an excess of the allosteric activator AMP or by working at a higher pH value.