The reaction velocity is directly proportional to the enzyme concentration showing a linear dependence, in contrast to the hyperbolic dependence on substrates and cofactors ( Figure 6B). Therefore, the reaction velocity can be regulated by varying the amount of enzyme, adding more if the reaction proceeds too slowly, and less if it is too fast. In general too low amounts of the enzyme are less a problem than too high amounts. The latter convert check details the substrate instantly, already during the mixing and starting procedure and, at the worst, the reaction will already

be finished at the onset of recording and no reaction can be observed. In such a case inexperienced experimenter add even more enzyme, supposing

a too low enzyme activity. Often a distinct enzyme amount is indicated in the assay protocol; it can also be calculated, as described in the following section. However, since the activity of enzyme preparations does not remain constant, but depends on different conditions, like mode and time of storage, preliminary tests for the control of the actual enzyme activity are strongly recommended. Directly related with the enzyme amount is the observation time. Although defined time periods (seconds or minutes) are specified for calculation of the enzyme activity, there exists no general rule for the time Erlotinib in vivo observing the reaction, only that it must be within the area of the initial linear progression Histidine ammonia-lyase of the velocity, while the following non-linear phase will yield erroneous results. It may be supposed, that the initial

phase should be rather short, but this is not indispensable. If in a special assay the linear initial phase lasts for only 10 s, this will be a barely observable period for the conventional assay methods. However, tenfold reduction of the enzyme amount will expand the linear period to 100 s, a hundredfold reduction even to about 17 min, a fairly long time for observation. But, on the other hand to obtain the same intensity for the signal the long observation time of 17 min, instead of 10 s, must be accepted. The reaction proceeds very slowly and, finally, with very low enzyme amounts the signal will not be detectable at all. To intensify the signal the sensitivity of the detection method can be increased, but only within a distinct range, until the basic noise of the method exceeds the signal intensity (Figure 2). Therefore a suitable combination of enzyme amount and observation time should be tried out; longer observation times save enzyme, but are time consuming. Computer-controlled instruments like spectrophotometers usually have available programs calculating the enzyme velocity immediately after the assay. This is convenient, but should not be used uncritically.