Stationary Methods of Enzyme Kinetics

Most aspects of enzyme kinetics and its quantification have been covered in Chapter 5. In this section, we will discuss several methods of obtaining the necessary data, with which the kinetic and mechanistic studies covered in Chapter 5 and in this chapter are performed. 

Despite the remarks above, in a well-defined reaction system on-line analysis methods are very valuable. The most important on-line methods are, in this order, UV-VIS spectrophotometry, fluorimetry, and polarimetry. All three methods are based on the validity of equivalent laws linking the measured optical parameter with concentration  

Before applying either the Lambert-Beer or the Biot law uncritically, one should check the range of linearity. As a rule of thumb, extinctions E above 1.5, but definitely those above 2.0, are to be discarded because linearity no longer holds. 

A short list of particularly important assays and their extinction coefficients or specific rotations [a]d20 is given in Table 9.1. 

The limit of stationary kinetics is reached when the reaction proceeds faster than data taking is possible with the above-mentioned equipment, on timescales faster than about 10 s. 

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