Theoretical basis for calculation
The hydrolysis of the chromogenic peptide substrate by the proteolytic enzyme follows in general the Michaelis-Menten kinetics. This means that, if the substrate is present at a sufficiently high concentration or if a comparatively small fraction of the substrate is hydrolized, the rate of product (color) formation is proportional to the activity of the enzyme. The rate of pNA formation, i.e. the increase in absorbance per second, is measured photometrically at 405 nm. At this wavelength the extinction coefficient of pNA is
9600 mol -1 • l • cm -1
The enzymatic activity can be quantified in two ways:
- By comparing the activity of an enzyme with that of a standard preparation, which is defined in terms of a specified number of units set by an international or national authority or society (WHO, NIH etc.), or by the activity present in 1 ml of activated pooled normal plasma (Plasma Equivalent Unit = PEU). The standardization is performed by using at standard curve obtained with at least five different concentrations, each performed in duplicate. The standard material must be of the same kind and of the same quality as the sample which is to be measured. This may be still more important for a secondary or domestic standard.
- By measuring the amount (mol) of substrate split, or rather product formed per unit time (absolute activity).
One unit of enzymatic activity, katal (kat) is defined as the amount of activity that converts one mole of substrate per second under standardized conditions. Such conditions as type of substrate, substrate concentration, buffer, pH, ionic strength and temperature should be given along with unit.
Thus, 1 nkat gives a conversion rate of:
1 x 10-9 mol/sec = 60 x 10-9 mol/min
If the total (measuring) volume used is V (ml), the increase in concentration per minute caused by 1 nkat is
If the absorbance is measured at 405 nm, in a 1 cm cuvette the difference in extinction coefficient is
e = 9600 mol-1 • l
The increase in absorbance/min can then be calculated by using Lambert-Beer’s law:
A = e x C
Thus, 1 nkat gives:
By using a sample volume v (ml):
For the end-point method, the incubation time t (min) with substrate is taken into account by the following formula:
According to nomenclature, one unit (U) is the amount of enzyme activity that converts one mol of substrate per minute under standardized conditions. By using the above formulas the units are: