Automated Fluorescence Polarization Method for Enzyme Activity Measurement

Hitachi High Technologies America, Inc.

Fluorescence polarization methods have been used in biochemical applications since the 1970's (1). Dedicated instruments that provide simple, speedy, and highly sensitive results have been used in applications ranging from the quantitative analysis of drugs in blood to PCR products (2). Here, enzyme activity of trypsin is measured using the Hitachi F-4500 fluorescence spectrophotometer equipped with the automated polarization accessory.

In the fluorescence polarization measurement system, the excitation beam is polarized and projected to the sample, and only molecules in the same orientation as the polarizer will fluoresce. The emitted fluorescence is measured in its parallel and perpendicular components separately relative to the incident polarization angle. The fluorescence polarization degree, P, is calculated by the formula in Figure 1. A smaller molecule is easier to rotate with a lower polarization degree, and a larger molecule takes a higher polarization degree.

Experimental

This experiment utilized the EnzCheck™ Polarization Assay Kit for Proteases by Molecular Probes containing a fuorescence-labeled casein (BODIPY FL-casein) and a dilution buffer (3). Pancreatic trypsin from cattle supplied by Wako Pure Chemical Industries was diluted with PBS (phosphate-buffered saline) (4). Using the conditions listed in Table I, the P value 20 min after start was measured consecutively five times with polarization degree measurements at intervals of 10–30 s. Trypsin concentrations ranged from 10–100 ng.

Figure 1. Formula for P value calculation, and a graphical representation of its relationship to enzymatic activity.

Results and Conclusions

The mean P value and standard deviation were plotted for trypsin concentrations, and a linear relationship was established as shown in Figure 2. A reduction in molecular weight with the incremental addition of trypsin was determined by measuring the decrease in P value; thus, the decrease in P value is directly proportional to the enzymatic activity of trypsin.

Table I. Experimental Conditions

Summary

The Hitachi F-4500 fluorescence spectrophotometer equipped with the automated polarization accessory accurately quantifies enzymatic activity using the fluorescence polarization method.

Figure 2. Plot of mean P value versus trypsin concentration.