Monitoring of soluble enzyme activity by enzyme flow microcalorimetry

The possibility of use of the flow microcalorimetry for the enzyme activity testing is demonstrated. Two model enzymes, urease and invertase, were stu died, when urea and sucrose were used as substrates. The technique is based on a measurement of temperature change in the sample stream provoked by th e reaction heat effect of enzyme reaction. The analysis is conducted so tha t a pre-cooled sample of enzyme solution is mixed with the substrate soluti on, and then immediately injected through an injection valve into a carrier buffer pumped continuously into the flow microcalorimeter column packed wi th glass particles. A temperature change in the column is observed as a hea t peak while the peak area depends on the sample volume and an enzymatic re action rate. The enzyme activity in the sample was determined from the peak height using a previous microcalorimeter calibration for the known enzyme activity. The set of calibrations was effectuated for both tested enzymes. Enzyme activi ty ranges of linearity and the influence of substrate concentration on the method sensitivity were investigated. The main advantage of the proposed te chnique is its versatility due to the versatility of the detection principl e.