Application of flow microcalorimetry to the study of dehydrofolate reductase activities in crude tissue homogenates

Abstract

Calorimetric methods are becoming important analytical tools in several areas of biochemical and biological research. In this work, a flow microcalorimetric method has been applied to the determination of dihydrofolate reductase (DHFR) activities in rat and human malignant tissue homogenates. In contrast to other commonly used DHFR analytical assays, the sensitivity of flow microcalorimetry allows direct measurements of this enzyme in crude tissue preparations. Our experimental data from rat tissue homogenates show that liver has the highest level of enzyme activity, while lung and brain have lower amounts of DHFR activity. The liver enzyme has a higher activity atpH 4.5, but the optimumpH for the lung and brain enzymes is 6.8. The substrate/cofactor molar ratio which gives the highest levels of DHFR activity is 1/1.5 for the liver and lung enzymes and 1/2.5 for brain DHFR. The DHFR in these rat tissue homogenates is activated by KCl or NaCl: in the presence of these salts (0.6M), the values of enzyme activity are 1.5–3 times higher than in their absence. Using flow microcalorimetry, very low levels of DHFR activity were also measured in human bone tumour homogenates, demonstrating the potential of the technique in the analysis of this enzyme in malignant tissues.