Development of a microcomputer-controlled, isothermal titration microcalorimeter

The ability of a calorimeter to directly measure heat makes it a very desirable instrument when performing thermodynamic studies. A titration microcalorimeter is particularly suited for measuring the heat evolved in biochemical reactions. Unfortunately there does not exist a commercially available instrument with sufficient sensitivity, so it was decided that a batch type microcalorimeter would be modified to allow the injection and mixing of reactants.

The microcalorimeter chosen was a LKB 2277 BioActivity Monitor. In addition to the modifications to the calorimeter a titration system was built so that the injection of reactants and data collection would be computer controlled. Computer software was written to manipulate the data and make the necessary calculations. The result of this work is a titration microcalorimeter approximately ten times more sensitive than commercially available instruments.

To check the feasibility of the instrument the interaction between cytochrome b₅ protein and phospholipid membrane vesicles was investigated. The measured molar enthalpy was -227 kcal/mole protein. The association and insertion of the protein into the membrane was found to be a slow process characterized by a half-time of 8.6 seconds.