A thin-layer gas-solution microcalorimeter for the determination of heat binding curves

A thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand binding macromolecules in solution. Gas partial pressures are accurately changed logarithmically by means of a precision dilution valve allowing for the stepwise determination of reaction heats. Heat binding curves are constructed in which the enthalpy per mole of reaction site is plotted versus the logarithm of the ligand activity. MicroJoule sensitivity is achieved through closed loop proportion computer control and precisely twinned highly isolated sample and reference geometry. The sample, typically 50 microliters and 1 to 4 mM heme protein, is placed on a filter paper membrane which acts as a matrix of support. This orientation allows for the rapid equilibrium of reacting ligand in approximately 10 min while not significantly altering the ligand activity. The system is controlled by computer measuring the heat of reaction as the partial pressure is changed automatically, typically by flushing the system with reacting ligand then reducing its partial pressure logarithmically with a nonreacting gas such as nitrogen. Binding curves can be constructed with as little as 20 nmol of oxygen binding sites.