Several new proteins developed at the NCI modify the human immune system response and are of intense interest in the areas of organ transplants, AIDS, and chemotherapy, but are now available only in small amounts. Investigators at the NCI and NIDDK consulted the BEIP about measuring the heats of these small samples. We recommended using our tantalum stopped-flow microcalorimeter, which can measure heats of 10 microjoules or less in 80-microliter samples. By mixing the proteins with carefully constructed molecules of DNA and measuring the enthalpy, we can determine the protein-DNA bond interactions. The first experiments showed that we could measure heats in the range of 5 microjoules with a standard error of less than 0.5 microjoules. We have now completed the preliminary study of one of these proteins with two different DNA hosts at 25~ and 37~C. We were able to measure a binding heat difference when one base pair on the DNA host was changed. Results show that the enthalpy is very low, indicating that the binding is almost exclusively entropy-driven. Based on the success of these measurements and the usefulness of the information acquired, we have constructed a refined version of the calorimeter with a reduced priming volume (250 microliters vs 1000) and an improved pre-equilibration scheme to increase usable sensitivity. Preliminary results indicate a threefold improvement in resolution (from 3 microjoules to 1) for enthalpy measurements. The reduced priming volume allows us to nearly double the number of useful runs obtainable from a 1-cc sample. We also discovered that the pressure-induced component of the flow artifact is not significant, and that pressure-relieving the sensors is of marginal value. The automated loading feature has not yet been incorporated into the instrument.
