MICROCALORIMETER MEASUREMENTS OF DNA-PROTEIN INTERACTIONS

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The use of various proteins to modify the human immune system response is of intense interest in the areas of organ transplants, AIDS, and chemotherapy. At the NCI, several new proteins have been developed for this purpose. At present, these proteins are available only in small amounts. By mixing these proteins with carefully constructed molecules of DNA and measuring the enthalpy, the protein-DNA bond interactions can be determined. 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 has the capability of measuring heats of 10 microjoules or less in 80-microliter samples. 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 this protein with two different DNA hosts at 25 degrees and 37 degrees C. Using the tantalum stopped-flow microcalorimeter, 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, which indicates that the binding is almost exclusively entropy-driven.