A NEW INSTRUMENT FOR BIOMEDICAL RESEARCH

BECAUSE OF THEIR EXTREME IMPORTANCE AND PREVALENCE IN NEARLY ALL BIOLOGICAL PROCESSES, MOST INDUSTRIAL AND ACADEMIC LABORATORIES ARE INVOLVED IN SOME WAY IN THE CHARACTERIZATION OF LIGAND-MACROMOLECULE OR MACROMOLECULE-MACROMOLECULE BINDING REACTIONS. UP TO THE PRESENT, THE MEASUREMENT OF EQUILIBRIUM CONSTANTS FOR THE DISSOCIATION OF SUCH COMPLEXES HAS USUALLY REQUIRED THE USE OF INCONVENIENT, TIME-CONSUMING METHODS BASED ON PARTITIONING (E.G., EQUILIBRIUM DIALYSIS OR GEL EXCLUSION). PHASE I OF THIS PROJECT INVOLVES THE CONSTRUCTION OF A RAPID TITRATION CALORIMETER, UTILIZING MUCH OF THE TECHNOLOGY AND HARDWARE EXISTING IN MICROCAL'S SCANNING CALORIMETER, WHICH WILL ACCURATELY DETERMINE BOTH THE DISSOCIATION CONSTANT, K, AND HEAT OF BINDING, H, WITHOUT THE NEED OF PARTI- TIONING. THE TECHNIQUE WILL BE POTENTIALLY APPLI- CABLE TO ALL LIGAND-MACROMOLECULE AND MACRO- MOLECULE-MACROMOLECULE INTERACTIONS HAVING A NON-ZERO H AND HAVING K VALUES IN THE RANGE 10-2 TO 10-8M, WHICH INCLUDES MOST BIOLOGICAL BINDING PROCESSES. IN MANY CASES, A COMPLETE BINDING ISOTHERM WILL BE OBTAINED IN A SINGLE EXPERIMENT LASTING ABOUT ONE MINUTE AND CONSUMING ONLY NANOMOLES OF BIOLOGICAL MACROMOLECULE. THE INSTRUMENT WILL BE INTERFACED TO AN IBM-PC AND, IN PHASE II, STATE-OF-THE-ART SOFTWARE WILL BE DEVELOPED FOR RAPID DATA ANALYSIS FOR SINGLE-SITE, MULTIPLE-SITE, AND ALLOSTERIC INTERACTIONS OF VARYING COMPLEXITY, USING DECONVOLUTION SUBROUTINES BASED ON LEAST-SQUARES MINIMIZATIONS. AFTER COMPLETION OF THE SOFTWARE, MODEST EVALUATION EXPERIMENTS WILL BE