Funds are requested for an automated protein sequencer to be used by seven investigators working on NIH-supported projects. No protein sequencing instrumentation is currently available on campus or within 150 miles. Ongoing research projects that require protein sequencing focus on the following topics: (1) A new ruthenium-labeling technique has been developed that allows biological electron transfer reactions to be initiated by a laser pulse and detected on a nanosecond time scale. This is a highly significant development because it has not been possible to directly detect biological electron transfer reactions except in a few instances. This is because the reactions are usually too rapid for stopped-flow techniques, and protein binding and dissociation steps are frequently rate-limiting. (2) The new ruthenium labeling technique is being applied to detect the electron transfer reaction between cytochrome c and cytochrome oxidize for the first time. A major goal will be to determine which redox center is coupled to proton pumping. (3) Cross-linking and ruthenium labeling techniques are being used to study the reaction of plastocyanin with cytochrome c and cytochrome f. (4) Cross-linking and laser kinetics techniques are being used to study the reaction between adrenodoxin and cytochrome P-450scc. (5) The interaction between photosynthetic reaction centers and cytochrome C2 is being studied by cross-linking and laser kinetics techniques. (6) The mechanism of cation transport by gramicidin A is being studied by the total synthesis of peptide analogues. (7) The effect of toxins on blood clotting and the regulation of blood flow is being studied.
