Several new classes of luminescent metal ion substitutional probe experiments for the study of the structure and function of calcium-binding proteins and nucleic acids are developed and exploited. A pulsed nitrogen laser-pumped dye laser excitation source allows the observation on dilute solutions of emission from the ordinarily weakly luminescent trivalent lanthanide ions. Measurement of the excited state lifetimes of these ions (principally europium(III) and terbium(III) in both H2O and D2O solution provides a direct measure of the number of water molecules coordinated to the metal ion. Lifetime measurements also monitor inter-ionic (lanthanide to transition metal and interlanthanide) Forster-type energy transfer which permits the estimation of distances between metal ion binding sites in macromolecules. Characterization of individual distinct binding sites is made possible by europium(III) excitation spectroscopy. This method involves the observation of the 7Fo yields 5Do transition between nondegenerate states by scanning the excitation laser source frequency and monitoring emission from the 5Do level. Different europium(III) binding sites give rise to distinct excitation peaks, each characterized by its excited state lifetime. Luminescence emissions will be examined under high resolution to probe the metal ion coordination site. The fluorescent amino acid or nucleotide base to lanthanide ion energy transer which sometimes sensitizes lanthanide emission will be investigated. Among the macromolecules we intend to probe by our techniques are: thermolysin, parvalbumin, alpha-amylase, phosphoglycerate kinase, prothrombin, troponin C, phospholipase A2, immunoglobulin, Ca-ATPase, calmodulin, staphylococcal nuclease and t-RNA.
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