ROTATIONAL DIFFUSION WITH 2 PHOTON FCS

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We are using one-photon confocal and two-photon fluorescence spectroscopy to detect protein reactions on the single molecule level. By measuring processes on single proteins, we are not subject to ensemble averaging. Thus, the degree of heterogeneity in proteins, and the variety of reaction pathways available can be monitored directly. Initial experiments are performed on the binding of 8-anilino-1 -naphthalene sulfonic acid (ANS) to apornyoglobin. The quantum yield of ANS increases by over 2 orders of magnitude when the ligand is bound to the protein, making it possible to distinguish between the ligated and unligated conformations of the protein. The proteins are singly labeled with oregon green to make them easy to locate and are immobilized in a silica gel. The ANS diffuses through the glass, and the binding events monitored with time. The rate coefficients of the reaction can then be extracted from the time sequence of binding events.