We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine Blabeled substrate by ribonuclease T1. A gapped heteroduplex substrate a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides was immobilized via biotin to a streptavidincoated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10[-21] mol 0.05 f of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).
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