COMMUNICATIONSL-Phenylalanine ethyl ester (0.4 g) and dicyclohexylcarbodiimide (0.4 g) were added 4-(-pyrenyI)butyric acid (0.5 g) in acetonitrile (50 mL). The mixture was stirred at morn temperature (4 h), and excess DCC deomposed. The solvent was removed under reduced pressure, and the reaction mixture subjected to acid hydrolysis (1 N HCI, 12 h, room temperature). Pure product was isolated after chromatography on silica gel. 'HNMR (270 MHz, DMSO): b = 8.06-8.34 (m, 9H), 7.2 (m, 5H), 4.5 (exchangeable NH, 1 H), 2.0 (m, 1 H), 2.1-2.5 (m, 6 aliphatic H); IR (KBr): G = 3300 (OH), 1703 (acid C=O), 1640 cm-' (amide C=O); the absorption maxiama at 313,326, and 343 nm as well as the fluorescence maxima at 376, 396, and 417 nm (340 nm excitation) correspond to those of the pyrenyl chromophore. The circular-dichroism maxima are at 336. 351, and 360 nm. A. Wolfe, G. H. Shimer, Jr., T. Mechan, Biochemistry 1987, 26, 6392; A. M. Pyle, R. M. Rehman, C. V. Kumar, N. J. Turro, J. K. Barton, J. Am. Chem. Soc. 1989, flf, 3051. The equation used is ([BSA]/A&.,) = ([BSA]/As) +(l/A&K) where Atap = I&, --E J , A& = [cb -E,], K is the binding constant, E. = (absorbance at 343 nm)/[Py-Phe], E,, = extinction coefficient of the bound form (2. 21 1 x 1 0 4~-' cin-I), and E( is the extinction coefficient of free Py-Phe. The reaction mirture (0.1 mL) was irradiated at 344nm for various lengths of time with a xenon lamp source and a monochromator ( 3 . 0~ lo-* einsternsrnin-' at 340 nm). Filters were used to remove stray UV light, and the band pass on the monochromator was adjusted to 10 nm. Irradiated solutions of the protein and probe were dried under reduced pressure, and the residue was redis!;olved in the sample buffer (0.024mL) made of glycerol (1 mL), sodium dodecyl sulfate (3 mL, 10% aqueous solution), tris(hydrox-ymethy1)aminomethane hydrochloride (1.25 mL. 0.5 M), bromophenol blue (0.6 mL, 0.1 % solution), and deionized distilled water (4.5 mL). The gels were run by applying .a voltage of 60V until the dye (Coomassie blue) passed through the stacking gel. The voltage was then increased to 110 V, and the gels were run for a total of2 h; see H. Schagger, G. V. Jagow, Anal. Biochem. 1987,