Photocross-linking is a useful technique for the partial definition of the nucleic acid-protein interface of nucleoprotein complexes. It can be accomplished by one or two photon excitations of wild-type nucleoprotein complexes or by one photon excitation of nucleoprotein complexes bearing one or more substitutions with photoreactive chromophores. Chromophores that have been incorporated into nucleic acids for this purpose include aryl azides, 5-azidouracil, 8-azidoadenine, 8-azidoguanine, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-iodocytosine. The various techniques and chromophores are described and compared, with attention to the photochemical mechanism.

An analogue of the replicase translational operator of bacteriophage R17, that contains a 5-bromouridine at position -5 (RNA 1), complexes with a dimer of the coat protein and photocrosslinks to the coat protein in high yield upon excitation at 308 nm with a xenon chloride excimer laser. Tryptic digestion of the crosslinked nucleoprotein complex followed by Edman degradation of the tryptic fragment bearing the RNA indicates crosslinking to tyrosine 85 of the coat protein. A control experiment with a Tyr 85 to Ser 85 variant coat protein showed binding but no photocrosslinking at saturating protein concentration. This is consistent with the observation from model compound studies of preferential photocrosslinking of BrU to the electron rich aromatic amino acids tryptophan, tyrosine, and histidine with 308 nm excitation.

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Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (BChE)-catalyzed hydrolysis. Solvent (D2O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D2O) KIEs are (D2O)k = 0.20 in 200 mM HCl, (D2O)k = 0.81 in 50 mM HCl, and (D2O)k = 4.2 in pure water. The formyl-H KIEs are (D)k = 0.80 in 200 mM HCl, (D)k = 0.77 in 50 mM HCl, (D)k = 0.75 in pure water, (D)k = 0.88 in 50 mM NaOH, and (D)(V/K) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of (18)O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be (18)k = 0.9917 under neutral conditions, (18)k = 1.0290 (water nucleophile) or (18)k = 0.989 (hydroxide nucleophile) under alkaline conditions, and (18)(V/K) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented.

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