No abstract

The conformations of 1-anilinonaphthalene-8-sulphonate in ,H,O and CaH302H have been determined by proton magnetic resonance spectroscopy, and its fluorescence spectra as a solid, in a methyl-cellulose matrix, and in solution in water, methanol and aqueous MgC1, have been studied.The aromatic rings of anilinonaphthalene-sulphonate are more nearly coplanar in C2H302H than in ,H,O. The N H proton fails to exchange with 2H from the solvent in C2H302H, and its chemical shift indicates that there is a strong interaction with the sulphonate group. The solvents in which rapid exchange of the NH proton occurs are those in which the quantum yield of fluorescence of anilinonaphthalene-sulphonate is quenched. The fluorescence emission is enhanced and blue-shifted in strong aqueous MgC1, solutions. Under some conditions the overall emission is composed of contributions from two separable spectra.It is argued that an efficient mechanism for quenching the first excited singlet of anilinonaphthalene-sulphonate is through vibrations of bonds to the nitrogen, and that the shifts of emission maxima in non-aqueous solvents may be partly a result of the molecule displaying different spectra in different conformations (rather than simply a solvent-polarity-induced change). There is no convincing reason for the assumption that when a fluorescence enhancement follows the binding of anilinonaphthalene-sulphonate to a biological structure the site of interaction must be hydrophobic.The fluorescence emissions of 1 -anilinonaphthalene-8-sulphonate and some close analogues depend critically on environment. Whereas the quantum yields of fluorescence can exceed 0.70 in a non-aqueous solvent (e.g. dioxane) the emissions are strongly quenched in water to give yields in the range 0.0032 to 0.011 [l]. The emission maximum is shifted as much as 100nm to longer wavelengths in water. The discoveries that emission spectra change when molecules of the anilinonaphthalene-sulphonate family interact with some proteins [2] and other biological structures [3] (quantum yields are enhanced and maxima shift to shorter wavelengths from their positions in simple aqueous solution) has led to the use of these effects to probe structure and biochemical function [4]. The causes of these effects are not fully understood.Experiments described in this paper were designed to suggest answers to the following questions. (a) Are the emissions of anilinonaphthalene-sulphonate in water and non-aqueous solvents caused by transitions between molecular states with different energies in the various solvents because of different solvation effects, or are the emissions different because the molecular conformations adopted by anilinonaphthalene-sulphonate are strongly solvent dependent ? MATERIALS AND METHODSI-Anilinonaphthalene-8-sulphonate was purchased as the ammonium salt from J. T. Baker Chemical Co. (Phillipsburg, N.J.). It was purified by recrystallization from either dilute aqueous MgCI, or from saturated aqueous MgC1,. The appearances of the products after repeated recr...

1. Flavines are photoreduced through their triplet states by amines and amino acids (e.g. EDTA and dl-phenylglycine). The anaerobic photoreduction of FMN and several other flavines with dl-phenylglycine was analysed in terms of a detailed kinetic scheme. 2. The reaction produces equimolar amounts of benzaldehyde, carbon dioxide and reduced flavine. 3. The sensitivity of the rates to substituents in the dl-phenylglycine can be described by a Hammett rho-value of -1.1. 4. Phenylacetic acid behaves differently from dl-phenylglycine or benzylamine towards a series of flavines. 5. The photoreductions are quenched by several aromatic compounds. From the effects of light-intensity and temperature, and by comparison with potassium iodide quenching, it is concluded that inhibition by the aromatic compounds is not simply a collisional process. 6. FAD reacts more slowly than FMN both in the photoreduction and in dark reduction by NADH. Urea and dimethyl sulphoxide decrease the intramolecular interaction in FAD, but they have no effect on the rate of dark reduction of FAD compared with FMN. In contrast, the photoreduction of FAD is quicker in urea.

1. When a mixture of FMN and a reducing substrate (e.g. unprotonated amine) is illuminated oxygen is consumed. 2. The rate of oxygen uptake increases as oxygen concentration falls with some substrates (type I reaction), but with other substrates (typically aromatic compounds) the rate falls as the oxygen concentration falls (type II reaction). 3. The kinetics of type I reactions with EDTA, dl-alpha-phenylglycine and diethanolamine are all consistent with a mechanism in which the rate-determining step, hydrogen abstraction by the FMN triplet, is followed by rapid reoxidation of reduced FMN by oxygen. The reaction is faster at low oxygen concentrations because oxygen quenches the triplet. 4. The sensitivity of reaction rates to substituents in dl-alpha-phenylglycine can be described by a Hammett rho value of -0.6. 5. Individual rate constants for quenching and reaction of the FMN triplet with substrate were calculated (2.4x10(8) and 2.1x10(7)m(-1)s(-1) respectively for EDTA) on the assumption that oxygen quenches the triplet in a diffusion-controlled reaction. 6. The pH-dependences of oxygen uptake rates with six natural amino acids as substrates were measured. 7. Photoinactivations of l-glutamate dehydrogenase and d-amino acid oxidase by FMN were demonstrated.

Fluorescence properties of purified isoleucyl-tRNA synthetase isolated from Escherichia coli B have been studied. No changes in the quantum yield, energy or polarisation of the emission were detected in the presence (either individually or in combinations) of the substrates and cofactors required for activation of L-isoleucine.In 2.5 M urea enzyme activity and intrinsic fluorescence intensity (at 340 nm) each decrease with time, showing similar kinetics and rate constants. The rate of this decay is reduced in the presence of ligands which can bind to the enzyme and the effect has been used to measure dissociation constants for enzyme-ligand complexes. Values have been obtained for the complexes between enzyme and L-isoleucine (&iSs = 25 pM), L-valine (KdiS8 = 300 pM), ATP (Kdiss = 150 pM) and PPi (&iss = 200 pM) at 25". The effects of ionic strength, and the temperature dependence and urea concentration dependence of L-isoleucine binding have also been studied. Magnesium ions, which are required for catalysis, do not greatly affect the binding of single substrates, but changes are seen in the presence of ATP and L-isoleucine together. The magnesium ion concentration dependence of this effect (half-point about 200 pM) and the equilibrium constant for L-isoleucine activation (2 pM) have both been measured.The reliability of the methods has been discussed. Results have been interpreted in terms of current theories of amino acid activation. The binding parameters are sufficient to explain the stability of enzyme bound L-isoleucyladenylate without invoking conformation changes. This is consistent with the absence of substrate induced fluorescence changes. Magnesium effects are explained in terms of reduced electrostatic repulsion between reactants bearing like charges.The reactions catalysed by isoleucyl-tRNA synthetase are usually represented by the following equilibria :Enzyme + ATP + Ile ,-' Enzyme-AMP-Ile Yarus and Berg, studying interactions between isoleucyl-tRNA synthetase and tRNAIle, have found that the rates a t which complexes between enzyme and tRNA form and dissociate are both enhanced in the presence of L-isoleucine [l,S]. They explain their results on the hypothesis that isoleucyl-tRNA synthetase adopts several conformations during a single catalytic cycle [l]. A problem in comparing and interpreting the results of these and other groups is that the physical conditions used are often widely different.