R. Bruce Dunlap

Correction for Light Absorption in Fluorescence Studies of Protein-Ligand Interactions

It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.

The development of room temperature phosphorescence into a new technique for chemical determinations : Part 1. Physical Aspects of Room Temperature Phosphorescence

Abstract The development and physical aspects of room temperature phosphorescence are reviewed in this first part of a two-part series. Certain fundamental aspects of phosphorescence are presented. The novel phenomenon of room temperature phosphorescence (strong phosphorescence for organic compounds adsorbed on appropriate substrates) is then treated in detail. Specific attention is given to the nature of the support-phosphor interaction and the quenching effects of moisture and oxygen on room temperature phosphorescence. Thorough coverage is also given to the heavy-atom effect. The effects of the sample matrix on certain phosphorescent properties, such as lifetime, intensity and spectral characteristics, are mentioned.

The development of room temperature phosphorescence into a new technique for chemical determinations: Part 2. Analytical considerations of room temperature phosphorimetry

Abstract The rapidly developing technique of room temperature phosphorimetry is discussed from a practical analytical standpoint in this second part of the review. Basic factors concerning the technique such as methods of sample preparation, special instrumentation employed, and quantitative capability are presented, together with a listing of the variety of organic compounds reported to display room temperature phosphorescence. Potential applications of room temperature phosphorimetry and the advantages of sensitivity and selectivity afforded by this technique are discussed.

Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS·drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180° relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 Å) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH2H4folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.

Folylpolyglutamates as substrates and inhibitors of folate-dependent enzymes

The true intracellular substrates for folate-dependent enzymes are folylpolyglutamates. We have used measurements of the Ki values of folylpolyglutamate dead end inhibitors to assess the relative affinities of folate-dependent enzymes for folate derivatives of different polyglutamate chain lengths. Studies of four enzymes from pig liver, methylenetetrahydrofolate reductase, serine hydroxymethyltransferase, methylenetetrahydrofolate dehydrogenase and thymidylate synthase, have indicated that folylpolyglutamate inhibitors are bound 3-500 fold more tightly than the corresponding monoglutamates. The individual enzymes differ in their selectivity for polyglutamate vs. monoglutamate inhibitors, and in the chain length associated with the greatest affinity of enzyme for inhibitor. We have also examined the effect of polyglutamate chain length on the catalytic parameters associated with folate substrates. Two enzymes, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase, show decreases in Km values for folylpolyglutamate substrates. Methylenetetrahydrofolate dehydrogenase shows no detectable differences in the catalytic parameters of polyglutamate vs. monoglutamate substrates and no change in the order of substrate addition or product release. Thymidylate synthase shows small effects of Km and Vmax values, but the order of addition of substrates and of release of products is reversed with polyglutamate as compared with monoglutamate substrates. Our studies with thymidylate synthase from L. casei have shown that the bacterial enzyme also exhibits a greatly increased affinity for polyglutamate vs. monoglutamate derivatives of folic acid, and that reversal in the order of substrate addition and product release also occurs with polyglutamate as compared with monoglutamate substrates. We have also studied the polyglutamate specificity of methionine synthase, which is responsible for the conversion of CH3-H4PteGlu1 into H4PteGlu1. This reaction is required for the incorporation of plasma folate into the cellular folate pool, because methyltetrahydrofolate is a poor substrate for folylpolyglutamate synthetase. Our studies demonstrate that CH3-H4PteGlu6, and suggest that incorporation of plasma CH3-H4PteGlu1 will only occur when methylenetetrahydrofolate reductase is inhibited by adenosylmethionine and cellular pools of CH3-H4PteGlu6 are at very low levels.

Isolation of the covalent binary complex of 5-fluorodeoxyuridylate and thymidylate synthetase by trichloroacetic acid precipitation

Strong chemical evidence for the existence of a covalent binary complex between 5-fluorodeoxyuridylate and thymidylate synthetase was provided by the isolation of the complex by trichloroacetic acid precipitation. This result together with that of a control experiment with N-ethymaleimide inactivated thymidylate synthetase demonstrated that only nucleotide covalently bound to the protein survived repeated washings of the precipitate. Under the conditions used, a maximum binding stoichiometry of about 0.9 was obtained for the covalent binary complex, Kd = 1.1 X 10(-5) M. Also, a binding ratio of 1.7 was obtained for the methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase ternary complex.

Characterization of the subunits of thymidylate synthetase

Abstract N-terminal analysis of thymidylate synthetase by the Edman procedure gives rise to a single spot when analyzed on three separate thin layer chromatographic systems. In each case this spot is consistent with only the phenythiohydantoin of methionine. Dansylation confirms the presence of only methionine as the N-terminus and the subtractive dansyl-Edman technique indicates that only leucine is found as the amino acid once removed from the N-terminus. Cyanogen bromide treatment of car☐ymethylated thymidylate synthetase results in the formation of five fragments as determined by sodium dodecyl sulfate gel electrophoresis and by dansylation of the cyanogen bromide peptides. All of the results are consistent with the conclusion that thymidylate synthetase is composed of two subunits with identical primary structures.

Synthesis of (4S,5R)-(–)-4-methyl-5-phenyloxazolidine-2-selone: a chiral auxiliary reagent capable of detecting the enantiomers of (R,S)-lipoic acid by 77Se nuclear magnetic resonance spectroscopy

(4S,5R)-(–)-4-Methyl-5-phenyloxazolidine-2-selone was constructed in 5 steps on the 10-gram scale. 77Se NMR spectroscropic studies revealed that this selone served as a remarkably sensitive chiral auxiliary agent which readily distinguished the selone-coupled (R,S)-lipoic acid (Δδ 0.119 ppm), even though its chiral centre was separated by 8 bonds from the observed nucleus.

Studies in selenium-77 and tellurium-125 nuclear magnetic resonance. Substituent effects and polarizability

Abstract Selenium-77 substituent-caused chemical shifts, where the substituents are alkyl groups, have been studied for a series of selenols, selenides and diselenides, respectively. An understanding of the origin of these shifts was obtained by examining solvent effects on 77 Se chemical shifts of dialkyl selenides and dialkyl diselenides in ten halocarbon solvents of varying molecular polarizabilities as well as in 2,2,2-trifluoroethanol. Based on these results of intermolecular polarizability, an intramolecular polarizability concept is proposed whereby dispersion forces within a molecule exerted by neighboring alkyl and haloalkyl groups influence selenium shielding. This effect along with the well-known “γ effect” offer a clear understanding of a large number of 77 Se chemical shifts. The same intramolecular polarizability concept can be used when examining the chemical shifts of tellurides and ditellurides. Here, even larger substituent caused chemical shifts are observed. Solvent effects on 125 Te chemical shifts of dimethyl telluride were also examined in solvents of varying polarizability. As in the case of several selenides and diselenides studied, the 125 Te chemical shift of dimethyl telluride can be correlated with the polarizability of the solvent.

Characterization of a new sulfhydryl group reagent: 6,6′-Diselenobis-(3-Nitrobenzoic acid), a selenium analog of Ellman's reagent

Abstract A new reagent, 6,6′-diselenobis-(3-nitrobenzoic acid) (DSNB) has been synthesized and is shown to be useful for quantitative estimation of sulfhydryl groups in proteins. This reagent, which is a selenium analog of Ellmans reagent, reacts specifically and quantitatively with thiol groups of proteins to yield a selenenyl sulfide and the dianion of 3-nitro-6-selenobenzoic acid. The molar absorption coefficient of the chromophoric dianion is 10,000 at 432 nm in dilute aqueous solutions. The titration can best be performed at pH 8.20 where >98% of 3-nitro-6-selenobenzoic acid is in the form of the intensely colored dianion. Sulfhydryl content determinations by this reagent of reduced and denatured ribonuclease, reduced and denatured lysozyme, native papain, and native and denatured thymidylate synthetase are compared with those from corresponding 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) titrations. The reagent was found to inactivate thymidylate synthetase, an enzyme with essential sulfhydryl groups. Unlike DTNB which undergoes alkaline decomposition of pH values greater than 9, DSNB was found to be stable to hydrolysis, even in 0.05 m NaOH.