Minoru Machida

Fluorescence and structures of proteins as measured by incorporation of fluorophore: IV. Synthesis and fluorescence characteristics of N-(p-(2-benzimidazolyl)phenyl) maleimide

Abstract 1. 1.|A sulfhydryl reagent N-(p-(2- benzimidazolyl)phenyl ) maleimide (BIPM) was synthesized and the fluorescence characteristics of its derivatives were studied. 2. 2.|The fluorescence behavior agrees with an empirical rule that maleimide derivatives are practically nonfluorescent, while their addition products with sulfhydryl compounds are fluorescent. 3. 3.|BIPM may be employed as a tracer of SH-containing peptides in structural studies of proteins. Further possible applications are discussed.

Fluorescent thiol reagents. VI: N-(1-Anilinonaphthyl-4)maleimide; a fluorescent hydrophobic probe directed to thiol groups in protein☆☆☆

N-(1-Anilinonaphthyl-4)maleimide (ANM) (m.p. 207-208.5 degrees C), nonfluorescent, reacts selectively with thiols to give addition products which are strongly fluorescent (excitation maximum 355 nm, emission maximum 448 nm, in ethanol). 0.7 mole of ANM was introduced into thiol groups of egg albumin without modifying any other amino acid residues. In the fluorescence spectra of the reaction products of ANM with thiols, the quantum yields increase and the emission maxima shift towards the blue as the solvent polarity decreases. This remarkable solvent dependence was described in terms ofZ value of the solvents. ANM is thus expected to be a useful fluorescent hydrophobic probe directed to thiol groups in protein.

Fluorescent thiol reagents. V. Microfluorometry of thiol compounds with a fluorescent-labeled maleimide.

Abstract 1. 1. N-(p-(2-Benzimidazolyl)phenyl)maleimide (BIPM) has been used for the quantitative determination of thiol compounds. The method is based on the particular fluorescence characteristics of this reagent: its adducts to thiol compounds have strong fluorescence (λmax, ex 315 mμ, λmax, em 365 mμ), while the reagent itself has no appreciable fluorescence. 2. 2. The addition reaction of 2 × 10−6M BIPM to thiols is completed within 30 min at pH 6.85 and 0°C. A linear relation was observed up to 10−5M of the reagent between concentration of adduct and intensity of fluorescence. 3. 3. A very sensitive fluorometric determination of thiol compounds has been developed based on the above results. The detection limit was 10−6M concentration of thiols, with 0.1 ml of sample, corresponding to 0.03 μg reduced glutathione, with an accuracy of 3%. 4. 4. Using the fluorometric method, the oxidation rate of glutathione catalyzed by metal ions was examined and the contents of thiols in human lacrimal fluid and seurm were determined.

Photoreaction of arenecarbothioamides with furans. The regioselective photoinduced acylation at the 3-position of furans☆☆☆

Abstract Irradiation of arenecarbothioamides ( 1 , 4 ) with furan ( 2 ) gave regioselectively 3-aroylfurans ( 3 , 5 ).

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry: I. Conformation changes around the fluorescence-labeled sulfhydryl group of actin

Whether the conformation of the region containing the maleimide-reactive sulfhydryl group(s) in actin is associated with the functional states of the molecule was examined by means of fluorescence-label technique using a fluorescent sulfhydryl reagent, N-(p-(2-benzimidazolyl)phenyl)maleimide. n1. n1.|When G-actin labeled with the maleimide was polymerized to F-actin, the fluorescence intensity and the efficiency of the energy transfer from tryptophan residue(s) to the attached dye increased to a certain extent without shift of the emission maximum. n n2. n2.|Nucleotides quenched the fluorescence of both maleimide-labeled G- and F-actins. Divalent cations, Mg2+ and Ca2+, antagonized this effect on G-actin, but not on F-actin. n n3. n3.|On addition of heavy meromyosin, the fluorescence intensity of BIPM-labeled F-actin increased by 20% with a slight blue shift of the emission maximum. The addition of ATP to this system put these changes in the fluorescence properties back, but not completely, to those of the free BIPM-actin. n n n n nBased on the results obtained, a possible role of the region containing BIPM-labeled sulfhydryl group(s) was discussed.

X. fluorimetric estimation of the reactivity of thiols; examples in small molecular compounds, taka-Amylase A and myosin A

Abstract The kinetic analysis on the reactivity of organic thiols was carried out using a maleimide derivative, N-(p-(2-benzimidazolyl)phenyl)maleimide, by means of a novel fluorimetric determination of the simple linear equations giving the dissociation constant of a thiol, pKa, and the pH-independent second-order rate constant, κo. 1. 1. Kinetic parameters, pKa and κo, for seven organic thiols including aromatic compounds were fluorimetrically determined. The values obtained were in good agreement with those reported using different methods. 2. 2. The Bronstead relation on the data obtained was found to be fairly linear except thiosalicylic acid, which has a strong intramolecular hydrogen bond. 3. 3. pKa and κo of thiophenol simultaneously shshowed a biphasic response to the increasing polarity of solvents, while the value of pKa for thiosalicylic acid increased with decreasing polarity, without an appreciable increase in κo. 4. 4. The parameters for the single thiol per mole of Taka-amylase A were on the Bronstead plot in spite of the very low pKa value, 7.9–8.0. In contrast, those for myosin A possessing S1 modified sith N-ethylmaleimide were far from this straight line, the rate being markedly low relative to the pKa values. Substrates affected the state of the thiols for the both enzymes, but in different ways. The above results, obtained with a very small amount (usually, few mg) of a protein sample, have revealed that the proposed fluorimetric determination of the reaction rate of thiolsis simple and easy, although N-(p-(2-benzimidazolyl)phenyl)maleimide is somewhat labile in alkaline pH. The Bronstead relation thus obtained is effective for searching the state of thiol in macromolecules.

Formation and reactivity of dilithiated N-furfurylbenzamides. Synthesis of α-substituted N-furfurylbenzamides

Abstract Dianion 3 can be efficiently and regioselectively generated from N-furfurylbenzamide with lithium diisopropylamide/tetrahydrofuran/−78°C and react with various electrophiles to give α-substituted N-furfurylbenzamide derivatives in good yields.

Chemical evidence showing involvement of sulfhydryl groups in protein synthesis with E. coli system

Abstract The activity of E. coli ribosomes to synthesize polyphenylalanine under the direction of polyuridylic acid is inhibited by certain maleimide derivatives. Using a fluorescence-labeled sulfhydryl reagent, N-( p -(2-benzimidazolyl)phenyl)maleimide, it is confirmed that the inhibition takes place through covalent bond formation of this reagent with cysteine-sulfhydryl groups of the ribosomal proteins.