Split-compartment mixing cells for difference spectroscopy

Yankeelov, John A.

doi:10.1016/0003-2697(63)90138-6

Cited by 56 publications

(10 citation statements)

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“…Unless otherwise specified, all runs were performed at 8". Matched Yankeelov cuvettes [32] (Pyrocell Manufacturing Co., Westwood, N.Y.) with an effective path length of 9 m m were employed. Into one compartment of a Yankeelov cuvette was placed 0.80ml of an appropriate concanavalin A solution; 0.80 ml of a saccharide solution was pipetted into the second compartment.…”

Section: Methodsmentioning

confidence: 99%

Ultraviolet Difference Spectral Studies on Concanavalin A

Hassing

Goldstein

1970

European Journal of Biochemistry

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The interaction of concanavalin A, the phytohemagglutinin of the jack bean, with a variety of glycosides has been studied by the technique of ultraviolet difference spectroscopy. Whereas methyl a-D-glUC0-and manno-pyranoside gave rise to relatively low intensity difference spectra, p-nitrophenyl a-D-mannopyranoside and a-D-glucopyranoside yielded large difference spectra upon interaction with concanavalin A. Using this technique as a measure of concanavalin A activity, it was demonstrated that the protein specifically binds low molecular weight carbohydrates at much lower pH values (e. g. pH 2.4) than previously believed. Although polysaccharides are also bound at these low pH values, they are not precipitated by concanavalin A. Molecular weight studies in acid media indicate that the protein does not dissociate and it is suggested that electrostatic repulsion of the protein molecules due to their high net positive charge prevents protein-polysaccharide lattice formation and hence failure of the complex to precipitate.The interaction of certain specific carbohydrates and carbohydrate-containing macromolecules with concanavalin A, the lectin present in jack bean, has been studied extensively. This protein has been shown t o interact to form it precipitate with various polysaccharides, e.g. glycogens, mannans, and dextrans, and with certain serum glycoproteins [I-121. Extensive inhibition studies, using mono-and oligosaccharides, have demonstrated that the specificity of the saccharide binding sites of concanavalin A are directed towards the unmodified hydroxyl groups at the (2-3, C-4 and C-6 positions of a-D-glucopyranosyl and a-D-mannopyranosyl residues [la-I?]. Equilibrium dialysis studies have indicated that one mole of monosaccharide (methyl a-D-ghcopyranoside or methyl a-D-mannopyranoside) is bound per 32000 to 34000 g of concanavalin A [IS, 191. Furthermore, the linearity of the Scatchard plots suggests a homogeneity of binding constants for a given ligand.Other studies have indicated the complexity of structure of concanavalin A. Olson and Liener [20] found that in 8 M urea at pH 7, concanavalin A dissociated into three fractions, with molecular weights corresponding to 16500, 42000 and in excess of 200000, each with the same peptide map, amino acid composition, and& N-terminal amino acid (alanine) . Recently Doyle et al.[30] demonstrated that when D-glUCOSe, an inhibitor of concanavalin A-polysaccharide precipitation, was added to concanavalin A, an ultraviolet difference spectrum was obtained. This report represents a detailed study of the carbohydrate-binding activity of concanavalin A, using the technique of ultraviolet difference spectroscopy. MATERIALS AND METHODSConcanavalin A, prepared as described previously [31] was stored in 1.0 M NaCl at 4". Heavy metalfree concanavalin A was prepared by dialysis of the native protein against 0.1 N HC1, followed by dialysis against 0.05M NaCl in the cold. The demetallized protein did not form a precipitate with dextran B-135543 [ll].All carbohydrates used wer...

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Section: Methodsmentioning

confidence: 99%

Ultraviolet Difference Spectral Studies on Concanavalin A

Hassing

Goldstein

1970

European Journal of Biochemistry

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“…Difference absorption spectra were obtained with a UVICON 810 double-beam spectrophotometer (Kontron) and tandem mixing cuvettes (Yankeelov, 1963). Titrations were performed with two 1 cm x 1 cm x 4.5 cm cuvettes, equipped with motor-driven stirrers and connected to two Hamilton syringes that deliver a solution of a non-binding sugar (sucrose) to the reference cuvette and a solution of the appropriate ligand to the sample cuvette (De Boeck et al, 1984).…”

Section: Binding Experimentsmentioning

confidence: 99%

Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei

Claeyssens

Tilbeurgh

Tomme

et al. 1989

Biochemical Journal

107

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Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1----4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.

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“…The cuvettes were mounted in a thermostated all-copper cuvette changer. The slit width used was 0.2 mm for obtaining difference spectra with 2 x 0.437-cm mixing cuvettes [15].…”

Section: Materials a N D Methodsmentioning

confidence: 99%

Binding of 4-Methylumberlliferyl beta-d-galactosyl-(13)-N-acetyl-beta-d-galactosaminide to Peanut Agglutinin. Characterisation and Application in Substitution Titrations

et al. 1983

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Binding of 4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐3‐O‐(β‐d‐galactopyranosyl)‐β‐d‐galactopyranoside [MeUmbβGal(β1→3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4–30°C range correspond to ΔG°=−(26.5 ± 0.1) kJ mol−1, ΔH°=−(58.4 ± 2) kJ mol−1 and ΔS°=−(107 ± 8) J mol−1 K−1. Values of the association constants are e. g. K= 2.5 × 105 M−1 at 4°C and K= 4.5 × 104 M−1 at 25°C. MeUmbβGal(β1→3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non‐ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K= 4.8 × 103 M−1 for methyl α‐d‐galactopyranoside [MeαGal], 2.0 × 103 M−1 for methyl β‐d‐galactopyranoside [MeβGal] and 4.7 × 103 M−1 for lactose [Gal(β1→4)Glc], all at 14.5°C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmbβGal(β1→3)GalNAc and MeUmbβGal(β1→4)Glc are larger than for MeUmbβGal and MeUmbαGal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.

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Split-compartment mixing cells for difference spectroscopy

Cited by 56 publications

References 1 publication

Ultraviolet Difference Spectral Studies on Concanavalin A

Ultraviolet Difference Spectral Studies on Concanavalin A

Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei

Binding of 4-Methylumberlliferyl beta-d-galactosyl-(13)-N-acetyl-beta-d-galactosaminide to Peanut Agglutinin. Characterisation and Application in Substitution Titrations

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