Frank G. Loontiens

Fluorogenic and chromogenic glycosides as substrates and ligands of carbohydrases

Publisher Summary Nitrophenylglycosides are frequently used substrates of carbohydrases. Alternatively, the 4-methylumbelliferyl (7-hydroxy-4-methylcoumaryl) derivatives offer a more sensitive (fluorometric) method of detection. Some of these compounds have become commercially available. This chapter describes the preparation and use of these glycosides in the study of some cellulolytic enzymes. The difference in spectral properties of free 4-methylumbelliferone and its carbohydrate conjugates allows sensitive and continuous assays of cellulolytic activities in absorption or fluorescence modes. The preparation and use of 1-thio derivatives with different chromophoric reporter groups are included. Due to their optical characteristics, the chromophoric (fluorochromic) derivatives offer distinct advantages over the use of classical substrates of cellulolytic enzymes, as they are sensitive tools for the determination of the number of binding sites, study of association modes, and binding kinetics, breakdown patterns, and inhibition characteristics. The chapter describes some applications of chromophoric.

Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand

Erythrina cristagalli agglutinin, a dimeric lectin [J.L. Iglesias, et al. (1982) Eur. J. Biochem. 123, 247-252] was shown by equilibrium dialysis to be bivalent for 4-methylumbelliferyl-beta-D-galactoside. Upon binding to the lectin, this ligand showed a difference absorption spectrum with two maxima (at 322 and 336 nm) of equal intensity (delta epsilon = 1.2 X 10(3) M-1 cm-1). A similar spectrum with a comparable value of delta epsilon was obtained with 4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide. Binding of methyl-alpha-D-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (delta epsilon = 2.8 X 10(2) M-1 cm-1) at 291.6 nm. Upon binding of N-dansyl-D-galactosamine to the lectin, there was a fivefold increase in fluorescence intensity of this ligand. The association constant for N-dansyl-D-galactosamine was caused by a very favorable delta S degree of the dansyl group without affecting the strictly carbohydrate-specific character of binding. N-Dansyl-D-galactosamine was employed as a fluorescent indicator ligand in substitution titrations. This involved the use of simple carbohydrates, N-acetyllactosamine, and oligosaccharides which occur in the carbohydrate units of N-glycoproteins; the latter were Gal(beta 1----4)GlcNAc(beta 1----2)Man, Gal(beta 1----4)GlcNAc(beta 1----6)Man, and Gal(beta 1----4)GlcNAc(beta 1----6)[Gal(beta 1----4)GlcNAc(beta 1----2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl-D-galactosamine, methyl-alpha-D-galactoside, and lactose, -delta H degrees increased from 24 to 41 kJ mol-1; it increased further for N-acetyllactosamine and then remained unchanged for the N-acetyllactosamine-containing oligosaccharides (55 +/- 1 kJ mol-1. This indicated that the site specifically accommodated the disaccharide structure with an important contribution of the 2-acetamido group in the penultimate sugar. Beyond this, no additional contacts seemed to be formed. This conclusion also followed from considerations of delta S degrees values which became more unfavorable in the above series (-23 to -101 +/- 4 J mol-1 K-1); the most negative value of delta S degrees was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.

The interaction of Ricinus communis hemagglutinin with polysaccharides and low molecular weight carbohydrates

Abstract The hemagglutinin from the castor bean (Ricinus communis) shows a precipitin-like like reaction with a series of branched galactomannas, dependent on their galactose: mannose ratio. Charged and neutral linear galactants fail to co-precipitate with the protein. Hapten inhibition of the turbidimetrically assayed hemagglutinin-Lucerne seed galactomannan system incidates that simple sugars such as D -galactose, D -fucose and L -arabinose bind to the protein. Of the glycosides tested, methyl β- D -galactopyranoside is a better inhibitor than the corresponding α-another. p- Nitrophenyl -2- acetamido -2- deoxy -β- D - galactopyranoside is about 10 tiems less effective than p- nitrophenyl -β- D - galactopyranoside , the best inhibitor tested. Equilibrium dialysis data obatined with the latter ligand are consistent with a protein containing two identical and independent binding sites with an intrinsic association constant equal to 1.65 ṡ 104 l/mole at 25 °C.

Binding of para-substituted phenyl glycosides to concanavalin a

Abstract The binding of para-substituted phenyl glycopyranosides of α- D -glucose, β- D -glucose, and α- D -mannose by concanavalin A has been related to the electronic and hydrophobic nature of the substituents by multiparameter regression analysis. Hydrophobicity is an important factor for the binding of the β- D -glucosides, especially in the p -alkyl series; a smaller but mutually comparable dependence on hydrophobicity is noted for each of the p -halogeno, p -alkoxy, and p -acyl substituent series. In the last two series, an additional substituent interaction with the protein might occur. The more tightly bound α- D -mannosides and α- D -glucosides show a constant binding ratio for all p -phenyl substituents. Here, hydrophobic contributions are negligible when compares with electronic effects. Hammett relations ( p −= 0.5) are valid for α- D -glucosides and α- D -mannosides, and can be improved by considering inductive and mesomeric contributions of the substituents. These observations are compatible with crystallographic data at a resolution of 2 A. Their relevance for the α- D -anomeric specificity, governed by a protein electrophile, is discussed.

Carbohydrate binding specificity of the lectin from the PEA (Pisum sativum)

Hapten inhibition measurements on the precipitin reaction between Pisum sativum lectin and Pichia pinus phosphomannan showed the lectin to bind D-mannose, D-glucose, D-fructose and L-sorbose. Unmodified hydroxyl groups at the C-4 and the C-6 positions of the D-glucopyranose ring were essential for binding to the protein. Modification of the C-2 hydroxyl group was allowed in the D-glucopyranose ring but not in the D-mannopyranose configuration. Substitution of the hydroxyl hydrogen atom at the C-3 position of D-glucose increased the binding efficiency. With the exception of gentiobiose, the beta-linked glycobioses tested were not bound to the lectin, whereas the alpha-linked glycobioses were potent inhibitorsmin general, the P. sativum lectin was found to be less sensitive to structural variation of inhibiting carbohydrates than concanavalin A, the lectin from Canavalia ensiformis.

Modification by site-directed mutagenesis of the specificity of Erythrina corallodendron lectin for galactose derivatives with bulky substituents at C-2

Examination of the three‐dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc‐specific lectin [(1991) Science 254, 862‐866], revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134‐Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C‐2 of the lectin‐bound galactose. Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C‐2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134‐Trp135 is replaced by Ser‐Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser‐Glu‐Tyr‐Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134‐Trp135 was replaced by Ser‐Glu‐Tyr‐Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T. They were expressed in Escherichia coli, as done for recombinant ECorL [(1992) Eur. J. Biochem. 205, 575‐581]. The mutants had the same hemagglutinating activity as native or rECorL. Their specificity for galactose, GalNAc and MeβGalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with MeαGalNDns and MeβGalNDns were measured by spectrofluorimetric titration. The results showed that Y108T had essentially similar specificity as the native and recombinant lectins. The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants. Our findings also provide an explanation for the inability of PNA to accommodate C‐2‐substituted galactose derivatives at its primary subsite.

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric concanavalin A Fluorescence temperature-jump relaxation study.

The kinetics of saccharide binding to the treatment form of concanavalin A have been studies at pH 7.2 with the temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside was used as a ligand; its fluorescence is totally quenched upon binding. A single relaxation of ligand fluorescence (tau = 20-400 ms) was observed and was investigated at three different temperatures, using kinetic titration and dilution types of experiments. The concentration dependence of the relaxation time and amplitude was consistent with a single-step bimolecular association and independent binding sites. In the temperature range 13-24 degrees C the association and dissociation rate parameters are in the range (6-10) X 10(4) M-1 s-1 and (1.4-3.2)s-1 respectively, corresponding to activation energies for the forward and reverse reactions equal to approx. 13 and 8 kcal/mol (54 and 33 kJ/mol) respectively. Two additional relaxations of protein fluorescence (3 ms and larger than 1 s at 25 degrees C) were unaffected by carbohydrate binding. Tetrameric concanavalin A shows carbohydrate binding parameters that are almost identical to those of native or derivatized dimeric concanavalin A.

Kinetics of binding of Hoechst dyes to DNA studied by stopped-flow fluorescence techniques.

Publisher Summary The dye-DNA structures determined both by X-ray crystallography and solution nuclear magnetic resonance (NMR) show the molecular details of the complex formed and make the p-OH Hoechst–DNA interaction an excellent model system with which to study strong and sequence-specific binding in the minor groove of DNA. To gain insight into the binding mechanism the structural information has been complemented with the kinetics of complex formation and dissociation. This chapter presents a working example of a kinetic analysis involving a ligand that binds strongly to specific sequences of DNA. The Hoechst dye–DNA interaction is an informative model system for the study of the highly A/T-specific minor groove binding to DNA. The details of a two-exponential analysis are discussed in order to demonstrate the complexities that can be expected when dye molecules interact strongly with specific DNA sequences. Unless the data are accurate the stopped-flow curves are well described by single exponentials and the kinetic analysis suggests a simple, single-step binding mechanism. The association rate is almost diffusion controlled and the specificity of the dye comes solely from the reverse rate parameter, possibly determined by the number of dye–DNA interactions in the minor groove that must be broken for the dye molecule to escape.

The use of 4-methylumbelliferyl glycosides in binding studies with the lectins BS I-A4, BS I-B4 and BS II from Bandeiraea (Griffonia) simplicifolia

Seeds of Bandeiraea simplicifolia (Griffonia simplicifolia) contain 12 lectins and isolectins distributed over the 4 groups BS I-IV [I]. This paper deals with the tetrameric and tetravalent lectins BS I and BS II [2]. BS II is a GlcNAc-specific lectin [3,4]. The BS I group is composed of 5 Gal-binding isolectins Aq, A3B, AzBz, AB3 and B4 with degrees of A or B bloodgroup specificity determined by the ratio of subunit A (GalNAc specific) over subunit B (Gal specific) [S-8]. The degree of (Yor p-anomeric binding preference is influenced by the nature of the aglycon; this forms the basis of a reproducible separation of the 5 BS I isolectins by affinity chromatography using aryl P-glycosides as ligand arms [9]. The ligands used here for binding studies in solution are 4-methylumbelliferyl-(MeUmb-) glycosides. Their versatility is apparent from: 6)

Wheat germ agglutinin: effects of limited reduction of the disulfide bonds and carboxymethylation on the properties of the lectin

Lectins are proteins mainly of plant origin which bind specifically to free sugars, or to the oligosaccharide moieties of glycoproteins and glycolipids (reviewed [ 1,2]). The lectin isolated from lliticum vulgare (WGA) consists of two protomers of 164 amino acid residues each [3-51. It specifically binds GlcNAc or its /31+4-linked diand trisaccharides [3] as well as sialic acid [6,7] and contains 4 binding sites for these ligands [5-91. Each protomer consists of 4 structural domains containing 41 amino acid residues, among them 8 cysteines forming 4 intradomain disulfide bridges [3,8,10]. In connection with the application of heterobifunctional crosslinking reagents to lectins [l 1 ,121, the possibility of limited reduction of disultide bonds in WGA was investigated. The lectin was treated with different amounts of DTT followed by carboxymethylation with IAA or iodoacetamide. We found that it is not possible to obtain a homogeneous preparation of partially reduced and carboxymethylated WGA. Rather, two populations of WGA are formed, one unmodified (or only slightly modified) and fully active, the other carboxymethylated almost to completion with no sugar binding capacity or hemagglutinating activity. We conclude