Determination of association and dissociation rate constants for bilirubin--bovine serum albumin.

The association rate constant for the binding of bilirubin to bovine serum albumin has been determined in a continuous-flow experiment. The value obtained is 0.9 x 10⁶m^?1S^?1. Furthermore the dissociation rate constant is determined from the rate of the peroxidase-catalyzed oxidation of bilirubin in a bilirubin-albumin solution. This figure is 3.1 × 10^?2s ¹. Calculation of the apparent binding equilibrium constant from the two rate constants gives 2.9 x 10⁷m^?1. The above mentioned peroxidase oxidation has also been used for a direct estimation of the binding equilibrium constant giving 2.7 × 10⁷m^?1. All experiments are carried out at 36 °C and pH 7.4.     

Specific and saturable binding of albumin to rat adipocytes: Modulation by epinephrine and possible role in free fatty acid transfer

Specific and saturable binding of ¹²⁵I-bovine albumin to rat adipocytes in suspension was observed (apparent K_d 2.09 ± 0.52 × 10^?6 M; 8.58 ± 2.49 × 10⁶ sites per cell; mean ± SEM). The binding was rapid and reversible for at least 10 min, suggesting that endocytosis of albumin was minor under assay conditions. Pre-incubation of cells with epinephrine bitartrate caused an apparent increase in number and decrease in affinity of the adipocyte binding sites for albumin. These findings suggest that a specific and saturable interaction of albumin with the adipocyte surface may play a role in the cellular uptake and release of free fatty acids.     

Forskolin-loaded human serum albumin nanoparticles and its biological importance

Abstract

In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were successfully prepared by incorporation and affinity-binding methods. FR-HSANPs were characterized by transmission electron microscope that most of them are circular in shape and size is around 340?nm. The drug loading was more than 88% and further sustained release profiles were observed as it is 77.5% in 24?h time. Additionally, the cytotoxicity results with HepG2 cells indicated that FR-HSANPs showed significantly higher cytotoxicity and lower cell viability as compared to free forskolin (FR). Furthermore, to understand the binding mechanism of human serum albumin (HSA) with forskolin resulted from fluorescence quenching as a static mechanism and the binding constant is 6.26?±?0.1?×?10⁴ M^?1, indicating a strong binding affinity. Further, association and dissociation kinetics of forskolin–HSA was calculated from surface plasmon resonance spectroscopy and the binding constant found to be K_forskolin = 3.4?±?0.24?×?10⁴ M^?1 and also fast dissociation was observed. Further, we used circular dichroism and molecular dynamics simulations to elucidate the possible structural changes including local conformational changes and rigidity of the residues of both HSA and HSA–forskolin complexes.

Communicated by Ramaswamy H. Sarma     

Characterization of a small apolar anion binding site of human serum albumin.

Small unbranehed fatty acid anions inhibit the fast reaction between p-nitrophenylacetate and human serum albumin. Plots of reactivity versus fatty acid anion-albumin ratios resemble simple binding isotherms from which corresponding dissociation constants have been calculated. For the homologous fatty acid anions, butyrate through decanoate, dissociation constants decrease from 3.2 × 10^?4 to 1 × 10^?7m, respectively, by uniform increments per methylene group according to the relationship ?ΔG °(kcal) = 0.804_n + 2.30, where n is the number of constituent methylene groups. Small fatty acid anions thus appear to interact primarily with a single, relatively uniform apolar binding site with a capacity sufficient for nine methylene groups. Fatty acid anions larger than decanoate interact significantly with other sites and do not obey the same relationship. The reactivity of diluted human serum with p-nitrophenylacetate was found to be one-third to one-half of that expected for its content of serum albumin, but as in vitro, it could be completely inhibited by small amounts of decanoate.     

Multispectroscopic studies of the interaction of folic acid with glycated human serum albumin

Abstract

The interaction between glycated human serum albumin (gHSA) and folic acid (FA) was investigated by various spectroscopic techniques, such as fluorescence, circular dichroism, UV–vis absorption spectroscopy and electrophoretic light scattering technique. These methods characterize the binding properties of an albumin–folic acid system. The binding constants values (K_a) at 300 and 310 K are about 10⁴ M^?1. The standard enthalpy change (ΔH) and the standard entropy change (ΔS) were calculated to be ～?20?kJ mol^?1 and ～16 J mol^?1 K^?1, respectively, which indicate characteristic electrostatic interactions between gHSA and folic acid. The CD studies showed that there are no significant conformational changes in the secondary structure of the protein. Moreover, the zeta potential measurements proved that under physiological conditions the gHSA–folic acid complex shows instability. No significant changes in the secondary structure of the protein and reversible drug binding are the desirable effect from pharmacological point of view.

Communicated by Ramaswamy H. Sarma     

Fluorescence spectrometric study on the interaction of tamibarotene with bovine serum albumin

The interaction between tamibarotene and bovine serum albumin (BSA) was studied using fluorescence quenching technique and ultraviolet–visible spectrophotometry. The results of experiments showed that tamibarotene could strongly quench the intrinsic fluorescence of BSA by a dynamic quenching mechanism. The apparent binding constant, number of binding site and corresponding thermodynamic parameters at different temperatures were calculated respectively, and the main interaction force between tamibarotene and BSA was proved to be hydrophobic force. Synchronous fluorescence spectra showed that tamibarotene changed the molecular conformation of BSA. When BSA concentration was 1.00 × 10^?6 mol L^?1, the quenched fluorescence ΔF had a good linear relationship with the concentration of tamibarotene in the range 1.00 × 10^?6 to 12.00 × 10^?6 mol L^?1 with the detection limit of 6.52 × 10^?7 mol L^?1. Copyright © 2010 John Wiley & Sons, Ltd.     

Probing the intermolecular interactions into serum albumin and anthraquinone systems: a spectroscopic and docking approach

Intermolecular interaction study of human serum albumin (HSA) with two anthraquinones i.e. danthron and quinizarin has been performed through fluorescence, UV-vis and CD spectroscopy along with docking analysis. The titration of drugs into HSA solution brought about the quenching of fluorescence emission by way of complex formation. The binding constants were found to be 1.51 × 10⁴ L mol^?1 and 1.70 × 10⁴ L mol^?1 at λ_exc = 280 nm while at λ_exc = 295 nm, the values of binding constants were 1.81 × 10⁴ L mol^?1 and 1.90 × 10⁴ L mol^?1 which hinted toward binding of both the drugs in the vicinity of subdomain IIA. Different temperature study revealed the presence of static quenching mechanism. Moreover, more effective quenching of the fluorescence emission was observed at λ_exc = 295 nm which also suggested that both the drug molecule bind nearer to Trp-214. Thermodynamic parameters showed that hydrophobic interaction was the major force behind the binding of drugs. The UV-vis spectroscopy testified the formation of complex in both the systems and primary quenching mechanism as static one. The changes in secondary structure and α-helicity in both the systems were observed by circular dichroism spectroscopy. Furthermore, molecular docking analysis predicted the probable binding site of drugs in subdomain IIA of HSA molecule. The types of amino acid residues surrounding the drug molecule advocated that van der Waals forces, hydrophobic forces and electrostatic forces played a vital role in the stabilization of drug-protein complex formed.     

Effect of salicylic acid on glucucorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma

The binding activity of [³H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [³H]dexamethasone of 3.64 · 10^?8 M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 · 10⁵.Total binding activity to [³H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid at 37°C. The maximum enhancement was seen at the concentration of 10^?3 M and in 3 h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicylic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin, also enhanced the total binding activity to [³H]dexamethasone of the cytoplasmic fraction at the concentration of 2 · 10^?5 M and 2 · 10^?7 M, respectively.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine and human serum albumin

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine (CECT) and human serum albumin (HSA) were investigated using fluorescence, UV absorption spectroscopic and molecular modeling techniques under simulative physiological conditions. The intrinsic fluorescence intensity of HSA was decreased with the addition of CECT. The fluorescence data handled by Stern–Volmer equation proved that the quenching mechanism of the interaction between CECT and HSA was a static quenching procedure. The binding constants evaluated utilizing the Lineweaver–Burk equation at 17, 27 and 37?°C, were 2.340?×?10⁴, 2.093?×?10⁴ and 1.899?×?10⁴?L?mol^?1, respectively. The thermodynamic parameters were calculated according to van’t Hoff equations. Negative enthalpy (ΔH) and positive entropy (ΔS) values indicated that both hydrogen bond and hydrophobic force played a major role in the binding process of CECT to HSA, which was consistent with the results of the molecular modeling study. In addition, the effect of other ions on the binding constant of CECT-HSA was examined.     

Binding of meso-tetra(4-sulfonatophenyl)porphine to haemopexin and albumin studied by spectroscopy methods

• 1.1. The interaction of haemopexin and albumin with TPPS₄ was studied by measuring the absorption and fluorescence spectra. Haemopexin was found to have one strong TPPS₄ binding center (K_a = 3 × 10⁷M⁻¹).
• 2.2. Haem-haemopexin complex appears to have no specific binding site for TPPS₄. Occupation of the specific binding center of haemopexin molecule by a haem abolishes TPPS₄ binding.
• 3.3. Albumin was found to possess one strong TPPS₄ binding center (K_a = 3 × 10⁶M⁻¹) besides two or three weak binding sites (K_a = 2 × 10⁵M⁻¹).
• 4.4. Haern-albumin complex possesses only one weak TPPS₄ binding site (K_a = 7 × lO⁵M⁻¹). These observations suggest identity of primary binding sites of TPPS₄ and haem on albumin molecule.

     

Unraveling the binding mechanism of asiatic acid with human serum albumin and its biological implications

Asiatic acid (AsA), a naturally occurring pentacyclictriterpenoid found in Centella asiatica, plays a major role in neuroprotection, anticancer, antioxidant, and hepatoprotective activities. Human serum albumin (HSA), a blood plasma protein, participates in the regulation of plasma osmotic pressure and transports endogenous and exogenous substances. The study undertaken to analyze the drug-binding mechanisms of HSA is crucial in understanding the bioavailability of drugs. In this study, we analyzed the cytotoxic activity of AsA on HepG2 (human hepatocellular carcinoma) cell lines and its binding, conformational, docking, molecular simulation studies with HSA under physiological pH 7.2. These studies revealed a clear decrease in the viability of HepG2 cells upon exposure to AsA in a dose-dependent manner with an IC50 of 45?μM. Further studies showed the quenching of intrinsic fluorescence of HSA by AsA with a binding constant of K_AsA?=?3.86?±?0.01?×?10⁴?M^?1, which corresponds to the free energy of (ΔG) ?6.3?kcal?M^?1 at 25?°C. Circular dichroism (CD) studies revealed that there is a clear decrease in the α-helical content from 57.50?±?2.4 to 50%?±?2.3 and an increase in the β-turns from 25?±?0.65 to 29%?±?0.91 and random coils from 17.5%?±?0.95 to 21%?±?1.2, suggesting partial unfolding of HSA. Autodock studies revealed that the AsA is bound to the subdomain IIA with hydrophobic and hydrophilic interactions. From molecular dynamics, simulation data (RMSD, Rg and RMSF) emphasized the local conformational changes and rigidity of the residues of both HSA and HSA–AsA complexes.     

Analysis of phenolic compounds in health care products by low‐pressure liquid‐chromatography with monolithic column and chemiluminescent detection

This paper presents a new application for monolithic columns with low‐pressure chromatographic separation using an flow injection analysis configuration with chemiluminescent detection for the determination of a mixture of phenolic compounds: phloroglucinol, 2,4‐dihydroxybenzoic acid, salicylic acid, methyl paraben and n‐propyl gallate. The procedure consists of the separation of these compounds on a reverse‐phase ultra‐short monolithic column with pH 3.0 acetate buffer and 5% acetonitrile as carrier phase. The detection is based on a chemiluminescence measurement coming from Ce(IV)–Rhodamine 6G chemistry with the incorporation of two different chemiluminescent chemical conditions in the chromatographic setup in order to enhance the sensitivity for the different phenolic compounds. All separation and detection variables were optimized to propose a determination method. The analysis is performed in 280?s, with the sampling frequency being some 13 h^?1. The calibration function is a double reciprocal function obtaining good results within two orders of magnitude. The limits of detection were 8.8 × 10 ^?8 m (phloroglucinol), 2.7 × 10 ^?8 m (2,4‐dihydroxybenzoic acid); 2.3 × 10 ^?8 m (salicylic acid); 5.2 × 10 ^?8 m (methyl paraben) and 4.1 × 10 ^?6 m (n‐propyl gallate), and the relative standard deviations at a medium level of the linear range were 4.4% (phloroglucinol), 2.8% (2,4‐dihydroxybenzoic acid), 5.2% (salicylic acid), 3.6% (methyl paraben) and 6.8% (n‐propyl gallate). The method was applied and validated satisfactorily for the determination of these compounds in healthcare products, comparing the results against an HPLC reference method. Copyright © 2009 John Wiley & Sons, Ltd.     

Binding of a chromen-4-one Schiff’s base with bovine serum albumin: capping with β-cyclodextrin influences the binding

This work deals with the synthesis of 6-methyl-3-[(4′-methylphenyl)imino]methyl-4H-chromen-4-one (MMPIMC), its binding to β-cyclodextrin, and the influence of the cyclodextrin complexation on the compound’s binding to bovine serum albumin (BSA). The 1:2 stoichiometry for the complexation of MMPIMC with β-cyclodextrin is determined with the binding constant of 1.90 × 10⁴ M^?2. The structure of host–guest complex plays a role in protein binding of MMPIMC. One- and two-dimensional NMR spectra are used to determine the mode of binding of the guest to β-cyclodextrin cavity and the structure of the inclusion complex is proposed. The binding of MMPIMC with BSA in the absence and the presence of β-cyclodextrin is studied. The binding strengths of MMPIMC–BSA (1.73 × 10⁵ M^?1) and β-cyclodextrin-complexed MMPIMC–BSA (9.0 × 10⁴ M^?1) show difference in magnitude. The Förster Resonance Energy Transfer efficiency and the proximity of the donor and acceptor molecules, are modulated by β-cyclodextrin. Molecular modeling is used to optimize the sites and mode of binding of MMPIMC with bovine serum albumin.     

Some factors stimulating and inhibiting pancreatic deoxyribonuclease

Для выяснения роли ДН-азы в реакции трансформации исследовали действие различных сред, применяемых при трансформации пневмококков, на активность панкреатической ДН-азы. Эти виды среды резко повышали активность фермента. Далее, было установлено, что это повышение вызывается yeast-экстрактом, входящим в состав этих сред. С помщяю спектрального анализа и пламенного спектрофотометра в yeast-экстракте были обнаружены Mg²⁺, Ca²⁺, K⁺ и Na⁺. Изучалось действие этих ионов на активность ДН-азы и было установлено, что в присутствии Mg²⁺ (5×10^?3 m)иCa²⁺(вконцентрации 2×10^?4 m) резко повышается активность фермента, тогда как прибавление к этой системе KCl в концентрации 2×10^?1, 2×10^?2, 2×10^?3 и 2×10^?4 m оказывает угнетающее действие.—Обсуждается возможное влияние этих ионов на peaкцию трансформации.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Determination on the binding of chlortetracycline to bovine serum albumin using spectroscopic methods

In this work, the interaction of chlortetracycline with bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. Results indicated that chlortetracycline quenches BSA fluorescence mainly by a static quenching mechanism. The quenching constants (K_SV) were obtained as 5.64 × 10⁴, 4.49 × 10^4/, and 3.44 × 10^4/ M_?1 at 283, 295, and 307 K, respectively. The thermodynamic parameters of enthalpy change Δ H°, entropy change Δ S°, and free energy change Δ G° were ?5.12 × 10^4/ J mol?1, ?97.6 J mol?1 K?1, and ?2.24 × 10^4/ J mol?1 (295 K), respectively. The association constant (K_A) and the number of binding sites (n) were 9.41 × 10^3/ M?1 and 0.86, respectively. The analysis results suggested that the interaction was spontaneous, and van der Waals force and hydrogen‐bonding interactions played key roles in the reaction process. In addition, CD spectra proved secondary structure alteration of BSA in the presence of chlortetracycline. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:331–336, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21424     

Spectrophotofluorometric evidence of negatively cooperative binding of warfarin and phenylbutazone to human serum albumin

Competition between drugs for common binding sites on plasma proteins is an often discussed mechanism of drug interaction. In this study, the validity of the concept that phenylbutazone displaces warfarin from human serum albumin (HSA) by direct competition for the same sites was investigated. The fluorescence enhancement titration procedure of Kolb & Weber, Biochemistry $\text{14}$, 4471–4476 (1975) was used to characterise the warfarin-HSA interaction under the following conditions: excitation and emission wavelenghts - 310 & 390 nm respectively; buffer 0.1 M phosphate, pH 7.0 (25°C) and [HSA] - 10^?6M. The titrations were then repeated in the presence of varying concentrations of phenylbutazone (10^?6M ? 5 × 10^?4M). Bjerrum plots of the data showed a shift of the warfarin-HSA binding curves to higher free levels of warfarin with increasing concentrations of phenylbutazone. However this shift was saturable, contrary to expectation for directly competitive antagonism. Together with indirect evidence from the literature, the results suggest that displacement of warfarin by phenylbutazone is via a negatively cooperative mechanism rather than by direct competition.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Binding of divalent copper ions to aspartic acid residue 52 in hen egg-white lysozyme

Divalent copper was found to inhibit non-competitively the lysis of Micrococcus lysodeikticus cells by hen egg-white lysozyme, with an inhibition constant K_a= 3.8 × 10²m^?1. The association constants of Cu²⁺ for lysozyme and for a derivative of lysozyme in which tryptophan residue 108 was selectively modified, were measured spectrofluorimetrieally and found to be 1.8 × 10²m^?1 and 1.0 × 10³m^?1, respectively. The electron spin resonance spectrum of Cu²⁺ was not affected by the addition of lysozyme, whereas many new lines appeared on addition of the modified protein. This was interpreted as evidence for the binding of Cu²⁺ in the neighbourhood of tryptophan 108. To unequivocally establish the site of ligation of Cu²⁺, crystals of lysozyme soaked in Cu²⁺ were examined by X-ray crystallography and the results compared to those obtained from crystals of native lysozyme. Cu²⁺ was found to be located 2 to 3 Å from the carboxyl side-chain of aspartic acid 52, 5 Å from the carboxyl of glutamic acid 35 and about 7 Å from tryptophan 108.The addition of a saccharide inhibitor to lysozyme was found to increase the association constant of Cu²⁺ for lysozyme from a value of 1.8 × 10²m^?1 to 6.0 × 10²m^?1. This finding was interpreted as indicative of a change in conformation around tryptophan 108 and glutamic acid 35 induced by the interaction of saccharides with the enzyme, which affects the metal binding properties of aspartic acid 52.