Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Stoichiometry and Preferential Interaction: Two Components of the Principle for Protein Structure Organization

Abstract

The effect of pressure on the conformational structure of amyloid β (1–40) peptide (Aβ(1–40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid Aβ(1–40) from 1655 cm^?1 (α-helix) to 1647–1643 cm^?1 (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm^?1 assigned to β-antipar- allel sheet structure was also evident. Furthermore, the peak at 1540 cm^?1 also shifted to 1527 (1529) cm^?1 in amide II band. The former was assigned to the combination of α-helix and random coil structures, and the latter was due to β-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed Aβ(1–40) samples were obtained from 33% to 22% for α-helix/random coil structures and from 47% to 57% for β-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from α-helix to random coil and to β-sheet structure. The structural transformation of the compressed Aβ(1–40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of β-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the β-sheet structure transformed. The thermal-dependent transition temperatures of solid Aβ(1–40) prepared by different pressures were near 55–60 °C.     

A mutant β-D-glucoside transport system of Escherichia coli resistant to catabolite inhibition

A mutant strain of Escherichia coli in which β-glucoside transport is resistant to catabolite inhibition by methyl α-glucoside was characterized. The mutation was probably within the gene, bglC, coding for the β-glucoside enzyme II. The mutant organism is shown to transport the β-glucoside substrate, salicin, in preference to methyl α-glucoside or fructose. Salicin also caused inducer exclusion of lactose in the mutant strain.     

Biogeochemistry of billabong sediments. I. The effect of macrophytes

SUMMARY.

• 1 We examined the effects of an emergent macrophyte (Eleocharis sphacelata R. Br., Cyperaceae) and a submerged macrophyte (Vallisneria gigantea Graeb., Hydrocharitaceae) on the biogeochemistry of the sediments of a billabong in south-eastern Australia.
• 2 Sediments from an E. sphacelata bed had significantly lower concentrations of exchangeable phosphorus than did sediments from a nearby bare area or a V. gigantea bed, but neither macrophyte had a measureable effect on their sediment's exchangeable ammonium content.
• 3 The redox potential in the upper 10cm of E. sphacelata sediments was about 100 mV higher than that of bare sediments, or of sediments colonized by V. gigantea.
• 4 There were few consistent differences between vegetated and bare sediments in terms of the activity of extracellular enzymes, such as α-amylase, protease, β-d glucosidase, lipase or alkaline phosphatase. Rates of alkaline phosphatase activity (235–306μmol (g dry wt)^?1 day^?1) were markedly higher than those commonly reported for sediments or soils.
• 5 Rates of gas release were higher from bare sediments (21–93 ml m^?2 h^?1) than from E. sphacelata or V. gigantea sediments (17–23 and 21-24ml m^?2 h^?1, respectively). Gas bubbles consisted mainly of methane (26–66%) and nitrogen (15–68%). Rates of methane ebullition varied from 5 to 60ml m^?2 h^?1.
• 6 In-vitro methanogenesis was most rapid in samples of the upper flocculent sediment. Methanogenesis was slower in V. gigantea sediments than in bare area or E. sphacelata sediments, but was markedly accelerated by additions of acetate and/or H₂/CO₂ in all sites.
• 7 Profiles of total extractable fatty acids and phospholipid fatty acids demonstrated that material derived from higher plants dominated the sediment organic matter in all sites. Bacteria were also a significant component of sediment organic matter, as fatty acids for which bacteria can be assumed the sole source accounted for 18–30% of total fatty acid content. Biomarkers for sulphate-reducing bacteria (Desulfobacter spp.) were detected, and for type II methanotrophic bacteria.

     

Co-permeability of 3H-labelled water and 14C-labelled organic acids across isolated Prunus laurocerasus cuticles: effect of temperature on cuticular paths of diffusion

Co‐permeability of ³H‐labelled water and ¹⁴C‐labelled benzoic acid or 2,4‐dichlorophenoxyacetic acid across isolated cuticular membranes of Prunus laurocerasus L. was measured at temperatures ranging from 15 to 50 °C. The water and benzoic acid permeances were highly correlated over the whole temperature range investigated, whereas water and 2,4‐dichlorophenoxyacetic acid permeances were only correlated between 15 and 30 °C. The activation energies of cuticular permeability calculated from Arrhenius plots were 40 kJ mol^?1 for water and benzoic acid and 115 kJ mol^?1 for 2,4‐dichlorophenoxyacetic acid. The slopes of the Arrhenius plots of 2,4‐dichlorophenoxyacetic acid were linear between 15 and 50 °C, whereas pronounced phase transitions around 30 °C were observed for water and benzoic acid permeability. However, with isolated polymer matrix membranes, where cuticular waxes forming the transport‐limiting barrier of cuticles have been extracted, phase transitions were not observed for water and benzoic acid. It is concluded that temperatures above 30 °C caused structural changes in the transport‐limiting barrier of the cuticles leading to additional paths of diffusion for water and benzoic acid but not for 2,4‐dichlorophenoxyacetic acid.     

Glycosylation of sesamol by cultured plant cells

The glycosylation of sesamol was investigated using cultured cells of Nicotiana tabacum and Eucalyptus perriniana. The cultured suspension cells of N. tabacum converted sesamol into its β-glucoside (7%) as well as the disaccharide, sesamyl 6-O-(β-D-glucopyranosyl)-β-D-glucopyranoside (β-gentiobioside, 30%). On the other hand, sesamyl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside (β-rutinoside, 56%), together with the β-glucoside (3%), was produced when sesamol was incubated with suspension cells of E. perriniana.     

Optimization of the composition of the nutrient medium for cellulase and protein biosynthesis by thermophilic Aspergillus fumigatus NRC 272

An active strain of Aspergillus spp. has been selected for the production of cellulolytic enzymes and proteins when grown on peracetic acid-treated wheat straw. This strain produced a considerable amount of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] in the extracellular supernatant and exhibited good overall cellulolytic activity, as measured using filter paper and Avicel as substrates. Also, under the same conditions the strain showed high activities of β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37). The maximum enzyme yields (carboxymethylcellulose activity 26.4 units ml^?1, filter paper activity 2.26 units ml^?1 and Avicel activity 16.82 units ml^?1; β-d-glucosidase 9.09 units ml^?1 and β-d-xylosidase 1.92 units ml^?1) were obtained after 96 h incubation at 45°C.     

Solid state fermentation,storage and viability of Streptomyces similanensis 9X166 using agro-industrial substrates against Phytophthora palmivora-induced black rot disease in orchids

Streptomyces similanensis 9X166 is known to be an antagonist of the black rot pathogen of orchids, Phytophthora palmivora. In this study, we investigated the production of highly viable S. similanensis 9X166 cells by solid state fermentation using agro-industrial substrates, and the shelf life of a S. similanensis 9X166 dried solid. Rice bran was found to be the most appropriate raw material for production of both viable cells and β-1,3-glucanase. A medium containing 12?g of rice bran and coconut husks at a ratio of 10:2, supplemented with 10?mL of mineral salts produced the highest number of viable cells and greatest level of β-1,3-glucanase. Ammonium sulfate was the most suitable nitrogen source, and an initial moisture content of 65% and a temperature of 30°C were found to be optimal conditions for the production of viable cells and β-1,3-glucanase. Storing the dried fermented solid under non-vacuum conditions resulted in the highest cell viability. The specific rate of degradation on viability increased as the temperature increased to 37°C, according to the Arrhenius equation. There was no difference between the storage time estimated by the Arrhenius equation from the specific rate of degradation compared to the validated storage time of S. similanensis 9X166 dried solids when maintained at the ambient temperature in Thailand. At 60 days, the product retained 10⁶ CFU/g of S. similanensis 9X166 in dried solid, which was the minimal effective amount for 100% inhibition of P. palmivora in living orchids.     

Characterization of β-hydroxybutyrate transport in rat erythrocytes and thymocytes

A method was developed for study of β-hydroxybutyrate transport in erythrocytes and thymocytes. Critical to the method was a centrifugal separation of cells from medium which took advantage of β-hydroxybutyrate transport's temperature dependence and inhibition by phloretin and methylisobutylxanthine, all of which are demonstrated in this work. These properties suggested mediated transport, as did saturation kinetics and inhibition by several agents including pyruvate and α-cyanocinnamate. Most conclusive in this regard was a 2-fold preference for d- over l-β-hydroxybutyrate. Entry was not Na⁺ dependent. It was stimulated by substitution of SO₄^2? for most of the Cl^?. The equilibrium β-hydroxybutyrate space was much higher than the Cl^? space of thymocytes, suggesting that β-hydroxybutyrate entry is not associated with net inward negative current and is not coupled to outward Cl^? or inward K⁺ movement (assuming that K⁺ is at electrochemical equilibrium). Coupling to H⁺ entry or OH^? exit is compatible with the result. These findings are consistent with β-hydroxybutyrate entry by the carboxylate transport site which has been studied extensively with pyruvate and lactate as permeants. The Cl^?/HCO₃^? exchange carrier did not appear to contribute significantly to β-hydroxybutyrate transport.     

The stability of almond β-glucosidase during combined high pressure–thermal processing: a kinetic study

The thermal and the combined high pressure–thermal inactivation kinetics of almond β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) were investigated at pressures from 0.1 to 600 MPa and temperatures ranging from 30 to 80 °C. Thermal treatments at temperatures higher than 50 °C resulted in significant inactivation with complete inactivation after 2 min of treatment at 80 °C. Both the thermal and high pressure inactivation kinetics were described well by first-order model. Application of pressure increased the inactivation kinetics of the enzyme except at moderate temperatures (50 to 70 °C) and pressures between 0.1 and 100 MPa where slight pressure stabilisation of the enzyme against thermal denaturation was observed. The activation energy for the inactivation of the enzyme at atmospheric pressure was estimated to be 216.2?±?8.6 kJ/mol decreasing to 55.2?±?3.9 kJ/mol at 600 MPa. The activation volumes were negative at all temperature conditions excluding the temperature–pressure range where slight pressure stabilisation was observed. The values of the activation volumes were estimated to be ?29.6?±?0.6, ?29.8?±?1.7, ?20.6?±?3.2, ?41.2?±?4.8, ?36.5?±?1.8, ?39.6?±?4.3, ?31.0?±?4.5 and ?33.8?±?3.9 cm³/mol at 30, 35, 40, 45, 50, 60, 65 and 70 °C, respectively, with no clear trend with temperature. The pressure–temperature dependence of the inactivation rate constants was well described by an empirical third-order polynomial model.     

The β-glucoside system of Escherichia coli III. Properties of A P–HPr: β-Glucoside phosphotransferase extracted from membranes with detergent

A P-HPr:β-glucoside phosphotransferase (enzyme II^bgl)

1 The nomenclautre of the enzymes II is that suggested by Lin (1)

has been extracted from membranes of a β-glucoside fermenting strain of Escherichia coli K 12 using the nonionic detergent Triton X–100. The extracted enzyme was rendered virtually free of both lipid and detergent by chromatography on DEAE-cellulose. At this stage, the partially purified enzyme had negligible activity, but activity was restored effectively by the addition of (1) nonionic detergents of the Tween or Triton series and (2) crude E. coli phospholipids or an anionic lipid enriched fraction, but not phosphatidylethanolamine. Detergent activators were most effective at or near the critical micelle concentration, but were inhibitory when added at concentrations above the critical micelle concentration. In order to obtain maximal initial rates of phosphotransferase activity, it was necessary to incubate the extracted, partially purified enzyme with detergent activator and HPr prior to the addition of the other assay system components. High detergent concentration inhibited the initial rate of phosphorylation by interfering with an essential step (or steps) that occur during this preliminary incubation. The activation occuring during the preliminary incubation was also highly temperature dependent; a precipitous decrease in activation was detected below 16° when Tween 40 was employed as the detergent activator. Phosphorylation mediated by the membrane associated form of the phosphotransferase was not influenced by the physical state of the lipid components of the membrane. This is in marked contrast to the properties of the phosphorylation reaction mediated by the phosphotransferase in intact cells.     

The influence of calcium and magnesium on sea bass (Dicentrarchus labrax) intestinal brush border membrane purification and activity

• 1.1. The activity of brush border enzymes (alkaline phosphatase, maltase, sucrase, trehalase, leucine amino peptidase) was higher in purified membranes prepared with calcium. The contamination of these membranes with basolateral membranes was also lower (1.27 for Na-K-ATPase activity ratio).
• 2.2. The extraction of brush border lipids was carried out according to Folch adapted method. Two dimensional thin layer chromatography was used to separate the phospholipidic fractions. Fatty acids of phospholipids were analysed using gas chromatography after acid transmethylation (column SP 2330).
• 3.3. Phospholipids are composed of phosphatidylcholine (PC: 33%), phosphatidylethanolamine (PE: 30%), sphingomyeline (SM: 21%), phosphatidylserine (PS: 14%) and phosphatidylinositol (PI: 2%). 4. PC, PE and PS are characterized by high levels of unsaturated fatty acids (monounsaturated MUFA: 21.5% and polyunsaturated PUFA: 34.9%). The most abundant PUFA belong to the (n-3) family [18:3 (n-3), 20:5 (n-3) and 22:6 (n-3)].
• 4.5. Fatty acids from sphingomyelin of purified membranes have low proportions of PUFA (13.5%) but higher proportions of MUFA (39.5%).
• 5.6. No specific differences were found between calcium and magnesium prepared membranes.
• 6.7. The low content in LPC and the absence of LPE confirmed the absence of major structural lipids transformation during the membrane purification with calcium or magnesium.
• 7.8. Glycine transport was measured during 10 sec at different temperatures using the rapid filtration technique. Glycine transport was higher with Na⁺ than with K⁺. In the presence of Na⁺, this transport increases with temperature.
• 8.9. Arrhenius curves were mono phasic without obvious breakpoint and indicated no phase transition in the lipid bilayer.
• 9.10. A significant Na⁺ dependent glycine transport has been characterized at low temperatures (0°C) which suggests a possible role of membrane polyunsaturated fatty acids in the control of glycine transport.

     

The beta-D-glucosidase system of an Actinoplanes sp

Actinoplanes sp. No. 1700, a sporangium-forming, filamentous, soil bacterium possesses a β-D-glucosidase (β-D-glucoside glucohydrolase, E.C. 3.2.1.21). The enzyme was induced to higher concentrations by addition of methyl or phenyl β-D-glucopyranoside, gentiobiose, or salicin to growing cultures. Addition of D-glucose, lactate, or acetate repressed enzyme induction back to the constitutive level, but never below it. The properties of this inducible system place it in the semi-constitutive category.Both the constitutive and the inducible enzyme were purified 60-fold; their properties were compared and found to be identical. Their pH optima lay between 5.8 and 6.0; the enzymes were stable for 2 h at 30° at pH 5.5 to 7.3. Rapid inactivation occurred at temperatures above 50°. The enzymes were inactivated by 100μM CU²⁺, Hg²⁺, Pb²⁺, and Ag⁺.Each of these β-D-glucosidases was inhibited by p-chloromercuribenzoate (100 μ/M); this effect was overcome by cysteine or 2-mercaptoethanol, indicating that the β-D-glucosidase is a sulfhydryl enzyme. Kinetic determinations with chromogenic p-nitrophenyl β-D-glucopyranoside established a K_m. of 2.5 x 10^-4 and an Arrhenius activation-energy of 8.5 kcal.mole^-1. The molecular weight of the induced enzyme was 165,000 as determined by elution from Sephadex G-200. Chromatographic studies showed the enzyme to be a hydrolase, not a transferase.     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

The effect of temperature on the velocity of exogenous auxin transport in intact chilling-sensitive and chilling-resistant plants

The velocity of exogenous indol-3yl-acetic acid ([1-¹⁴C]IAA) transport from the apical buds of intact pea, sunflower and cotton plants was determined from 0.5° C to 47° C. The minimum temperature at which transport occurred varied from 2° C (pea and sunflower) to 7° C (cotton). Above these temperatures the velocity of transport increased steadily to maxima near 44° C in all three species. Further increase in temperature resulted in a complete cessation of transport, suggesting a sudden high-temperature breakdown of the auxin transport system. Temperature coefficients (Q₁₀) for transport velocity calculated from Arrhenius plots were low (1.36 to 1.41 between 15° C and 30° C).Arrhenius plots for the chilling-sensitive cotton and sunflower plants exhibited abrupt discontinuities at 14.6° C and 8.7° C respectively. An Arrhenius plot for the chilling-resistant pea exhibited no such discontinuity over the whole temperature range at which transport occurred.Abbreviation IAA indol-3yl-acetic acid     

Vitamin E and Hippophea rhamnoides L. extract reduce nicotine‐induced oxidative stress in rat heart

The effects of vitamin E and Hippophea rhamnoides L. extract (HRe‐1) on nicotine‐induced oxidative stress in rat heart were investigated. There were eight rats per group and supplementation period was 3 weeks. The groups were: nicotine [0.5 mg kg^?1day^?1, intraperitoneal (i.p.)]; nicotine plus vitamin E [75 mg kg^?1day^?1, intragastric (i.g.)]; nicotine plus HRe‐1 (250 mg kg^?1day^?1, i.g.); and the control group (receiving only vehicles). Nicotine increased the malondialdehyde level, which was prevented by both vitamin E and HRe‐1. Glutathione peroxidase (GPx) activity in nicotine plus vitamin E supplemented group was higher than the others. Glutathione S‐transferase (GST) activity in nicotine plus HRe‐1 supplemented group was increased compared with the control group. Catalase activity was higher in nicotine group compared with others. GPx activity in nicotine plus vitamin E supplemented group was elevated compared with the others. Total and non‐enzymatic superoxide scavenger activities in nicotine plus vitamin E supplemented group were lower than nicotine plus HRe‐1 supplemented group. Superoxide dismutase (SOD) activity was higher in nicotine plus HRe‐1 supplemented group compared with others. Glutathione reductase activity and nitric oxide level were not affected. Increased SOD and GST activities might have taken part in the prevention of nicotine‐induced oxidative stress in HRe‐1 supplemented group in rat heart. Flavonols such as quercetin, and isorahmnetin, tocopherols such as α‐tocopherol and β‐tocopherol and carotenoids such as α‐carotene and β‐carotene, reported to be present in H. rhamnoides L. extracts may be responsible for the antioxidant effects of this plant extract. Copyright © 2010 John Wiley & Sons, Ltd.     

Biogenesis of microbial transport systems: evidnce for coupled incorporation of newly synthesized lipids and proteins into membrane

The biogenesis of the independent β-galactoside and β-glueoside transport systems of Escherichia coli K12 has been studied using an unsaturated fatty acid auxotroph. The response of transport rate to temperature was determined for cells grown with different fatty acid supplements. A change in the slope of an Arrhenius plot for transport rate was obtained at transition temperatures unique for each of the five fatty acid supplements tested. Both of the transport systems studied here had identical transition temperatures when the cells were grown with the same fatty acid supplement, indicating that the transport temperature characteristics are determined primarily by the properties of the lipid phase of the membrane.     

Synthesis of glucosylceramide by purified calf spleen beta-glucosidase

Radioactive glucocerebroside was formed when purified calf spleen β-glucosidase was incubated in the presence of 4-methylumbelliferyl-β-$\text{D}$-glucoside and (¹⁴C) labeled ceramide. The product was identified by cochromatography on thin layer plates and carrier dilution and crystallization to constant specific activity. The radioactive glucocerebroside was also a substrate for hydrolysis by this same β-glucosidase preparation employed for its synthesis resulting in the liberation of labeled ceramide. Neither methyl-β-$\text{D}$-glucoside nor free D-glucose were effective in producing radioactive glucocerebroside when incubated with labeled ceramide in this system.     

Thermodynamic Study of Iodocyanopindolol β-Adrenoceptors Interactions with Rat Lung and Cerebral Cortex

Abstract

(±)¹²⁵ I-cyanopindolol (±) I CYP) was used to characterize β-adrenoceptors on rat lung and cerebral cortex membranes. The affinity of (±) ICYP was higher for lung (K^d = 64.3 pM) at 37°C. The association reaction of (±) ICYP was faster with lung (k₊₁ = 1.52 × 10⁹ M^?1.min^?1) than with cerebral cortex β-adrenoceptors (k₊₁ = 1.75 × 10⁸ M^?1.min^?1). In both tissues, the dissociation reaction followed a biphasic process with a fast (t ½ = 15.4 min and 5.6 min for lung and cerebral cortex respectively) and a slow component (t ½ = 474 min and 255 min for lung and cerebral cortex respectively). The thermodynamic parameters for (±) ICYP - β-adrenoceptors binding have been determined from kinetics and equilibrium studies, for the two tissues, at several temperatures between 0° and 44° C. For lung and cerebral cortex, Arrhenius plots were linear with different energies of activation. Van't Hoff plot was not linear for lung and the standard enthalpy and entropy changes of (±) ICYP - β-adrenoceptors interaction decreased linearly with temperature : the binding occured with a negative heat capacity change (ΔCp° = -368.9 cal. moles^?1. K^?1) at 25° C. Thermodynamic and kinetic results show that binding of (±) ICYP to lung β-adrenoceptors could involve two successive equilibria with a conformational change of the β-adrenergic receptor.     

Biophysical insights into the binding characteristics of bovine serum albumin with dipyridamole and the influence of molecular interaction with β cyclodextrin

Abstract

The binding characteristic of anti-platelet drug dipyridamole has been investigated with a transport protein, serum albumin. A multi-spectroscopic approach has been employed, and the results were well supported by in silico molecular docking and simulation studies. The fluorescence quenching of serum albumin at three different temperatures revealed that the mechanism involved is static and the binding constant of the interaction was found to be of the order of 10⁴ M^?1. The reaction was found to be spontaneous and involved hydrophobic interactions. Synchronous, 3D fluorescence and CD spectroscopy indicated a change in conformation of bovine serum albumin (BSA) on interaction with DP. Using site-selective markers, the binding site of DP was found to be in subdomain IB. Molecular docking studies further corroborated these results. Molecular dynamic (MD) simulations showed lower RMSD values on interaction, suggesting the existence of a stable complex between DP and BSA. Furthermore, since β-Cyclodextrin (βCD) is used to improve the solubility of DP in ophthalmic solutions, therefore, the effect of (βCD) on the interaction of BSA and DP was also studied, and it was found that in the presence of βCD, the binding constant for BSA-DP interaction decreased. The present study is an attempt to characterize the transport of DP and to improve its bioavailability, consequently helping in dosage design to achieve optimum therapeutic levels.

Communicated by Ramaswamy H. Sarma