The mechanisms underlying the effect of alpha-cyclodextrin on the aggregation and stability of alcohol dehydrogenase.

High concentrations of proteins and enzymes have to be stored for extended periods of time. Under such conditions, at least three major factors contribute to aggregation and loss of protein function: hydrophobicity, propensity to form non-native beta-sheet structure and net charge of the polypeptide chain. Here we evaluate these thermal aggregation factors for horse liver ADH (alcohol dehydrogenase) and the effect of alpha-CyD (alpha-cyclodextrin) on the ADH aggregation, by using fluorescence, CD, UV-visible spectrophotometry, the DLS (dynamic light scattering) technique and the enzymatic activity assay.

Fundamental studies of the cytochrome c immobilization by the potential cycling method on nanometer-scale nickel oxide surfaces.

This work describes the performance of cytochrome c/nickel oxide nanoparticles/glassy carbon electrode, prepared by the electrochemical deposition of the nickel oxide nanoparticles (NiO NPs) on the glassy carbon (GC) electrode surface and the cytochrome c immobilization on the nickel oxide nanoparticle surfaces. An extensive sample examination with the help of the SEM and AFM presented the existence of different geometrical shapes of the nickel oxide particles. These geometrical structures could lead to the better immobilization of proteins on their surfaces.

Thermal dissection of lentil seedling amine oxidase domains by differential scanning calorimetry.

The relationships between the structural and energetic domains of lentil seedling amine oxidase (LSAO) were investigated using modifiers that target the active site and the carbohydrate moiety of the enzyme. An irreversible inhibitor, aminoguanidine, specifically modified the active site of the lentil enzyme, whereas sodium metaperiodate cleaves carbohydrate moieties covalently bound to the native enzyme. Differential scanning calorimetry (DSC) measurements were made on the modified LSAOs.

Competitive inhibitory effects of acetazolamide upon interactions with bovine carbonic anhydrase II.

Sulfonamide drugs mediate their main therapeutic effects through modulation of the activity of membrane and cytosolic carbonic anhydrases. How interactions of sulfonamide drugs impact structural properties and activity of carbonic anhydrases requires further study. Here the effect of acetazolamide on the structure and function of bovine carbonic anhydrase II (cytosolic form of the enzyme) was evaluated.

Electrochemical behavior of caffeic acid at single-walled carbon nanotube:graphite-based electrode.

The performance of a single-walled carbon nanotube:graphite-based electrode, prepared by mixing single-walled carbon nanotubes (SWCNTs) and graphite powder, is described. The resulting electrode shows an excellent behavior for the redox of caffeic acid (CA), an important biological molecule. Due to the existing resemblance between electrochemical and biological reactions, it can be assumed that the oxidation mechanisms on the electrode and in the body share similar principles.

Formation of the molten globule-like state during prolonged glycation of human serum albumin.

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA (r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively.

Alginate as an antiglycating agent for human serum albumin.

Hyperglycemia and the accumulation of advanced glycation endproducts (AGEs) in tissues and serum have important roles in diabetic complications. Therefore, the identification of anti-glycation compounds is attracting considerable interest. In this study, the interaction of human serum albumin (HSA) with fructose, in the absence and presence of alginate, was studied by circular dichroism, absorbance and fluorescence techniques.

A new method for the determination of haemoglobin concentration using a bromide-modified silver electrode.

A bromide-mediated silver electrode was applied to the measurement of haemoglobin. Bromide ions showed very good redox behaviour with the silver electrode. The cathodic and anodic peak potentials were related to the concentration of bromide ions involved in making the bromide-modified silver electrode.

Guanidinium chloride and urea denaturations of beta-lactoglobulin A at pH 2.0 and 25 degrees C: the equilibrium intermediate contains non-native structures (helix, tryptophan and hydrophobic patches).

We have carried out guanidinium chloride (GdmCl) and urea denaturations of bovine beta-lactoglobulin A (beta-lgA) at pH 2.0 and 25 degrees C, using far-UV and near-UV circular dichroism, near-UV absorption and tryptophan fluorescence spectroscopies. The stable intermediate state that occurs during GdmCl denaturation has been characterized by the far- and near-UV circular dichroism, tryptophan difference absorption, tryptophan fluorescence and 8-anilino-1-naphthalene sulphonic acid binding measurements.

Spectroscopic studies of the effects of glycation of human serum albumin on L-Trp binding.

Modification of proteins by nonenzymatic glycation is one of the underlying factors that contribute to the development of the complications of diabetes. Human serum albumin (HSA) is one of the major targets of interaction with glucose through the Maillard reaction. The effects of 1 and 5 mg/ml glucose concentrations, which are consistent with blood glucose levels found in diabetic patients, on human serum albumin were studied by circular dichroism and fluorescence spectroscopy in sodium phosphate buffer, pH 7.

Role of electrostatic interactions in 2,2,2-trifluoroethanol-induced structural changes and aggregation of alpha-chymotrypsin.

It has been recently demonstrated that alpha-chymotrypsin (CT) can be driven toward amyloid aggregation by addition of 2,2,2-trifluoroethanol (TFE), at intermediate concentrations. In the present article, the process of TFE-induced CT aggregation was investigated in more detailed kinetic terms where the effects of medium conditions, such as temperature, presence of kosmotropic and chaotropic salts, pH and chemical modification of lysine residues were examined. Various techniques, including light scattering, fluorescence and circular dichroism spectroscopy, were used to follow and characterize this process.

A theoretical elucidation of bilirubin interaction with HSA's lysines: first electrostatic binding site in IIA subdomain.

The electrostatic interaction of amino acid lysines 190, 195 and 199 of human serum albumin (HSA) with bilirubin have been investigated using molecular dynamic simulations, QM and QM/MM minimization methods. In this study two methodological approaches have been employed. In the first approach X-ray structure and the structure obtained from the molecular dynamic simulation of subdomain IIA of HSA in vacuum have been utilized.

Two-state irreversible thermal denaturation of Euphorbia characias latex amine oxidase.

Thermal denaturation of Euphorbia latex amine oxidase (ELAO) has been studied by enzymatic activity, circular dichroism and differential scanning calorimetry. Thermal denaturation of ELAO is shown to be an irreversible process. Checking the validity of two-state it really describes satisfactorily the thermal denaturation of ELAO.

Molten globule-like state of bovine carbonic anhydrase in the presence of acetonitrile.

We have evaluated the effects of acetonitrile on the structure and function of bovine carbonic anhydrase II. The potential structural and functional changes in carbonic anhydrase in the presence of different acetonitrile/buffer ratios (0%, 17.5% and 47.

A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization.

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222.

Stability of proteins in the presence of polyols estimated from their guanidinium chloride-induced transition curves at different pH values and 25 degrees C.

We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.

A statistical mechanical deconvolution of the differential scanning calorimetric profiles of the thermal denaturation of cyanomethemoglobin.

A differential scanning calorimetric study of the thermal unfolding of horse cyanomethemoglobin (as an irreversible protein system) was carried out in phosphate-EDTA buffer (20 mM phosphate, 1 mM EDTA) pH 7.2. The calorimetric rescanning of the protein solution was found to be irreversible and the process unfolded state --> final state appears to follow first order kinetic.

Testing polyols' compatibility with Gibbs energy of stabilization of proteins under conditions in which they behave as compatible osmolytes.

It is generally believed that compatible osmolytes stabilize proteins by shifting the denaturation equilibrium, native state <--> denatured state toward the left. We show here that if osmolytes are compatible with the functional activity of the protein at a given pH and temperature, they should not significantly perturb this denaturation equilibrium under the same experimental conditions. This conclusion was reached from the measurements of the activity parameters (K(m) and k(cat)) and guanidinium chloride-induced denaturations of lysozyme and ribonuclease-A in the presence of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at pH 7.

Effect of polyol osmolytes on DeltaG(D), the Gibbs energy of stabilisation of proteins at different pH values.

Thermal denaturation curves of lysozyme and ribonuclease-A were determined by measuring their far-UV circular dichroism (CD) spectra in the presence of different concentrations of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at various pH values in the range 7.0--1.9.

Carbapenem-based prodrugs. Design, synthesis, and biological evaluation of carbapenems.

Syntheses of racemic trans-3-hydroxycarbonyl-6-(phenylacetamido)carbapenem (13), trans-3-phosphono-6-(phenylacetamido)carbapenem (17), and beta-lactam based prodrugs 19 and 22 were accomplished. Carbapenem 13 was found to possess antibacterial activity, comparable with imipenem (+)-3, against Staphylococcus aureus FDA 209P, S. aureus 95, Escherichia coli ATCC 39188, Klebsiella pneumoniae NCTC 418, Pseudomonas aeruginosa 1101-75, P.

Modification of substrate inhibition of synaptosomal acetylcholinesterase by cardiotoxins.

Different types of cardiotoxin (I-V and n) were isolated and purified from the venom of the Taiwan cobra (Naja naja atra). The effects of these cardiotoxins were studied on membrane-bound acetylcholinesterase, which was isolated from a sheep's brain cortex. The results showed that cardiotoxins I-III, V, and n activated the enzyme by modification of substrate inhibition, but cardiotoxin IV's reaction was different.

Binding properties of adenosine deaminase interacted with theophylline.

Thermodynamic studies were carried out to evaluate the binding of theophylline on adenosine deaminase (ADA) in 50 mM sodium phosphate buffer pH 7.5, at 300 K, using isothermal titration calorimetry (ITC). A simple method for determination of binding isotherm in the drug--ADA interaction was applied using ITC data.

The structural and functional studies of His119 and His12 in RNase A via chemical modification.

The histidyl residues of bovine pancreatic ribonuclease A (RNase A) play a crucial role in enzymatic activity. Diethylpyrocarbonate (DEPC) is a potent inhibitor of RNase A, and its precise sites of action on the imidazole rings of the four histidyl residues of RNase A are not clearly defined. We have used a multidisciplinary approach including enzyme assay, calculation of accessible surface area (ASA), isoelectric pH gradient technique, fluorescence investigations, circular dichroism spectroscopy, differential scanning calorimetry, and 1H NMR analysis to study the sites of DEPC interaction with the imidazole rings of the four histidyl residues.

Interaction of glucose oxidase with alkyl-substituted Sepharose 4B.

Glucose oxidase (GOD) is often used in immobilized forms for determination of glucose. To examine the possibility of its adsorption by hydrophobic interactions, palmityl-substituted Sepharose 4B (Sepharoselipid) was employed as an adsorptive matrix. Various conditions were used in tests to improve the limited immobilization of the enzyme observed under normal (native) conditions, including use of high concentrations of denaturing agents.