Study on the Interaction Between {\mathrm{Cu}}{\left( {{\mathrm{phen}}} \right)}^{{2 + }}_{3} and Bovine Serum Albumin by Spectroscopic Methods

In this work, the interaction between ${\text{Cu}}\left( {{\text{phen}}} \right)_3^{\,\,2 + } In this work, the interaction between Cu(phen)(2+)(3) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy combined with UV-vis absorption and circular dichroism (CD) spectroscopic techniques under physiological conditions. The fluorescence data proved that the fluorescence quenching of BSA by Cu(phen)(2+)(3) was the result of the Cu(phen)(2+)(3) -BSA complex formation. The binding constants (K (a)) between Cu(phen)(2+)(3) and BSA at four different temperatures were calculated according to the modified Stern-Volmer equation. The enthalpy change (DeltaH) and entropy change (DeltaS) were calculated to be 10.74 kJ mol(-1) and 54.35 J mol(-1) K(-1), respectively, which indicated that electrostatic interactions played a major role in the formation of Cu(phen)(2+)(3) -BSA complex. The distance r between the donor (BSA) and acceptor[Cu(phen)(2+)(3)] was obtained to be 3.55 nm based on F?rster's energy transfer theory. The synchronous fluorescence and CD spectroscopy results showed that the polarity of the residues increased and the lost of the alpha-helix content of BSA (from 59.84 to 53.70%). These indicated that the microenvironment and conformation of BSA were changed in the presence of Cu(phen)(2+)(3).     

Studies on the interaction between tetraphenylporphyrin compounds and bovine serum albumin.

The interaction of three porphyrin compounds with bovine serum albumin (BSA) was examined by fluorescence emission spectra at the excitation wavelength 280 nm and in UV-Vis absorption spectra. Through fluorescence quenching experiments, it was confirmed that the combination of three porphyrin compounds with BSA was a single static quenching process. The binding constant K(A), the thermodynamic parameters enthalpy change (DeltaH(0)), Gibbs free energy change (DeltaG(0)) and entropy change (DeltaS(0)) were obtained. It was found that hydrophobic interaction played a main role in tetraphenylporphyrin (TPP) or tetraparacholophenylporphyrin (TClPP) binding to BSA, while tetraparamethoxyphenylporphyrin (TMEOPP) mainly based on van der Waals' force. According to F?ster energy transfer, the separate distance r, the energy transfer efficiency E and F?ster radium R(0) were calculated. The results obtained from the above experiments showed that three porphyrin compounds were tightly bound to BSA.     

In vitro study on the binding of anti-coagulant vitamin to bovine serum albumin and the influence of toxic ions and common ions on binding

The mechanism of binding of vitamin K(3) (VK(3)) with bovine serum albumin (BSA) was investigated by fluorescence, absorption and circular dichroism (CD) techniques under physiological conditions. The analysis of fluorescence data indicated the presence of static quenching mechanism in the binding. Various binding parameters have been evaluated. Thermodynamic parameters, the standard enthalpy change, DeltaH(0) and the standard entropy change, DeltaS(0) were observed to be -164.09 kJ mol(-1) and -465.08 J mol(-1)K, respectively. The quantitative analysis of CD spectra confirmed the change in secondary structure of the protein upon interaction with VK(3). The binding average distance, r between the donor (BSA) and acceptor (VK(3)) was determined based on the F?rster's theory and it was found to be 3.3 nm. The effects of toxic ions and common ions on VK(3)-BSA system were also investigated.     

Lanthanide Salts of Heteropoly Molybdotungstosilicate LnHSiMo10W2O40·xH2O (Ln?=?Pr, Nd, Sm, Gd, Tb, Dy, Yb) Binding to Bovine Serum Albumin: A Fluorescence Quenching Study

In the present work, the interaction between a series of novel lanthanide salts of heteropoly molybdotungstosilicate LnHSiMo(10)W(2)O(40)·xH(2)O (LnW(2); Ln = Pr (x = 23), Nd (x = 24), Sm (x = 26), Gd (x = 20), Tb (x = 23), Dy (x = 21), Yb (x = 25)), and bovine serum albumin (BSA) was investigated by spectroscopic approach at different temperatures under imitated physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by LnW(2) is a result of the formation of LnW(2)-BSA complex. Binding affinity between LnW(2) and BSA was determined using Scatchard equation and the modified Stern-Volmer equation, and the corresponding electronic structure-affinity relationship were discussed. The results of thermodynamic parameters ?G, ?H, ?S at different temperatures indicate that the electrostatic interactions play a major role in LnW(2)-BSA binding process. Moreover, the enthalpy change (?H) and entropy change (?S) were in accordance with the "enthalpy-entropy compensation" equation obtained from this and previous work. Furthermore, the distance r between donor (BSA) and acceptor (LnW(2)) was obtained according to fluorescence resonance energy transfer.     

Multi-spectroscopic study on interaction of bovine serum albumin with lomefloxacin-copper(II) complex

The binding reactions of lomefloxacin-copper(II) complex (LMF-Cu) or LMF to bovine serum albumin (BSA) in physiological solution were investigated by multi-spectroscopy. The binding constant, the number of binding sites and the binding distance between LMF-Cu or LMF and BSA were obtained by a fluorescence quenching method and according to the mechanism of Forster-type dipole-dipole non-radioactive energy-transfer, respectively. Enthalpy and entropy changes for two systems were calculated to be -7.970 kJ mol(-1) and 47.438 J mol(-1)K(-1) for LMF-BSA, -12.469 kJ mol(-1) and 33.542 J mol(-1)K(-1) for LMF-Cu-BSA, respectively. The highly positive values observed for the entropy give evidence for a strong interaction. The values of DeltaH and DeltaS in two systems are similar, indicating that electrostatic interactions in two systems play major role. The effect of LMF-Cu or LMF on the conformation of BSA was also analyzed by synchronous fluorescence, three-dimensional fluorescence and circular dichroism spectra. The results showed that the presence of Cu ion in LMF-Cu can affect the conformation of BSA to some degree. All the results revealed that the addition of copper ion promotes the interaction of lomefloxacin with bovine serum albumin.     

Characterization of the interaction between reserpine and bovine serum albumin: spectroscopic approaches

The characteristics of the interaction between reserpine and bovine serum albumin (BSA) were studied by fluorescence, UV-vis absorption and Fourier transform infrared (FT-IR) spectroscopy. Spectroscopic analysis revealed that fluorescence quenching of BSA by reserpine was through a static quenching procedure. The binding constant K(A) of reserpine with BSA at 293, 301 and 309 K was 1.63, 1.78 and 2.35 x 10(5) moL(-1) L respectively, which indicated degree of binding force between reserpine and BSA. There was one binding site between reserpine and BSA. The entropy and enthalpy changes were positive, indicating that interaction of reserpine and BSA was driven mainly by hydrophobic forces. The average binding distance between the donor (BSA) and the acceptor (reserpine) was about 3.84 nm based on the Forster non-radiation energy transfer theory. Results of synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of BSA were changed by the binding of reserpine. The results may provide important insights into the physiological activity of reserpine.     

Quenching of the intrinsic fluorescence of bovine serum albumin by phenylfluorone-Mo(VI) complex as a probe

In this paper, the binding characteristics of bovine serum albumin (BSA) and phenylfluorone (PF)-molybdenum (Mo(VI)) complex have been studied by fluorophotometry. The binding constants are calculated at different temperatures. The binding distance and the energy transfer efficiency between PF-Mo(VI) complex and protein are obtained on the basis of the theory of Forster energy transfer. DeltaH and DeltaS are calculated to be -7.11 kJ mol-1 and 70.30 J mol-1 K-1, which indicate that electrostatic force plays major role in the interaction of PF-Mo(VI) complex and BSA. The experimental results show that BSA and PF-Mo(VI) complex have strong interactions and the mechanism of quenching belongs to static quenching.     

Evaluation of binding and thermodynamic characteristics of interactions between a citrus flavonoid hesperitin with protein and effects of metal ions on binding

The mechanism of interaction of a non-glycosidic citrus flavonoid, hesperitin (HES) with bovine serum albumin (BSA) was studied by UV-vis absorption, fluorescence, FT-IR, circular dichroism, fluorescence anisotropy and synchronous fluorescence spectroscopy in phosphate buffer of pH 7.4. Fluorescence data revealed that the fluorescence quenching of BSA by HES was the result of the formed complex of HES-BSA. The binding constants and thermodynamic parameters at four different temperatures, the location of binding, and the nature of binding force were determined. The hydrogen bonds interactions were found to be the predominant intermolecular forces to stabilize the complex. The conformation of BSA was discussed by synchronous fluorescence and CD methods. The alterations of protein secondary structure upon complexation with HES were evident from the gradual decrease in α-helicity. The distance between the donor (BSA) and acceptor (flavonoid) was calculated from the fluorescence resonance energy transfer and found to be 1.978 nm. Common ions viz., Zn(2+), K(+), Cu(2+), Ni(2+), Mn(2+) and Co(2+) were found to influence the binding of flavonoid to protein.     

Quantification and characterization of P-glycoprotein-substrate interactions

It is generally accepted that P-glycoprotein binds its substrates in the lipid phase of the membrane. Quantification and characterization of the lipid-transporter binding step are, however, still a matter of debate. We therefore selected 15 structurally diverse drugs and measured the binding constants from water to the activating (inhibitory) binding region of P-glycoprotein, K(tw(1)) (K(tw(2))), as well as the lipid-water partition coefficients, K(lw). The former were obtained by measuring the concentrations of half-maximum activation (inhibition), K(1) (K(2)), in living NIH-MDR-G185 mouse embryo fibroblasts using a Cytosensor microphysiometer, and the latter were derived from surface activity measurements. This allowed determination of the membrane concentration of drugs at half-maximum P-glycoprotein activation (C(b(1)) = (0.02 to 67) mmol/L lipid), which is much higher than the corresponding aqueous concentration (K(1) = (0.02 to 376) microM). Moreover we determined the free energy of drug binding from water to the activating binding region of the transporter (DeltaG degrees (tw(1)) = (-30 to -54) kJ/mol), the free energy of drug partitioning into the lipid membrane (DeltaG degrees (lw) = (-23 to -34) kJ/mol), and, as the difference of the two, the free energy of drug binding from the lipid membrane to the activating binding region of the transporter (DeltaG degrees (tl(1)) = (-7 to -27) kJ/mol). For the compounds tested DeltaG degrees (tl(1)) was less negative than DeltaG degrees (lw) but varied more strongly. The free energies of substrate binding to the transporter within the lipid phase, DeltaG degrees (tl(1)), are consistent with a modular binding concept, where the energetically most efficient binding module comprises two hydrogen bond acceptor groups.     

Effects of oxidation on changes of compressibility of bovine serum albumin

The methods of ultrasound velocity and density measurements were used to study the adiabatic compressibility of bovine serum albumin (BSA) during its oxidation by the prooxidants Cu2+ and 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). We did not find changes of compressibility of BSA in the presence of copper ions at rather high molar ratio Cu2+/BSA = 0.66 mol/mol. This can be explained by binding of the Cu2+ to the binding site of BSA and thus protecting the prooxidant action of the copper. However, AAPH-mediated oxidation of BSA resulted in an increase of its apparent specific compressibility (psik/beta0). These changes could be caused by the fragmentation of the protein.     

Characteristics of complex-formation of chlorine e6 with human and bovine serum albumins

Pigment-protein complexes of chlorin e6 (Chl e6) with human (HSA) and bovine serum albumines (BSA) have been investigated by spectral-luminescent methods. Fluorescence quenching of tryptophan residues caused by the inductive-resonance energy transfer to pigment molecules and the rise of the polarization degree of Chl e6 emission were observed upon incorporation of Chl e6 in the protein globula. The obtained data on spectral-energetic parameters of protein tryptophanyls and Chl e6 permitted us to calculate the energy transfer critical distances R0 in complexes of Chl e6 with HSA (R0 = 32 A) and BSA (R0 = 35A). The binding constants (K) and the number of binding sites (N) of Chl e6 with HSA and BSA have been obtained from the experiments on tryptophanyl fluorescence quenching of the investigated proteins and polarization measurements of pigment emission (KHSA = 1.2.10(6) mole-1, KBSA = 3.6.10(6) mole-1, NHSA = NBSA = 1). On the basis of the measured values of electronic excitation energy transfer efficiency (phi greater than or equal to 99%) the average distances between the protein chromophores and the incorporated Chl e6 molecules have been calculated (RHSA = 15-17 A, RBSA = 16.5-18 A). The questions connected with pigment localization sites in the protein globula and specific features of pigment-protein interaction are discussed.     

Kinetics and mechanism of the decomposition of S-nitrosoglutathione by l-ascorbic acid and copper ions in aqueous solution to produce nitric oxide.

S-Nitrosothiols serve as a good source of nitric oxide ((*)NO) mainly due to the ease of cleavage of the S-N bond which consequently produces (*)NO. The reductive decomposition of S-nitrosoglutathione (GSNO) by l-ascorbic acid (vitamin C) yields (*)NO which was monitored both electrochemically (using NO-probe) and spectrophotometrically. The rate of reaction and (*)NO release was found to be pH dependent in a manner which drastically increases with pH demonstrating that the l-ascorbic acid dianion (A(2-)) is by far the most reactive species of l-ascorbic acid (H(2)A). The derived rate expression (measuring the disappearance of the absorption at ca. 336 nm due to GSNO) was established as rate = -d[GSNO](t)/dt = ((k(a)[H(+)](2) + k(b)[H(+)]K(1) + k(c)K(1)K(2))/([H(+)](2) + K(1)[H(+)] + K(1)K(2)))[GSNO](t)[H(2)A](t). k(a), k(b), and k(c) are second-order rate constants via the H(2)A, HA(-), and A(2-) pathways, respectively, while K(1) and K(2) represent the first and second equilibrium dissociation constants of l-ascorbic acid. There is little or no reaction at low pH (below 5.5), where H(2)A is a predominant species, and as a result the rate constant (k(a)) via this route was found to be negligible. At 25 degrees C, k(b) = 5.23 +/- 1.47 x 10(-3) dm(3) mol(-1) s(-1) and k(c) = 1.22 +/- 0.04 x 10(3) dm(3) mol(-1) s(-1), activation parameters DeltaH(double dagger)(b) = 54.4 +/- 4.3 kJ mol(-1), DeltaS(double dagger)(b) = -106 +/- 16 J K(-1) mol(-1), DeltaH(double dagger)(c) = 80.5 +/- 7.5 kJ mol(-1), DeltaS(double dagger)(c) = 84 +/- 7 kJ mol(-1). The experimental rate and activation parameters suggest that this redox process follows an outer-sphere electron transfer mechanism. GSNO is relatively stable in the dark, aqueous medium and even in the presence of trace quantities of Cu(2+). Induced catalytic decomposition of GSNO only becomes significant above ca. 10 microM Cu(2+), but after this it shows linear dependency. To nullify any catalysis by Cu(2+) or any other transition metal ions, EDTA was added to all experimental reactions except those where catalysis by Cu(2+) was studied.     

Blocking nonspecific adsorption of native food-borne microorganisms by immunomagnetic beads with iota-carrageenan

We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av DeltaG0=-20 +/- 4 kJ mol(-1)) even in the presence of casein [1% (w/v): DeltaG0=-18 +/- 3 kJ mol(-1); DeltaDeltaG0 approximately -2 kJ mol(-1)]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; DeltaG0=-19 +/- 2 kJ mol(-1)) was tested for nonspecific binding in the presence of iota-carrageenan (0.03-0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency xi (DeltaG0=-11 +/- 4 kJ mol(-1); DeltaDeltaG0 approximately -8 kJ mol(-1)). Other anionic polysaccharides (0.1% kappa-carrageenan and polygalacturonic acid) had no significant effect (av DeltaG0=-19 +/- 1 kJ mol(-1); DeltaDeltaG0 approximately 0 kJ mol(-1)). Varying iota-carrageenan from 0% to 0.02% resulted in xi significantly diminishing from 0.69 (e.g., 69% of the cells captured; DeltaG0=-19 +/- 3 kJ mol(-1)) to 0.05 (DeltaG0=-11 +/- 2 kJ mol(-1); DeltaDeltaG0 approximately -9 kJ mol(-1)) at about 0.03% iota-carrageenan where xi leveled off. An optimum blocking ability was achieved with 0.04% iota-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of iota-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.     

Spectroscopic investigation into the interaction of a diazacyclam‐based macrocyclic copper(ii) complex with bovine serum albumin

Cyclam‐based ligands and their complexes are known to show antitumor activity. This study was undertaken to examine the interaction of a diazacyclam‐based macrocyclic copper(II) complex with bovine serum albumin (BSA) under physiological conditions. The interactions of different metal‐based drugs with blood proteins, especially those with serum albumin, may affect the concentration and deactivation of metal drugs, and thereby influence their availability and toxicity during chemotherapy. In this vein, several spectral methods including UV–vis absorption, fluorescence and circular dichroism (CD) spectroscopy techniques were used. Spectroscopic analysis of the fluorescence quenching confirmed that the Cu(II) complex quenched BSA fluorescence intensity by a dynamic mechanism. In order to further determine the quenching mechanism, an analysis of Stern–Volmer plots at various concentrations of BSA was carried out. It was found that the K_SV value increased with the BSA concentration. It was suggested that the fluorescence quenching process was a dynamic quenching rather than a static quenching mechanism. Based on Förster's theory, the average binding distance between the Cu(II) complex and BSA (r) was found to be 4.98 nm; as the binding distance was less than 8 nm, energy transfer from BSA to the Cu(II) complex had a high possibility of occurrence. Thermodynamic parameters (positive ΔH and ΔS values) and measurement of competitive fluorescence with 1‐anilinonaphthalene‐8‐sulphonic acid (1,8‐ANS) indicated that hydrophobic interaction plays a major role in the Cu(II) complex interaction with BSA. A Job's plot of the results confirmed that there was one binding site in BSA for the Cu(II) complex (1:1 stoichiometry). The site marker competitive experiment confirmed that the Cu(II) complex was located in site I (subdomain IIA) of BSA. Finally, CD data indicated that interaction of the Cu(II) complex with BSA caused a small increase in the α‐helical content. Copyright © 2016 John Wiley & Sons, Ltd.     

Purification and biochemical characterization of ionically unbound polyphenol oxidase from Musa paradisiaca leaf

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77?min. The inactivation energy (Ea) value of PPO was estimated to be 91.3?kJ mol(-1). It showed higher specificity with catechol (K(m)?=?8?mM) as compared to 4-methylcatechol (K(m)?=?10?mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Free energy of the binding of uridylic acid oligomers with double stranded poly(A) x poly(U)

The binding parameters (K, omega) and the free energy (DeltaG(0)) of triple helix formation have been estimated for complexes of oligo(U)(n) (n = 5, 7-10) with poly(A) . poly(U) on the basis of hypochromicity measurements. The data were treated according to the formula of McGhee and von Hippel [J. Mol. Biol. 86 (1974) 469] by a computer program ALAU [H. Schütz et al., Stud. Biophys. 104 (1984) 23] which takes absorbancies and total concentrations as input. In 1 mM cacodylate buffer pH 7.0 with 10 mM NaCl and 10 mM MgCl(2) at 5 degrees C the free energy of contiguous binding was found to be a linear function of the oligomer length with a slope of DeltaG(c,U)(0) = -0.72 (+/-0.03) kcal x mol(-1) per nucleotide. The mean cooperativity coefficient (omega) was 24.5 (+/- 5.6), and the corresponding free energy of interaction between the neighbouring oligonucleotides in the third strand was DeltaG(0(omega)) = -1.74 (+/-0.13) kcal x mol(-1).     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Binding energy and catalysis by D-xylose isomerase: kinetic, product, and X-ray crystallographic analysis of enzyme-catalyzed isomerization of (R)-glyceraldehyde

D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and d-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D(2)O was monitored by (1)H nuclear magnetic resonance spectroscopy, and a k(cat)/K(m) of 0.034 M(-1) s(-1) was determined for this isomerization at pD 7.0. This is similar to the k(cat)/K(m) of 0.017 M(-1) s(-1) for the TIM-catalyzed carbon deprotonation reaction of DGA in D(2)O at pD 7.0 [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (k(cat)/K(m) = 490 M(-1) s(-1)) versus that for the TIM-catalyzed isomerization of DGAP (k(cat)/K(m) = 9.6 × 10(6) M(-1) s(-1)) is due to (i) the barrier to conversion of cyclic d-xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol) and (ii) the intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219-410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) being smaller than that of the phosphodianion group of DGAP (~12 kcal/mol). The XI-catalyzed isomerization of DGA in D(2)O at pD 7.0 gives a 90% yield of [1-(1)H]DHA and a 10% yield of [1-(2)H]DHA, the product of isomerization with incorporation of deuterium from solvent D(2)O. By comparison, the transfer of (3)H from the labeled hexose substrate to solvent is observed only once in every 10(9) turnovers for the XI-catalyzed isomerization of [2-(3)H]glucose in H(2)O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994) Biochemistry 33, 1481-1487]. We propose that truncation of the terminal ethylene glycol fragment of d-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high-resolution (0.97 ?) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI·sugar complexes.     

Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P(1B)-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E(A)=38kJ?mol(-1)) over the whole concentration range while it increased from 17 to 42kJ?mol(-1) with rising copper concentration and decreased from 39 to 23kJ?mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.     

Study of the interaction between doxepin hydrochloride and bovine serum albumin by spectroscopic techniques

The binding of doxepin hydrochloride (DH) to bovine serum albumin (BSA) was investigated by spectroscopic (fluorescence, UV–vis absorption and circular dichroism) techniques. The binding parameters have been evaluated by fluorescence quenching method. The thermodynamic parameters, ΔH°, ΔS° and ΔG° calculated at different temperatures indicated that the hydrogen bond and hydrophobic forces played a major role in the interaction of DH with BSA. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (DH) was evaluated and found to be 2.7 nm. Spectral results observed showed that the binding of DH to BSA induced conformational changes in BSA. The effect of common ions on the binding of DH to BSA was also examined.