UV Difference Spectroscopy Research Articles

Effect of urea on the structure and functional activity of proteins. Chymotrypsinogen A and α-chymotrypsin

The effect of urea on the structural stability and functional activity of globular proteins,viz., chymotrypsinogen A (ChtG) and α-chymotrypsin (Cht), was studied over a wide range of concentrations (0.5–6 rnol L-1), and the existence of two different mechanisms of the action of urea on these proteins was demonstrated. No changes in the spatial structure of ChtG were observed in the concentration range from 0,5 to 3 mol L−1 (region 1). Differential UV spectroscopy shows tile redistribution of aromatic groups between the inner volume and the outer surface of a protein molecule (protein denaturation) at concentrations >3 mol L−1 (regionII), In regionI, urea changes the kinetic parameters of enzymatic reactions involving Cht, which is explained on the basis of millimeter spectroscopy data by its action on the structure and nucleophilic reactivity of water.

Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin.

We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change to the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.

2,7-Diaminomitosene, a Monofunctional Mitomycin C Derivative, Alkylates DNA in the Major Groove. Structure and Base-Sequence Specificity of the DNA Adduct and Mechanism of the Alkylation

Reductive activation of the bifunctional alkylator and DNA cross-linking agent mitomycin C (MC) yields 2,7-diaminomitosene (2,7-DAM) as the major product of its activation both in cell-free systems and in vivo. 2,7-DAM lacks one of the alkylating functions of MC, the aziridine. We show that 2,7-DAM itself alkylates DNA monofunctionally upon reductive activation, to form a heat-labile adduct. A guanine-N7−2,7-DAM adduct was isolated from the drug−DNA complex upon heating. Nuclease digestion yielded this adduct both in the nucleoside (dG-2,7-DAM) and dinucleoside phosphate [d(GpG)-2,7-DAM] forms. The structures of all three forms were determined by LC−ESIMS and differential UV spectroscopy. The adduct is sequence-specific to guanines in (G)n tracts of DNA. A guanine-N7−2,7-DAM adduct in DNA was indirectly observed previously by Prakash et al. (Prakash, A. S.; Beall, H.; Ross, D.; Gibson, N. W. Biochemistry 1993, 32, 5518−5525). The results indicate that selective removal of the aziridine function of MC resu...

Preparation and physico-chemical study of inclusion complexes between idebenone and modified?-cyclodextrins

The inclusion properties of modifiedβ-cyclodextrins (trimethyl-β-cyclodextrin, dimethyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin) towards idebenone were compared with naturalβ-cyclodextrin. The inclusion complexes were prepared by different methods (coprecipitation, kneading, and freeze-drying) and characterized by differential scanning calorimetry, X-ray diffractometry, UV, CD and NMR spectroscopy. The results obtained by CD and NMR spectroscopy indicate a different orientation of idebenone in dimethyl-β-cyclodextrin with respect to other cyclodextrins. Stability constants of the complexes were determined in water at various temperatures and consequently thermodynamic parameters were obtained. All cyclodextrins are able to significantly increase the water solubility of idebenone, particularly dimethyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin, as a result of complexation. Consequently, they enhance the dissolution rate of the complexed drug compared to the free drug.

Chemoenzymatic synthesis of [formula omitted]: their binding to Aspergillus niger glucoamylase G1 and its starch-binding domain

A coupling reaction of cyclodextrin glucosyltransferase (CGTase) with glucose and 6-deoxy-6-iodo-cyclomaltoheptaose ( 1), in the presence of glucoamylase, followed by acetylation, led to a convenient synthesis of acetylated 6 III-deoxy-6 III-iodo-maltotriose ( 2) and 6 IV-deoxy-6 IV-iodo-maltotetraose ( 3). Nucleophilic displacement of the iodine atom of these protected maltotriose and maltotetraose analogs by the activated form of 2,3,4,6-tetra-O- acetyl-1-S- acetyl-1-thio-α- d-glucose ( 4) afforded peracetylated 6 III -S- α- d-glucopyranosyl-6 III-thiomaltotriose ( 5) and 6 IV -S- α- d-glucopyranosyl-6 IV-thiomaltotetraose ( 6) in high yield. The interaction of OH-free tetra- and penta-saccharides ( 7 and 8) with both glucoamylase G1 from Aspergillus niger as well as its isolated starch-binding domain fragment were studied by UV difference spectroscopy. It was found that the starch-binding domain has higher affinity for 7 and 8 than for maltotetraose and maltopentaose.

Cis/Trans Isomerization in Azobenzene-Chain Liposomes

Pseudoglyceryl cationic azobenzene-chain lipid 1 undergoes efficient trans → cis and cis → trans photoisomerization, as well as smooth cis → trans thermalisomerization in hololiposomes and in coliposomes with dioctadecyloxy lipid 2. Rate constants and activation parameters for the cis → trans intraliposomal thermal isomerization of 1 are reasonably comparable to those measured in homogeneous solution. Azobenzene lipid domain formation in coliposomes of 1 and 2 is explored by differential scanning calorimetry and UV spectroscopy.

Thermodynamics of denaturation of alpha-chymotrypsinogen A in aqueous urea and alkylurea solutions.

The effects of pH, urea, and alkylureas on the thermal stability of alpha-chymotrypsinogen A (alpha-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition of alpha-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25 degrees C were calculated. Comparison of these values shows that at 25 degrees C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein-molecules. At pH lower than 2.0, alpha-ctg A still exists in a very stable form, probably the acid-denatured from (A-form).

Identification of a stable complex of trichosanthin with nicotinamide adenine dinucleotide phosphate

Trichosanthin, a type I ribosome-inactivating protein with RNA N-glycosidase activity, forms a stable complex with nicotinamide adenine dinucleotide phosphate, a substrate analog. Difference UV spectroscopy, fluorescence spectroscopy, and 31P NMR are used to identify the formation of the complex, followed by a crystal structure analysis carried out to elucidate the active-site structure of trichosanthin. The determination of germinal vesicle breakdown indicates that the complex does not, at least for abortion-inducing activity, result in competitive inhibition to the protein.

Complexes of human lactoferrin with vanadium in oxidation states +3, +4 and +5

Complexes of human lactoferrin with vanadium in the +3, +4 and +5 oxidation states have been characterised, as biological models for vanadium–protein interactions, by UV difference spectroscopy. The results indicate that the metal binds to lactoferrin in the specific iron(III) binding sites with a metal: protein stoichiometry of 2 : 1. Electron spin resonance spectroscopy shows oxygen or hydrogen peroxide repidly oxidise vanadium(III)–lactoferrin to the vanadium(IV) complex which is then itself oxidised to vanadium(V)–lactoferrin. The metal-binding sites of the three vanadium–lactoferrin complexes have been modelled assuming a common protein–ligand donor set. The results show that the metal ion can be accommodated by the protein in the oxidation states +3, +4 and +5 as the V3+, VO2+ and VO2+ ions respectively, the increase in V–O(oxo) co-ordination being compensated for by decreasing the V–O(carbonate) interaction.

Determination of the binding constant of imidazole and histidine with immobilized Cu(II) by differential UV spectroscopy.

The binding constants of immobilized Cu(II) on PEG-IDA with imidazole and histidine at various solution pH values, salt concentrations and temperatures were determined by differential UV spectrophotometer. The bimodal binding behavior of basic solute was observed by the study of the salt effect. However, the formation of the coordinated compound is the dominated binding mechanism at the pH value higher than pKa of the deprotonation of imidazole nitrogen. There was no obvious effect of temperature on binding constant because of the complexity of binding mechanism. The binding behavior of several dipeptides and tripeptides with histidine at C- or N-terminal was also investigated and the results were explained by the “metal ion transfer” (MIT) hypothesis. Furthermore, the binding constants of synthetic heptapeptides with two histidine residues separated by different number of glycine residues were investigated to demonstrate the effect of histidine residues distance on the binding affinity. This study provides basic information of binding behavior of protein to immobilized metal ion.

1,3-Diphenylpropane-1,3-diamines, VI): DNA-interaction, estrogen receptor affinity, and cytostatic activity of 1,3-diphenylpropane-1,3-diamine-Pt(II) complexes.

The Pt(II) complexes of 1,3-diphenylpropane-1,3-diamines (part IV2)) were tested for DNA interaction (UV-difference spectroscopy, Tables 1 and 2), for affinity to the estrogen receptor (calf uterus cytosol; scarcely any affinity), and for cytostatic activity at estrogen independent MDA-MB 231 cells (Tables 3 and 4) and estrogen dependent MCF-7 cells (Tables 5 and 6). The data are compared with those reported for the analogous 1,2-diphenylethane-1,2-diamine-Pt(II) complexes: most probably, the cytostatic activity is not mediated by the estrogen receptor.

Thermally induced unfolding of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium

Abstract

Investigation of complexes between some glycopeptide antibiotics and bacterial cell‐wall analogues by electrospray‐ and capillary zone electrophoresis/electrospray‐mass spectrometry

Complexation in aqueous solutions between vancomycin, ristocetin A, teicoplanin and two bacterial cell-wall analogues, Ac2-L-Lys-D-Ala-D-Ala (tripeptide) and Ac-D-Ala-D-Ala (dipeptide) has been examined by positive-ion electrospray mass spectrometry (ES-MS) and capillary zone electrophoresis (CZE)/ES-MS. The ES-MS data demonstrate that as well as complexes between monomeric antibiotics and either peptide, the investigated solutions contained complexes ranging from the simple homodimer of the antibiotic to a more complex association giving ions of the type [2(antibiotic) + 3(tripeptide) + 3H]3+. The same data also demonstrate that the homodimers of the investigated antibiotics are significantly suppressed in the presence of the tripeptide. The use of CZE/ES-MS made it evident that the complexes between the antibiotic and the tripeptide were present in the solution prior to their introduction into the ion source. The two sets of data are compared with existing UV difference spectroscopy and nuclear magnetic resonance data on complexation and dimerization.

Fatty acid binding to bovine serum albumin prevents formation of intermediate during denaturation.

Urea-induced denaturation of defatted BSA and BSA containing six mol palmitic acid/mol protein (fatted BSA) has been studied by the techniques of UV difference spectroscopy and fluorescence spectroscopy. The notable differences between the two transitions are a shift in the transition of fatted BSA to a higher urea concentration and a three-state denaturation transition in defatted BSA compared to an apparent two-state denaturation transition in fatted BSA. The stable denaturation intermediate in defatted BSA occurs at 4.5-5.0 M urea, a urea concentration at which denaturation in fatted BSA has not yet started. These results are further supported by the difference spectral results obtained at 4.5 M urea in the two albumin preparations. The occurrence of denaturation intermediate only in defatted BSA and the presence of the two strong fatty-acid-binding sites in domain III lead us to conclude that this domain is relatively unstable in the absence of fatty acids and is responsible for the formation of intermediate.

Solubilization of a Tripeptide HIV Protease Inhibitor Using a Combination of Ionization and Complexation with Chemically Modified Cyclodextrins

Kynostatin (KNI-272), an experimental HIV protease inhibitor, is currently undergoing preclinical testing for the treatment of AIDS. This transition state mimetic tripeptide exhibits extremely low aqueous solubility (4μg/mL) making target concentrations (5-50 mg/mL) for parenteral solution formulations difficult to achieve. The presence of an ionizable (5-isoquinolinyloxy)acetyl moiety makes solubilization via pH adjustment possible, but a solubility > 5 mg/mL requires an adjustment in pH below 2.0, which would be physiologically unacceptable. This study examines and compares two approaches for solubilizing kynostatin: (1) inclusion complex formation at chemically distincthydrophobic binding sites using (2-hydroxypropyl)-β-cyclodextrin (HPCD) and a sulfobutyl ether derivative of β-cyclodextrin (β-CD-SBE) and (2) a combined strategy utilizing ionization of the isoquinoline moiety coupled with inclusion complex formation at the remaining binding site(s). Macroscopic binding constants determined from solubility profiles as a function of pH and HPCD concentration have been compared with the microscopic binding constant for formation of the isoquinoline-HPCD inclusion complex determined by UV difference spectroscopy to examine the independence of binding domains within KNI-272. As demonstrated in this report, combination strategies tailored to the properties of different domains within the molecule may be highly effective in solubilizing compounds such as poorly soluble peptides.

Calcium Binding Properties of Rabbit Liver Protein Disulfide Isomerase

We have characterized the Ca 2+ binding properties of protein disulfide isomerase (PDI). It binds 19 mol Ca 2+/mol protein with low affinity, which is reduced by increasing the ionic strength. Ca 2+ induced conformational changes detected by UV difference spectroscopy. These Ca 2+ binding properties resemble those of a sea urchin egg 58 kDa protein.

Starch-binding domain of Aspergillus glucoamylase-I. Interaction with beta-cyclodextrin and maltoheptaose.

The characterization is reported of two peptide fragments (SBD106 and SBD122) containing the starch-binding domain (SBD) of Aspergillus sp. glucoamylase I. The starch-binding peptides were produced in Escherichia coli as fusion proteins of the maltose-binding protein (MBP). SBD106 (11.9 kDa) and SBD122 (13.8 kDa) were purified from the factor Xa digest of MBP fusion proteins. The amino acid compositions were similar to those deduced from their amino acid sequences. The interactions of beta-cyclodextrin and maltoheptaose with purified SBD peptides were investigated by UV difference spectroscopy. SBD106 and SBD122 bound specifically beta-cyclodextrin with a dissociation constant (Kd) of 34 microM and 23.5 microM, respectively. Maltoheptaose binding to SBD106 and SBD122 was weaker than that of beta-cyclodextrin; dissociation constants were 0.57 and 0.50 mM, respectively. The results indicate that the intramolecular disulfide bonding is not required for the domain functioning and that O-glycosylation is not critical for the functioning of the starch-binding domain, but may affect its conformation and dynamics.

Comparison of the results of thermal denaturation of β-lactoglobulin obtained by DSC and UV-spectroscopy

The thermal denaturation of β-lactoglobulin in the presence of urea and alkylurea solutions were measured. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. For studying the effect of temperature two methods were used, differential scanning calorimetry (DSC) and UV-spectroscopy. DSC provides direct model-independent determination of the transition enthalpy in comparison with UV-spectroscopy, which gives only apparent or van't Hoff enthalpy of transition. The UV-melting curves were analyzed on the basis of a two-state approximation. The apparent standard enthalpies of thermal denaturation, ΔHapp.o, were compared with calorimetric ones.

The dynamic behavior of annexin V as a function of calcium ion binding: a circular dichroism, UV absorption, and steady-state and time-resolved fluorescence study.

The binding of calcium ions to annexin V in the absence of phospholipids has been studied by UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence. In the absence of calcium, the unique tryptophan 187, located in domain III of annexin V, is surrounded by a strongly hydrophobic environment, as indicated by its "blue" fluorescence emission maximum (325 nm). This corresponds well with the description of the structure determined by X-ray crystallography of several crystal forms. The Trp187 time-resolved fluorescence decay shows the existence of a fast (picosecond) excited-state reaction which can involve the formation of an H-bond between the indole NH group and the proximate epsilon-OH and/or alpha-carbonyl groups of Thr224. Titration with calcium tends to stabilize the overall structure, as shown by circular dichroism, while leading to large modifications of the local structure around Trp187 making it accessible to the solvent as shown by UV-difference spectra, circular dichroism spectra, and the displacement of its fluorescence emission maximum at saturating concentrations of calcium (350 nm). A rapid (picosecond) formation of an excited-state complex, probably involving one or a few water molecules of the solvation shell, is observed. These observations correlate well with the conformational change observed in crystal structures obtained in high calcium concentrations, involving the removal of Trp187 from the buried position to the surface of the molecule [Sopkova, J., Renouard, M., & Lewit-Bentley, A. (1993) J. Mol. Biol. 234, 816-825; Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., & Seaton, B. A. (1993) Science 261, 1321-1324]. In the solvent-exposed conformation, the indole ring becomes mobile in the subnanosecond and nanosecond time range. This conformational change and the increase in local flexibility can be important for the accommodation of the protein on the surface of phospholipid membranes.

Electron paramagnetic resonance and difference ultraviolet studies of Mn 2+ binding to serum transferrin

Serum transferrin is the mammalian protein whose normal function is to transport ferric ions through the blood among sites of absorption, storage, and utilization. It has two specific metal-binding sites that bind a variety of metal ions in addition to ferric ion. The macroscopic equilibrium constant for the binding of the first equivalent of Mn 2+ to apotransferrin has been determined by electron paramagnetic resonance spectroscopy (EPR) to be logK M1 = 4.06 ± 0.13 at pH 7.4 in 0.1 M N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (Hepes). An equilibrium constant for nonspecific binding of Mn 2+ to apotransferrin of logK ns = 2.93 ± 0.13 has also been obtained by using EPR. Binding of Mn 2+ to apotransferrin and to both C- and N-terminal nonferric transferrin has also been studied by difference UV spectroscopy. The second stepwise macroscopic equilibrium constant for the formation of Mn 2Tf is logK M2 = 2.96 ± 0.13. The site-specific microconstants for Mn 2+ binding are logK N = 3.13 ± 0.09 for the N-terminal site and logk C = 3.80 ± 0.09 for the C-terminal site. There does not appear to be any significant cooperativity between the two sites with respect to metal binding. An equilibrium model for the speciation of Mn 2+ in serum has been developed which estimates that almost 90% of Mn 2+ is bound to serum proteins, but only ∼ 1% is bound to transferrin. The weak binding of Mn 2+ to apotransferrin and the obvious inability of transferrin to compete with albumin indicates that the appearance of Mn-transferrin as a major serum species in vivo must involve oxidation of the metal to form the much more stable Mn 3+-transferrin complex. The computer model confirms that albumin has a sufficient binding affinity to complex most of the Mn(II) in serum in competition with the common low molecular weight ligands in serum. However, there is insufficient data to rule out the possibility that some other protein, such as α 2-macroglobulin, may compete with albumin for Mn(II).