Enzyme immunoassay for serum dexamethasone using 4-(carboxymethylthio)dexamethasone as a new hapten.

A sensitive and simple enzyme immunoassay for direct quantitation of serum dexamethasone was established. An antiserum with high specificity was produced by the immunization of rabbits with a newly synthesized 4-(carboxymethylthio)dexamethasone-bovine serum albumin conjugate. Alkaline phosphatase was used as a labeling enzyme. The minimum amount of dexamethasone detected was 2 pg per tube on the basis of B/Bo 100 - 2 SD (%) of standard curve. However, taking into account the cross-reaction with steroids such as cortisol in dexamethasone-free serum, the measurable range was from approximately 0.13 to 10 micrograms/dl. Intra- and interassay coefficients of variation were 1.5 - 5.4% and 0.6 - 6.5%, respectively. Serum levels of dexamethasone and cortisol in four normal subjects after an oral administration of 1 mg of dexamethasone are also reported.     

Spectroscopic study on the interaction of bovine serum albumin with zinc(II) phthalocyanine

The interaction between the photosensitive antitumour drug, 2(3),9(10),16(17),23(24)‐tetra‐(((2‐aminoethylamino)methyl)phenoxy)phthalocyaninato‐zinc(II) (ZnPc) and bovine serum albumin (BSA) has been investigated using various spectroscopic methods. This work may provide some useful information for understanding the interaction mechanism of anticancer drug–albumin binding and gain insight into the biological activity and metabolism of the drug in blood. Based on analysis of the fluorescence spectra, ZnPc could quench the intrinsic fluorescence of BSA and the quenching mechanism was static by forming a ground state complex. Meanwhile, the Stern–Volmer quenching constant (K_SV), binding constant (K_b), number of binding sites (n) and thermodynamic parameters were obtained. Results showed that the interaction of ZnPc with BSA occurred spontaneously via hydrogen bond and van der Waal's force. According to Foster's non‐radioactive energy transfer theory, the energy transfer from BSA to ZnPc occurred with high possibility. Synchronous fluorescence and circular dichroism (CD) spectra also demonstrated that ZnPc induced the secondary structure of and conformation changes in BSA, especially α helix. Copyright © 2015 John Wiley & Sons, Ltd.     

Tb(III) as a fluorescent probe for the structure of bovine serum albumin

Tb(III) was used as a fluorescent probe in the study of the calcium-binding sites on Bovine Serum Albumin (BSA). The fluorescence of Tb(III) is enhanced markedly when bound to BSA and nonradiative energy transfer between two fluorescent tryptophan(Trp) residues and Tb(III) bound to calcium-binding sites on BSA occurred. Experimental results show that the major groups in BSA bound to metal ion are the carboxyl side groups of glutamic acid (Glu) and aspartic acid (Asp). The average distance between the bound Tb(III) and the two tryptophan residues in BSA calculated by a F?ster dipole-dipole nonradiative energy transfer mechanism is 1.48 nm.     

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.     

Short-wavelength sensitive opsin (SWS1) as a new marker for vertebrate phylogenetics

Background  

Vertebrate SWS1 visual pigments mediate visual transduction in response to light at short wavelengths. Due to their importance in vision, SWS1 genes have been isolated from a surprisingly wide range of vertebrates, including lampreys, teleosts, amphibians, reptiles, birds, and mammals. The SWS1 genes exhibit many of the characteristics of genes typically targeted for phylogenetic analyses. This study investigates both the utility of SWS1 as a marker for inferring vertebrate phylogenetic relationships, and the characteristics of the gene that contribute to its phylogenetic utility.     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

N-(2-Amino-phenyl)-4-(heteroarylmethyl)-benzamides as new histone deacetylase inhibitors

A variety of N-(2-amino-phenyl)-4-(heteroarylmethyl)-benzamides were designed and synthesized. These compounds were shown to inhibit recombinant human HDAC1 with IC₅₀ values in the sub-micromolar range. In human cancer cells growing in culture these compounds induced hyperacetylation of histones, induced the expression of the tumor suppressor protein p21^WAF1/Cip1, and inhibited cellular proliferation. Certain compounds of this class also showed in vivo activity in various human tumor xenograft models in mice.     

Modification of human serum albumin with N-(2,5-dinitro-4-fluorophenyl)-4-amino-2,2,6,6-tetramethyl-piperidinooxy radical.

Human serum albumin has been treated with the spin-labeling reagent indicated in the title. Ultraviolet spectral studies of the protein so modified suggest that reaction takes place at lysine and tyrosine sidechains; kinetic experiments indicate that there are two especially reactive amino groups of the protein which are preferentially modified. Evidence is presented that these groups include the one acetylated by aspirin (Lys-199) or those arylated by 2.6-dinitro-4-trifluoromethylbenzenesulfonate. Esr experiments show that bound spin labels have about the same correlation time expected for overall tumbling of the protein; ESR observations indicate that molecular freedom near the spin labels is not increased when the protein is transferred to 8 M urea.     

Study on the interaction between 4-(1H-indol-3-yl)-2-(p-tolyl)quinazoline-3-oxide and human serum albumin

An organic small-molecular drug, 4-(1H-indol-3-yl)-2-(p-tolyl)quinazoline-3-oxide 1a was synthesized. It was employed to investigate the binding interaction and mechanism with human serum albumin (HSA). The experimental results indicated that the fluorescence quenching of HSA by 1a is a static quenching process and formation 1a-HSA complex. The site competition experiments revealed that the combination of 1a on HSA are hydrophobic interactions in the IIA domain and hydrogen bonds in IIIA domain of HSA, and the hydrophobic interactions of 1a on HSA are stronger than that of hydrogen bonds. These results were also confirmed by molecular docking theoretic analysis and ANS-hydrophobic fluorescent probe experiment. Synchronous fluorescence experiments showed that the polarity of HSA microenvironment was increase in the interaction process of 1a with HSA. The results of binding distance explored indicated that the combination distance between 1a and HSA is 3.63 nm, which is between 0.5R₀ and 1.5R₀, revealing the energy transfer between HSA and 1a is non-radiative. These results are very helpful for people to screen out high efficient indoloquinazoline drugs.     

Giant unilamellar vesicles containing Rhodamine 6G as a marker for immunoassay of bovine serum albumin and lipocalin-2

Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml⁻¹, respectively.     

Intermolecular interaction of nickel (ii) phthalocyanine tetrasulfonic acid tetrasodium salt with bovine serum albumin: A multi-technique study

The interaction of nickel (II) phthalocyanine tetrasulfonic acid tetrasodium salt with bovine serum albumin (BSA) has been investigated by combination of fluorescence, UV-vis absorption, Fourier transform infrared (FT-IR), and circular dichorism (CD) spectroscopies as well as through molecular docking. Fluorescence quenching and absorption spectra were investigated as a mean for estimating the binding parameters. Analysis of fluorescence quenching data at different temperatures was performed in order to specify the thermodynamics parameters for interactions of phthalocyanine complex with BSA. According to experimental data it was suggested that phthalocyanine had a significant binding affinity to BSA and the process was entropy driven. Based on the results of molecular docking it was indicated that the main active binding site for this phthalocyanine complex is site I in subdomain IIA of BSA. The results provide useful information for understanding the binding mechanism of anticancer drug-albumin and gives insight into the biological activity and metabolism of the drug in blood.     

Fluorimetric studies on the binding of 4-(dimethylamino)cinnamic acid with micelles and bovine serum albumin

The constrained photophysics of intramolecular charge transfer (ICT) probe 4-(dimethylamino)cinnamic acid (DMACA) was studied in different surfactant systems as well as in presence of model water soluble protein bovine serum albumin (BSA). Binding of the probe in ionic micelles like sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) causes an increase in ICT fluorescence intensity, whereas, in non-ionic TritonX-100 (TX-100) the intensity decreases with a concomitant increase in emission from locally excited (LE) state. The observations were explained in terms of the different binding affinity, location of the probe and also the nature of specific hydrogen bonding interaction in the excited state nonradiative relaxation process of DMACA. The ICT fluorescence emission yield decreases in BSA due to the locking in of the probe buried in the hydrophobic pocket of the protein structure. SDS induced uncoiling of protein and massive cooperative binding between BSA and SDS is manifested by the release of probe molecules in relatively free aqueous environment.     

A new proalbumin variant: albumin Jaffna (-1 Arg----Leu)

Albumin Jaffna is an electrophoretically slowly moving genetic variant of human serum albumin found in two members of a Tamil family from Jaffna (Northern Sri Lanka), both heterozygous for the abnormal protein. Sequential analysis of albumin Jaffna, purified from serum by ion exchange chromatography on DEAE Sephadex and Mono Q columns, revealed that this variant is a new abnormal proalbumin, arising from a -1 Arg----Leu substitution, which prevents the proteolytic removal of the N-terminal hexapeptide and allows the mutated proalbumin to enter the circulation. The presence of two additional positive charges is in keeping with the decreased electrophoretic mobility of albumin Jaffna, as well as with its isoelectric point of 5.01, determined by chromatofocusing on a Mono P column. The variant is selectively cleaved by trypsin in vitro, leaving leucin -1 as N-terminal residue.     

Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation

Abstract

(-)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10–100 mM during a 21-day incubation at 37°C and pH: 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation.