A novel method for specific visualisation of serum albumin in polyacrylamide gels by iodine staining.

Bovine serum, bovine serum albumins (delipidated or globulin free or Fraction V), rabbit serum, rabbit serum albumin, Atlantic salmon serum, purified Atlantic salmon serum albumin, human plasma alpha 2-macroglobulin, hemocyanin, trypsin inhibitor, bovine transferrin and bovine lactoferrin were examined by a novel method for specific visualisation of albumins. In native polyacrylamide gel electrophoresis the albumins were visualised by iodine staining as a transparent spot against a brown background whilst the other proteins could not be visualised. It is suggested that the brown background was due to penetration of the gel by iodine while the chemical binding of iodine by albumin produced a decolourisation reaction. This novel method provides a fast and simple approach to identifying serum albumin in polyacrylamide gels.     

Microcalorimetry investigation of synthetic hemoprotein (albumin‐heme)

Recombinant human serum albumin (rHSA) incorporating the iron(II) complex of the tetraphenylporphyrin derivative (FepivP or FecycP) is a synthetic O₂‐carrying hemoprotein [albumin‐heme (rHSA‐FepivP or rHSA‐FecycP)], which acts as a red blood cell substitute. The association and dissociation behavior of FepivP and FecycP with rHSA has been initially investigated by isothermal titration calorimetry. A strong heat release appeared after the injection of albumin‐heme into a large molar excess of rHSA. This exothermic enthalpy change was due to the transference of hemes to the other free albumins. The difference in the heme binding affinity to rHSA can be manifested in the enthalpy term. Copyright © 2003 John Wiley & Sons, Ltd.     

一种转基因猪血中重组人血清白蛋白分离纯化新方法

基因工程技术已经成为研究和生产重组人血清白蛋白(rHSA)替代人血清白蛋白(HSA)的重点技术,而白蛋白的纯化则是该技术的关键。本文主要介绍了从转基因猪血中纯化rHSA的一种新方法,即热乙醇沉淀与多级色谱分离相结合的rHSA纯化方法。热乙醇沉淀法可从猪血浆中获得rHSA粗提取液,此时rHSA的纯度可达69.5%,回收率达51.3%。进一步采用多级色谱分离法,即阴离子交换色谱和反相色谱法进一步纯化,得到rHSA的最终纯度约为100.0%,总回收率为41.1%。该方法为从转基因猪血浆中大规模纯化用于临床和生化研究的高纯度rHSA提供可能,同时也为rHSA替代HSA奠定了基础。     

(EZ)-Cyclobilirubin formation from bilirubin in complex with serum albumin derived from various species

We determined the specificity of photochemical changes of bilirubin in complex with serum albumin from various species. There were no general trends in the configurational photoisomerizations of (ZE)-bilirubin/(ZZ)-bilirubin and (EZ)-bilirubin/(ZZ)-bilirubin associated with the albumins from various species as compared to those associated with human serum albumin. The absorbance spectra of bilirubin in complex with albumins from various species differed, indicating that the three-dimensional structures of (ZZ)-bilirubin bound to the various serum albumins, which are substrates of (ZE)- and (EZ)-bilirubin, differ among species. The rates of conversion of the (EZ)-bilirubin isomer into the structural cyclobilirubin isomer were similar for the albumins of chicken, rat, rabbit, dog, bovine, and pig, and were significantly slower than the rate for human serum albumin. This suggests that the three-domain human albumin has evolved to allow ready conversion of (EZ)-bilirubin to (EZ)-cyclobilirubin. Cyclobilirubin formation in a bilirubin-alpha-fetoprotein solution was much lower than that in a bilirubin–human serum albumin solution. It is believed that the ability of human serum albumin to facilitate the photochemical change of bilirubin was evolutionarily selected in response to neonatal jaundice in humans.     

Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors

The separation of drug enantiomers using proteins as the chiral selectors in capillary electrophoresis (CE) is considered in this review. The proteins used include albumins such as bovine serum albumin, human serum albumin and serum albumins from other species, glycoproteins such as alpha1-acid glycoprotein, crude ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as fungal cellulase, cellobiohydrolase I, pepsin and lysozyme and other proteins such as casein, human serum transferrin and ovotransferrin. Protein-based CE is carried out in two modes: in one proteins are immobilized or adsorbed within the capillary, or protein-immobilized silica gels are packed into the capillary (affinity capillary electrochromatography mode), and in the other proteins are dissolved in the running buffer (affinity CE mode). Furthermore, the advantages and limitations of the two modes and the factors affecting the chiral separations of various drugs by protein-based CE are discussed.     

Fluorescence Spectroscopic Studies on the Interaction of Oleanolic Acid and its Triterpenoid Saponins Derivatives with Two Serum Albumins

The interaction of oleanolic acid (OA) and its glycosylated derivatives (LL-2 and LL-4) with human and bovine serum albumins were investigated using the methods of fluorescence spectroscopy. The spectroscopic analysis of the fluorescence quenching that occurs when OA and its derivatives interact with serum albumin indicates that these quenching constants are inversely correlated with temperature and the quenching process involves static interactions. The binding affinity of OA and OA-derived compounds to bovine serum albumin (BSA) and human serum albumin (HSA) follow the trend LL-4 > LL-2 > OA, suggesting that glycosylation of OA can facilitate its binding to serum albumins. Additionally, the binding affinity of these compounds to HSA is stronger than it is to BSA. The calculated thermodynamic parameters suggest that hydrophobic interactions dominate these interaction processes. We also found that only a single type of binding site exists for OA and its derivatives to HSA and BSA. Synchronous fluorescence results indicate that the binding of OA, LL-2 and LL-4 to BSA and HSA can lead to the conformational changes around the tryptophan residues of the two serum albumins. These results provided valuable clues to the pharmacokinetics and the pharmacologic activities of OA and its types of triterpenoid saponins derivatives.     

Studies on the interaction of colloidal gold and serum albumins by spectral methods

The interactions of colloidal gold and serum albumins, including bovine serum albumin (BSA) and human serum albumin (HSA), were studied by fluorescence and absorption spectrometry. Fluorescence quenching spectrometry was applied to study the interactions between colloidal gold and serum albumins. At pH 7.4 phosphate-buffered saline (PBS), the intensity of fluorescence emission spectrum of serum albumins decreased in the presence of colloidal gold, which indicated that colloidal gold quenched the fluorescence of serum albumins. Experimental results indicated that the combination reactions of colloidal gold and serum albumins were static quenching processes. Based on the effect of colloidal gold on fluorescence intensity, the binding constants, the numbers of binding sites and the acting forces between colloidal gold and serum albumins were found.     

Molecular spectroscopic study on the interaction of tetracyclines with serum albumins

A molecular spectroscopic investigation of the interaction between tetracyclines antibiotics and human serum albumin or bovine serum albumin was reported. The influences of some metal ions on the interaction were also studied. When tetracyclines drugs were added into the solution containing serum albumins, the fluorescence intensity of serum albumins decreased with the increasing of the drugs concentrations, which is due to the formation of new non-fluorescence complexes of drug-serum albumin. The tetracyclines acted as quenchers and quenched the fluorescence of the serum albumins. The binding constants and the number of the binding sites of the reaction of tetracyclines and serum albumins were obtained. The main sorts of acting force between the drugs and serum albumins were found and the action distances and the energy transfer efficiencies between donor-acceptor were calculated based on the Foster energy transference.     

Spectral and fluorescent study of the noncovalent interaction of a meso-substituted cyanine dye with serum albumins

A comparative study of the noncovalent interaction of the cyanine dye probe 3,3′-di-(γ-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethylthiacarbocyanine betaine with serum albumins of different vertebrates: rat, rabbit, bovine, and human serum albumins (RSA, TSA, BSA, and HSA, respectively) has been performed by spectral and fluorescent methods. It has been shown that, the dye forms only one product, the trans-monomer bound to HSA, by interacting with HSA, whereas other binding products are also formed with other albumins. This is probably explained by a higher interaction energy of the dye with HSA than with other serum albumins.     

Fatty acid binding sites of human and bovine albumins: Differences observed by spin probe ESR

Bovine and human serum albumins and recombinant human albumin, all non-covalently complexed with 5- and 16-doxyl stearic acids, were investigated by ESR spectroscopy in solution over a range of pH values (5.5–8.0) and temperatures (25–50 °C), with respect to the allocation and mobility of fatty acid (FA) molecules bound to the proteins and conformation of the binding sites. In all proteins bound FA undergo a permanent intra-albumin migration between the binding sites and inter-domain residence. Nature identity of the recombinant human albumin to its serum-derived analog was observed. However, the binding sites of bovine albumin appeared shorter in length and wider in diameter than those of human albumin. Presumably, less tightly folded domains in bovine albumin allow better penetration of water molecules in the interior of the globule that resulted in higher activation energy of FA dissociation from the binding site. Thus, the sensitive technique based on ESR non-covalent spin labeling allowed quantitative analysis and reliable comparison of the fine features of binding proteins.     

Properties of an albumin inhibiting lysosomal acid cholesteryl ester hydrolase in rat liver.

Lysosomal acid cholesteryl ester hydrolase (acid CEH, EC. 1.1.1.13) activity was inhibited by addition of an increasing amount of d = 1.21 bottom fraction from rat serum (Lipids in press). To clarify the mechanism of this inhibition, rat native and modified albumin were added to the assay mixture and their effects on acid CEH activity were examined. The inhibitory effect on acid CEH activity was dependent on the concentration of rat albumin. Albumin of various mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. This inhibitory activity was markedly increased by heat treatment, the effect increasing in parallel with the prolongation of the treatment. Moreover, this albumin-dependent inhibition of acid CEH activity was also markedly increased by methylation of albumin. In contrast, the inhibition of acid CEH activity by modified albumins, such as acetyl albumin, succinyl albumin and glycine methyl ester albumin, was much lower than that of albumin, and no stimulatory effect of heat treatment on the albumins was observed. The heat-treated albumin-dependent inhibition of acid CEH activity was not abolished in the presence of sodium deoxycholate. The values of Vmax obtained were similar with or without heat-treated albumin. These results suggest that the inhibitory effects of heat-treated albumin may be due to an intrinsic and characteristic property of the lipid/water interface, and that the stimulatory effects of heat treatment on albumin-dependent inhibition may be due to heat-induced changes in the affinity and conformation of albumin.     

Protein binding of quinolonecarboxylic acids. I. Cinoxacin, nalidixic acid and pipemidic acid

Binding of cinoxain (CINX), nalidixic acid (NA) and pipemidic acid (PPA) to serum proteins was investigated by equilibrium dialysis, ultrafiltration and circular dichroism (CD) spectroscopy. CINX and NA were found to bind mainly to albumin in human serum, the latter interacting with the protein about ten times as strongly as CINX at pH 7.4 and 37 degrees C. PPA showed little or no significant binding to human serum albumin (HSA), alpha 1-acid glycoprotein, and globulins, but showed 20-30% binding to protein in human serum. The CD results were suggestive of some weak interaction of PPA with human apotransferrin. Binding of the three drugs to HSA was found to depend on the lipophilicity of their substituents at the 7-position. The degree of protein binding for human, dog and rat sera at 37 degrees C was in the order of NA (92-97%) greater than CINX (68-90%) greater than PPA (20-30%) at drug concentrations of 10-30 micrograms/ml. CINX showed relatively large species dependence in serum protein binding, which seemed to be due to different affinities of this drug to the respective albumins. CINX was found to bind to rat serum albumin as strongly as NA.     

Human Serum Albumin Hybrid Incorporating Synthetic Hemes:A Novel O_2-Carrying Hemoprotein

It has long been known that homologous blood transfusion will result in a lot ofserious problems such as viral infections,for example AIDS,hepatitis,antigenicsensitization and GVHD;therefore aggressive testing of donor blood has beenadopted[1 ,2 ] .Even after this introduction,which is time-consuming and expensive,wecould not eliminate all the risks. In the wake of these kinds of pitfalls,production andclinical use of the blood substitutes have emerged.The essential aim of blood substitutei…     

O2 and CO binding properties of artificial hemoproteins formed by complexing iron protoporphyrin IX with human serum albumin mutants

The binding properties of O2 and CO to recombinant human serum albumin (rHSA) mutants with a prosthetic heme group have been physicochemically and kinetically characterized. Iron(III) protoporphyrin IX (hemin) is bound in subdomain IB of wild-type rHSA [rHSA(wt)] with weak axial coordination by Tyr-161. The reduced ferrous rHSA(wt)-heme under an Ar atmosphere exists in an unusual mixture of four- and five-coordinate complexes and is immediately autoxidized by O2. To confer O2 binding capability on this naturally occurring hemoprotein, a proximal histidine was introduced into position Ile-142 or Leu-185 by site-directed mutagenesis. A single mutant (I142H) and three double mutants (I142H/Y161L, I142H/Y161F, and Y161L/L185H) were prepared. Both rHSA(I142H/Y161L)-heme and rHSA(I142H/Y161F)-heme formed ferrous five-N-coordinate high-spin complexes with axial ligation of His-142 under an Ar atmosphere. These artificial hemoproteins bind O2 at room temperature. Mutation at the other side of the porphyrin, Y161L/L185H, also allowed O2 binding to the heme. In contrast, the single mutant rHSA(I142H)-heme could not bind O2, suggesting that removal of Y161 is necessary to confer reversible O2 binding. Laser flash photolysis experiments showed that the kinetics of CO recombination with the rHSA(mutant)-heme were biphasic, whereas O2 rebinding exhibited monophasic kinetics. This could be due to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O2 binding affinities of the rHSA(mutant)-heme were significantly lower than those of hemoglobin and myoglobin, principally due to the high O2 dissociation rates. Changing Leu-161 to Phe-161 at the distal side increased the association rates of both O2 and CO, which resulted in enhanced binding affinity.     

Spectroscopic studies on the interaction between riboflavin and albumins

The interactions between riboflavin (RF) and human and bovine serum albumin (HSA and BSA) were studied by using absorption and fluorescence spectroscopic methods. Intrinsic fluorescence emission spectra of serum albumin in the presence of RF show that the endogenous photosensitizer acts as a quencher. The decrease of fluorescence intensity at about 350 nm is attributed to changes in the environment of the protein fluorophores caused by the ligand. The quenching mechanisms of albumins by RF were discussed. The binding constants and binding site number were obtained at various temperatures. The distance between albumins and RF in the complexes suggests that the primary binding site for RF is close to tryptophan residue (Trp214) of HSA and Trp212 of BSA. The hydration process of albumins has also been discussed.     

Dual temperature/pH-sensitive drug delivery of poly(N-isopropylacrylamide-co-acrylic acid) nanogels conjugated with doxorubicin for potential application in tumor hyperthermia therapy

The interaction of the amphiphilic drugs, i.e., amitriptyline hydrochloride (AMT) and promethazine hydrochloride (PMT), with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)), has been examined by the various spectroscopic techniques, like fluorescence, UV-vis, and circular dichroism (CD). Fluorescence results indicate that in case of HSA-drug complexes the quenching of fluorescence intensity at 280 nm is less effective as compared to at 295 nm while in case of BSA-drug complexes both have almost same effect and for most of drug-serum albumin complexes there is only one independent class of binding. For all drug-serum albumin complexes the quenching rate constant (K(q)) values suggest the static quenching procedure. The UV-vis results show that the change in protein conformation of PMT-serum albumin complexes was more prominent as compared to AMT-serum albumin complexes. The CD results also explain the conformational changes in the serum albumins on binding with drugs. The increase in α-helical structure for AMT-serum albumin complexes is found to be more as compared to PMT-serum albumin complexes. Hence, the various spectroscopic techniques provide a quantitative understanding of the binding of amphiphilic drugs with serum albumins.     

2,5-di-[2-(3,5-bis(2-pyridylmethyl)amine -4-hydroxy-phenyl) ethylene] pyrazine zinc complex as fluorescent probe for labeling proteins

The binding characteristics between 2,5-di-[2-(3,5-bis(2-pyridylmethyl)amine -4-hydroxy-phenyl) ethylene] pyrazine (1) or its complex (1-Zn) and serum albumins were studied by fluorescence spectroscopy in pH 7.4 aqueous solution. 1-Zn emitted weak fluorescence at 580 nm in a pH 7.4 Tris-HCl buffer solution when excited at 435 nm, however, the fluorescence intensity increased upon addition of serum albumins with the blue shift of emission peak to 524 nm. The binding constants were estimated as 8.40 x 10(7) and 3.03 x 10(6)mol(-1)L for bovine serum albumin (BSA) and human serum albumin (HSA) respectively, and the number of binding sites was 1 for each. The quenching mechanism of fluorescence of serum albumins by 1-Zn was considered as a static quenching process. The binding distance between 1-Zn and serum albumins and the energy transfer efficiency were obtained based on the theory of F?rester spectroscopy energy transfer. The effect of 1-Zn on the conformation of serum albumins was further analyzed using synchronous fluorescence spectrometry. The experiment results clearly showed that 1-Zn is a highly sensitive protein sensor.     

光谱法研究噻菁染料与血清蛋白的相互作用

本文通过吸收和荧光光谱法研究了一种噻菁染料与人血清蛋白及牛血清蛋白的相互作用。吸收光谱数据表明,与血清蛋白结合后,噻菁染料单体的吸收峰发生红移,同时强度也有很大变化;还通过吸收光谱计算确定了噻菁染料与血清蛋白的结合位点数（ n ）。与人血清蛋白或牛血清蛋白结合后,噻菁染料的荧光量子产率增加。分析噻菁染料的荧光强度随溶液中血清蛋白浓度的变化得到了二者反应的表观结合常数（ K _a）和自由能变化（ ΔG ）。根据表观结合常数（ K _a）可以判断,人血清蛋白比牛血清蛋白与噻菁染料的结合更强。     

Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 degrees C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the F?ster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.     

Artificial immunoglobulin G-binding protein mimetic to staphylococcal protein A. Its production and application to affinity purification of immunoglobulin G.

Staphylococcal protein A consists of a single polypeptide with five immunoglobulin G (IgG)-binding domains, which are linked as E-D-A-B-C in this order from the amino terminal. The DNA coding domains A-B were polymerized one to six times linearly, taking advantage of the non-palindromic nucleotide sequence of the AccI recognition site and the resultant DNAs were inserted in pTRP vector carrying trp promoter. The artificial IgG-binding proteins [pA(AB)1-6], which had been expressed in Escherichia coli JM109, were purified by methods involving IgG-Sepharose affinity chromatography. Among pA(AB)1-6 immobilized on cyanogen bromide-Sepharose, pA(AB)4-Sepharose was the highest in IgG-binding capacity at the same level of mg protein per ml gel, about 30% higher than protein A-Sepharose. At 8 mg protein per ml gel, it bound and eluted about 24 mg of IgG from rabbit serum. Its IgG-binding capacities were the highest with porcine, rabbit, human and guinea pig sera, intermediate with bovine, horse and sheep sera and the lowest with mouse, goat, rat and chicken sera.