Ayaka Suenaga

In vitro and in vivo properties of recombinant human serum albumin from pichia pastoris purified by a method of short processing time

AbstractPurpose. Recombinant human serum albumin (rHSA), secreted by a Pichia pastorisexpression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed. Methods. Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with 125I-labeled albumin. Results. Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations. Conclusion. The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding

Binding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5′-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation.

A comparative study of the interaction of warfarin with human α1‐acid glycoprotein and human albumin

The interaction of warfarin with human α1‐acid glycoprotein (α1‐AGP) and human albumin (HA) has been investigated using fluorescence and circular dichroism techniques. The fluorescence of warfarin is greatly enhanced following binding to α1‐AGP or HA, the binding constant for a single site being estimated by the Scatchard method. The binding constants for the two serum proteins are similar, but the thermodynamic parameters differ. The binding constants increase as the pH is raised to 9ṁ0. Various basic drugs, such as chlorpromazine, propranolol and imipramine, markedly inhibited the binding of warfarin to α1‐AGP. But, some acidic drugs, including phenylbutazone, effectively displaced warfarin bound to HA. The difference in CD spectra observed for α1‐AGP and HA indicated that the drug‐binding sites of the two proteins might have different asymmetries. It thus appears that the mode of interaction of warfarin with the two proteins differs.

Structural Insights into Differences in Drug-binding Selectivity between Two Forms of Human α1-Acid Glycoprotein Genetic Variants, the A and F1*S Forms

Human α1-acid glycoprotein (hAGP) in serum functions as a carrier of basic drugs. In most individuals, hAGP exists as a mixture of two genetic variants, the F1*S and A variants, which bind drugs with different selectivities. We prepared a mutant of the A variant, C149R, and showed that its drug-binding properties were indistinguishable from those of the wild type. In this study, we determined the crystal structures of this mutant hAGP alone and complexed with disopyramide (DSP), amitriptyline (AMT), and the nonspecific drug chlorpromazine (CPZ). The crystal structures revealed that the drug-binding pocket on the A variant is located within an eight-stranded β-barrel, similar to that found in the F1*S variant and other lipocalin family proteins. However, the binding region of the A variant is narrower than that of the F1*S variant. In the crystal structures of complexes with DSP and AMT, the two aromatic rings of each drug interact with Phe-49 and Phe-112 at the bottom of the binding pocket. Although the structure of CPZ is similar to those of DSP and AMT, its fused aromatic ring system, which is extended in length by the addition of a chlorine atom, appears to dictate an alternative mode of binding, which explains its nonselective binding to the F1*S and A variant hAGPs. Modeling experiments based on the co-crystal structures suggest that, in complexes of DSP, AMT, or CPZ with the F1*S variant, Phe-114 sterically hinders interactions with DSP and AMT, but not CPZ.

Quantitative evaluation of the role of cysteine and methionine residues in the antioxidant activity of human serum albumin using recombinant mutants

The importance of cysteine (Cys) and methionine (Met) residues for the antioxidant activity of human serum albumin (HSA) was investigated using recombinant HSA mutants, in which Cys34 and/or the six Met residues had been mutated to Ala. The scavenging activities of the mutants against five reactive oxygen and nitrogen species were evaluated by a chemiluminescence assay, electron paramagnetic resonance spectroscopy, or a HPLC‐flow reactor assay. Our results showed that the contributions of Cys34 and the Met residues to the antioxidant activity of HSA were 61% and 29% against O2 •−, 68% and 61% against H2O2, 38% and 6% against HO•, 36% and 13% against HOCl, and 51% and 1% against •NO, respectively. Thus, the findings propose in a direct way that Cys34 plays a more important role than the Met residues in the antioxidant activity of HSA.

Mode of Interaction of Loop Diuretics with Human Serum Albumin and Characterization of Binding Site

AbstractPurpose. The purpose of this study was to investigate the binding mechanism of loop diuretics with HSA and to characterize the binding site on HSA. Methods. Quantitative analysis of potential interaction between ligands bound to HSA was performed by equilibrium dialysis and data for binding of the two ligands to HSA were analyzed on the basis of a theoretical model of simultaneous binding of two ligands. Results. The binding of loop diuretics is dependent upon the N-B transition, conformational change of albumin. Furthermore, from the results of binding of the drugs to modified HSA, the lysine residue seems to be involved in the binding of loop diuretics to HSA. Conclusions. Analysis using models describing independent, competitive, cooperative and anti-cooperative binding led to the conclusion that loop diuretics bind to site I, particularly to the warfarin region on HSA.

Molecular-weight-dependent pharmacokinetics and cytotoxic properties of cisplatin complexes prepared with chondroitin sulfate A and C

In order to screen out an optimum complex for reducing the nephrotoxicity of cisplatin (CDDP), we investigated and compared CDDP-chondroitin sulfate complexes to CDDP in terms of in vivo pharmacokinetics and in vitro cytotoxicity. The polymeric carriers used in the study were chondroitin sulfate A (CSA, 4-sulfate) with mean molecular weights of 10 kDa (CSA-1) and 23 kDa (CSA-2), and chondroitin sulfate C (CSC, 6-sulfate) with mean molecular weights of 8 kDa (CSC-1) and 25 kDa (CSC-2). The resultant complexes (CDDP-CSA-1, CDDP-CSA-2, CDDP-CSC-1 and CDDP-CSC-2) were administered intravenously to rats. The obtained plasma concentration-time curves during the 3 h period studied for all complexes are biphasic. The plasma dispositions of complexes were dependent on the molecular sizes with urinary excretion as main elimination pathway. CDDP-CSA-1 and CDDP-CSC-1 were unable to effectively increase the plasma retention of platinum due to rapid renal excretion. Furthermore, CDDP-CSA-1 disappeared from plasma more quickly than CDDP-CSC-1. CDDP-CSA-2 and CDDP-CSC-2, with similar urinary excretion as CDDP, gave rise to approximately five and four-fold increase in AUC(0-3 h) values, respectively, than that was achieved with native CDDP treatment. Biodistribution was compared between CDDP-CSA-2 and CDDP-CSC-2. Both complexes effectively suppressed the extensive distribution of CDDP into most tissues, especially kidney. However, CDDP-CSC-2 showed less reduction effect than CDDP-CSA-2. In addition, a significantly higher accumulation in tumor tissue was found with the administration of CDDP-CSA-2 than CDDP. Moreover, CSA complexes displayed an IC(50) of 6 microM Pt-equivalents against SW4800 human colon cancer cells, similar to that of CDDP, whereas CSC complexes were less active than CDDP. These studies indicate that the complex prepared with CSA, which is greater than 20 kDa of molecular size, is superior to that of CSC, exhibiting improved pharmacokinetics and similar pharmacological activity to the native drug.

Nitrosylated human serum albumin (SNO-HSA) induces apoptosis in tumor cells.

Recently, nitric oxide has been investigated as a potential anti-cancer therapy because of its cytotoxic activity. Previously, we found that S-nitrosylated human serum albumin (SNO-HSA) induced apoptosis in C26 cells, demonstrating for the first time that SNO-HSA has potential as an anti-cancer drug. In the present study, the anti-tumor activity of SNO-HSA in another tumor type of cancer cell was investigated using murine tumor LY-80 cells. Mitochondrial depolarization, activation of caspase-3 and DNA fragmentation were induced in LY-80 cells by SNO-HSA treatment in a dose-dependent manner. Inhibition of caspase activity resulted in complete inhibition of DNA fragmentation induced by SNO-HSA. The cytotoxic effects of SNO-HSA on LY-80 were concentration-dependent. Tumor growth in LY-80-tumor-bearing rats was significantly inhibited by administration of SNO-HSA compared with saline- and HSA-treatment. These results suggest that SNO-HSA has potential as a chemopreventive and/or chemotherapeutic agent because it induces apoptosis in tumor cells.

Drug Binding to α1-Acid Glycoprotein Studied by Circular Dichroism

The interactions of acidic and basic drugs with α1-acid glycoprotein (α1-AGP) were investigated using circular dichroism (CD) measurements. Extrinsic Cotton effects were generated by the binding of drugs to α1-AGP. The CD data suggested the presence of a single binding site on the α1-AGP molecule. The induced ellipticities of the acidic drug–α1-AGP system decreased with increasing pH, while the ellipticities for the basic drugs increased with pH. The ellipticities for all drugs were reduced by the addition of fatty acids. Furthermore, the induced ellipticities decreased in the presence of cesium chloride for basic drugs bound to α1-AGP. The extrinsic Cotton effects therefore appear to result from hydrophobic interaction with α1-AGP for the acidic drugs and from hydrophobic and electrostatic interactions for the basic drugs.

Interactions of aldosterone antagonist diuretics with human serum proteins

AbstractPurpose. The purpose of this study was to investigate the binding mechanism of aldosterone antagonist diuretics with human serum proteins, human serum albumin (HSA) and α1-acid glycoprotein (AGP), as well as to identify the binding sites of the drugs on these proteins. Methods. Binding activities of spironolactone (SP) and its pharmacologically active metabolite canrenone (CR) to serum and serum protein were examined by ultrafiltration and spectroscopic techniques. The data for the binding of these drugs to HSA were analyzed on the basis of a theoretical model of simultaneous binding of the ligands. Results. The binding percentages of antagonist diuretics SP and CR to human serum proteins were 88.0% and 99.2%, respectively, at therapeutic concentrations. SP bound strongly and almost equally to both HSA and AGP, but CR bound strongly only to HSA. In addition, the displacement results found using fluorescent probes and ultrafiltration methods demonstrated that SP bound to site I, particularly to the warfarin region on HSA, and to the basic binding site on AGP, while CR bound to the warfarin region on HSA. Conclusions. The limited results presented here stress the need for caution on coadministration of acidic drugs which bind to the warfarin region on HSA and basic drugs which bind to AGP with SP and its metabolite CR.