Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

Dye removal, catalytic activity and 2D crystallization of chloroplast H(+)-ATP synthase purified by blue native electrophoresis

The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.     

The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation

The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO(2)(-). The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions.     

In vitro antioxidant activities of the polysaccharides from Tricholoma lobayense

The antioxidant activities of three polysaccharide components (TLH-1, TLH-2, TLH-3) extracted from Tricholoma lobayense were evaluated by three different in vitro methods, namely superoxide radical (O(2)(-)) scavenging activity, inhibition of mice erythrocyte hemolysis (MEH) and malondialdehyde (MDA) mediated by hydrogen peroxide (H(2)O(2)) and investigation of oxidative modification of human serum albumin (HSA) induced by 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH) through fluorescence spectroscopy. The antioxidant experiments showed that the polysaccharides had a notable activity in scavenging O(2)(-) in a concentration-dependent manner; H(2)O(2)-induced MEH and formation of MDA were effectively inhibited; by fluorescence spectroscopy, it was demonstrated that the polysaccharides could obviously inhibit AAPH-induced oxidative modification of HSA. The experimental data obtained from the in vitro models clearly revealed that TLH-3 had stronger antioxidant potency than TLH-1 and TLH-2, which indicated that TLH-3 might be exploited as effective natural antioxidant to alleviate oxidative stress.     

Photoinduced reactions of anthraquinone antitumor agents with peptides and nucleic acid bases: an electron spin resonance and spin trapping study

The photoexcitation (lambda = 313 +/- 10 nm) of adriamycin, daunomycin, and mitoxantrone in the presence of peptides or pyrimidine nucleic acid bases was investigated. In air-saturated and air-free solutions, peptides are decarboxylated by the photoexcited drug molecules. The decarboxylation reactions were shown to occur specifically at the C-terminal amino acid of the peptide. The decarboxylated peptide radicals were spin-trapped using 2-methyl-2-nitrosopropane (MNP) and identified by electron spin resonance (ESR). In air-free solutions, nucleic acid bases are oxidized by the photoexcited drug molecules predominantly generating C(5)-carbon-centered radicals in the pyrimidine rings of uracil, cytosine, and thymine. However, spin adducts of MNP and thymine were also obtained at the N(1) or N(3) positions of the pyrimidine ring. In air-saturated adriamycin and daunomycin solutions, the spin adducts of MNP with uracil or thymine are similar to those obtained following hydroxyl radical reactions with these pyrimidines. This suggests that in the presence of oxygen, the photoexcited adriamycin and daunomycin transfer an electron to oxygen generating the superoxide anion radicals (O2-.), which are precursors of hydroxyl radicals. O2-. was also formed when O2-saturated DNA solutions were photoirradiated (lambda = 313 +/- 10 and 438 +/- 10 nm) in the presence of adriamycin and daunomycin, indicating that the photodegradation of DNA in the presence of these drugs caused by hydroxyl radicals is mediated by dissolved oxygen.     

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.     

Model studies on the analysis of pyruvic acid acetal-containing polysaccharides by the reductive-cleavage method

The 4,6-O-(1-methoxycarbonylethylidene), -(hydroxyisopropylidene), and -(methoxyisopropylidene) acetals of methyl 2,3-di-O-methyl-alpha-D-glucopyranoside were subjected to reductive cleavage in the presence of triethylsilane and trimethylsilyl methanesulfonate-boron trifluoride etherate (Me3SiOMs-BF3.Et2O), BF3.Et2O, or trimethylsilyl trifluoromethanesulfonate (Me3SiOSO2CF3) and the mole fractions of products were determined as a function of reaction time. The 4,6-(1-methoxycarbonylethylidene) acetal was quite stable to reductive-cleavage conditions but isomerization of the initial R,S mixture of diastereomers to the more-stable S diastereoisomer was noted. In addition, a slow, regiospecific, reductive ring-opening of the acetal was observed to give 6-O-[1-(methoxycarbonyl)ethyl] derivatives. The 4,6-(hydroxyisopropylidene) acetal was very unstable under reductive-cleavage conditions. Both Me3SiOMs-BF3.Et2O and Me3SiOSO2CF3 catalyzed complete removal of the group, via the intermediate 6-[1-(hydroxymethyl)ethyl] ether, but BF3.Et2O gave a mixture of products. The 4,6-(methoxyisopropylidene) acetal was also very labile under reductive-cleavage conditions; Me3SiOMs-BF3.Et2O catalyzed complete removal of the acetal, via the intermediate 6-[1-(methoxymethyl)ethyl]ether, but the intermediate ether was quite stable in the presence of either BF3.Et2O or Me3SiOSO2CF3. It is concluded from these studies that polysaccharides bearing 4,6-O-(1-carboxyethylidene) substituents can be analyzed directly by sequential permethylation and reductive cleavage. It is proposed that the identity of the substituted monomer and the positions of substitution of the acetal can be determined by sequential permethylation, ester reduction, and reductive cleavage.     

On the modulation of photoinduced fluorescence enhancement and conformational stability of albumin-bound bilirubin: effect of epsilon-NH(2) groups blocking and chloroform binding

Photoinduced fluorescence enhancement of bilirubin bound to primary binding site on human serum albumin (HSA) was completely ceased when epsilon-NH(2) groups of its internal lysine residues were covalently blocked by acetylation or succinylation though the pigment bound to these derivatives in a folded conformation akin to that bound to HSA. These photoinduced fluorescence modulations cannot be ascribed to the binding of bilirubin to secondary low affinity sites as the CD spectrum of bilirubin bound to these derivatives showed complete inversion upon addition of chloroform which binds to subdomain IIA in HSA where high affinity bilirubin binding site is located. Presence of chloroform reconciled the photoinduced alterations in the CD spectrum observed in its absence, suggesting that chloroform stabilized the bound ligand against light but the fluorescence properties of bilirubin complexed with acetylated or succinylated derivatives remained unchanged. Guanidination of internal epsilon-NH(2) groups in HSA by O-methylisourea did not alter the spectral properties of the bound ligand. These results suggest that salt linkage(s) existing between epsilon-NH(2) groups of lysine residues in HSA and carboxyl groups of bilirubin, act(s) as a potential barrier during conformational rotation of the bound ligand assisted by photoactivation and their abolishment can alter its dynamics and stereoselectivity, a hitherto unnoticed implication of salt linkage(s) in BR-HSA complex.     

Influence of fluoro, chloro and alkyl alcohols on the folding pathway of human serum albumin

Urea-induced equilibrium unfolding of human serum albumin (HSA) when studied by mean residue ellipticity at 222 nm (MRE(222)) or intrinsic fluorescence measurements showed a two-step, three-state transition with a stable intermediate around 4.6-5.2 M urea. The presence of 2,2,2-trifluoroethanol (TFE) resulted in a single-step, two-state transition with a significant shift towards higher urea concentration, suggesting the stabilizing effect of TFE. The free energy of stabilization (DeltaDeltaG(D)(H(2)O)) in the presence of 3.0 M TFE was determined to be 2.68 and 2.72 kcal/mol by MRE(222) and fluorescence measurements, respectively. The stabilizing potential of other alcohols on the refolding behavior of HSA at 5.0 M urea (where the intermediate exists) as studied by MRE(222) and intrinsic fluorescence measurements showed the following order: 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) > TFE > 2-chloroethanol > tert-butanol > iso-propanol > ethanol > methanol. Further, the extent of refolding at the highest concentration of alcohol was similar in all cases. The stabilizing effect of TFE on guanidine hydrochloride (GdnHCl)-induced unfolding of HSA was nearly equal to that found for urea denaturation, as reflected in the DeltaDeltaG(D)(H(2)O) value (2.38 kcal/mol). Taken together, these results suggest that the stabilizing effect of TFE and other alcohols on urea/GdnHCl-induced unfolding of HSA is higher for alcohols that contain bulky groups or fluorine atoms.     

Interaction between hesperetin and human serum albumin revealed by spectroscopic methods

Hesperetin (5,7,3'-trihydroxyl-4'-methoxyl-flavanone) is an important bioactive compound in Chinese traditional medicine and has multiple biological and pharmacological activities. The interaction of hesperetin with human serum albumin (HSA) has been investigated by UV absorption, fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of drug at the molar ratio of drug to HSA ranging from 0.3 to 7 and the binding affinity (K(A)) was 8.11x10(4) M(-1). The primary binding site was most likely located on subdomain IIA. The binding ability of the drug to protein decreased from pH 6.4 to 8.4 in the drug to protein molar ratio of 1. Combining the curve-fitting results of infrared amide I band in D2O and H2O phosphate buffers, the alterations of protein secondary structure after drug complexation were estimated. With increasing the drug concentration, the percentage of protein alpha-helix structure decreased gradually. The reduction of protein alpha-helix structure reached about 7-9% after the protein interacted with hesperetin in D2O and H2O buffer solution at pH 7.4 when the drug to protein molar ratio was 10. This indicated a partial unfolding of HSA in the presence of the drug. From the results of UV absorption, fluorescence and Fourier transformed infrared spectrometry, the binding mode was discussed. The main mechanism of protein fluorescence quenching was a static quenching process and the hydroxyl groups of the drug in its neutral part played an important role in the binding process.     

Kinetics of hemoprotein reduction and interprotein heme transfer

The transfer of hemin from one protein to another is an event biologically important for the conservation of heme iron. Hemin entering the circulation (or added to serum) is mainly bound by albumin and then transferred to hemopexin [Morgan, W.T., Liem, H.H., Sutor, R.P., & Muller-Eberhard, U. (1976) Biochim. Biophys. Acta 444, 435-445], and we are now investigating which mechanisms may be operative in enhancing this process. The presence of imidazole has been demonstrated to accelerate hemin transfer from albumin to hemopexin [Pasternack, R.F., Gibbs, E.J., Hoeflin, E., Kosar, W.P., Kubera, G., Skowronek, C. A., Wong, N.M., & Muller-Eberhard, U. (1983) Biochemistry 22, 1753-1758]. The present work is an examination of the effect of the reduction of albumin-bound hemin on the rate of its transfer to hemopexin. Hemin (HmIII., ferriprotoporphyrin IX) was reduced to HmII (ferroprotoporphyrin IX) by the addition of sodium dithionite under argon. The reduction kinetics of HmIII to HmII were studied separately in the two complexes: with human serum albumin (HSA), which binds up to 20 mol of heme/mol (the first mole with K congruent to 10(8)), and with hemopexin (HHx), which binds heme equimolarly (K congruent to 10(13)). The rate of reduction of HmIII to HmII on HSA was first order over several half-lives and linearly dependent on [S2O4(2-)]1/2. At [HSA]0/[HmIII] = 3, the kobsd was (5 X 10(-3) + 0.75[S2O4(2-)]1/2, and with [HSA]/[HmIII] approximately 25, the kobsd was (2 X 10(-3)) + 0.25[S2O4(2-)]1/2. The reduction of HmIII to HmII on human hemopexin (HHx) is much more rapid with kobsd = (2.5 X 10(3))[S2O4(2-)]1/2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart

The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by Nomega-nitro-L-arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihydroguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n=18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during control conditions, in the presence of NO synthesis inhibitor L-NAME (30 micromol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered separately or in combination. CF varied in autoregulatory range from 4.12+/-0.26 ml/min/g wt at 50 cm H2O to 5.22+/-0.26 ml/min/g wt at 90 cm H2O. In autoregulatory range, nitrite outflow varied from 2.05+/-0.17 nmol/min/g wt at 50 cm H2O to 2.52+/-0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22+/-0.12 ml/min/g wt and 2.90+/-0.25 ml/min/g wt, respectively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56+/-0.11 nmol/min/g wt at 40 cm H2O to 1.45+/-0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42% at 50 cm H2O to 12% at 90 cm H2O, respectively) with no significant changes in nitrite outflow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34% at 50 cm H2O to 50% at 90 cm H2O, respectively) with parallel changes in nitrite outflow (from 40% at 50 cm H2O to 51% at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective LOX inhibitor (NDGA) decreased control values of CF only at lower values of CPP but did not change nitrite outflow indicating antioxidant properties of NDGA. In addition, L-NAME decreased the effects induced by NDGA on CF and nitrite outflow indicating the role of NO.     

Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes

Fluorotelomer alcohols (FTOHs; CF(3)(CF(2))(x)C(2)H(4)OH; where x=3, 5, 7, 9) are a novel class of polyfluorinated contaminants, recently detected in the North American atmosphere, that are possible precursors to the series of perfluoroalkyl carboxylates (PFCAs) in human blood. An in vivo rat study validated earlier independent work that poly- and per-fluoroalkyl carboxylates were metabolites of FTOHs, but our detection of several novel metabolites prompted us to examine their pathways in greater detail using isolated rat hepatocytes. Using 8:2 FTOH (i.e. where x=7) as a model compound, the metabolic products formed by isolated rat hepatocytes were identified, and three synthesized intermediates were incubated separately to elucidate the metabolic pathways. For 8:2 FTOH, a major fate was direct conjugation to form the O-glucuronide and O-sulfate. Using 2,4-dinitrophenylhydrazine (DNPH) trapping, the immediate oxidation product of 8:2 FTOH was identified as 8:2 fluorotelomer aldehyde (8:2 FTAL; CF(3)(CF(2))(7)CH(2)C(H)O). 8:2 FTAL was transient and eliminated HF non-enzymatically to yield 8:2 fluorotelomer alpha,beta-unsaturated aldehyde (8:2 FTUAL; CF(3)(CF(2))(6)CFCHC(H)O) which was also short-lived and reacted GSH and perhaps other endogenous nucleophiles. Four polyfluorinated acid intermediates were also detected, including 8:2 fluorotelomer carboxylate (8:2 FTCA; CF(3)(CF(2))(7)CH(2)C(O)O(-)), 8:2 fluorotelomer alpha,beta-unsaturated carboxylate (8:2 FTUCA; CF(3)(CF(2))(6)CFCHC(O)O(-)), tetrahydroperfluorodecanoate (CF(3)(CF(2))(6)(CH(2))(2)CO(2)(-)), and dihydroperfluorodecenoate (CF(3)(CF(2))(6)CHCHCO(2)(-)). The pathways leading to 8:2 FTCA and FTUCA involve oxidation of 8:2 FTAL, however, the pathways leading to the latter two polyfluorinated acids remain inconclusive. The fate of the unsaturated metabolites, 8:2 FTUAL and FTUCA, included conjugation with GSH and dehydrofluorination to yield alpha,beta-unsaturated GSH conjugates, and GS-8:2 FTUAL which was subsequently reduced to the corresponding alcohol. Perfluorooctanoate (PFOA) and minor amounts of perfluorononanoate (PFNA) were confirmed as metabolites of 8:2 FTOH, and the respective roles of beta- and alpha-oxidation mechanisms are discussed. The analogous acids, aldehydes, and conjugated metabolites of 4:2, 6:2, and 10:2 FTOH (i.e. where x=3, 5, and 9, respectively) were also detected, and metabolite profiles among FTOHs generally differed only in the length of their perfluoroalkyl chains. Preincubation with aminobenzotriazole, but not pyrazole, inhibited the formation of metabolites from all FTOHs, suggesting that their oxidation was catalyzed by P450, not alcohol dehydrogenase.     

Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P.

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.     

Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene

The glyceraldehyde-3-phosphate dehydrogenase promoter, P(GAP), was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha. A set of integration vectors containing the HSA cDNA under the control of P(GAP) was constructed and the elemental parameters affecting the expression of HSA from P(GAP) were analyzed. The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P(GAP). Glycerol supported a higher level of HSA expression from P(GAP) along with a higher cell density than either glucose or methanol. The growth at high glycerol concentrations up to 12% did not cause any significant repression of the cell growth. A high cell density culture, up to 83 g l(-1) dry cell weight with a HSA production of 550 mg l(-1), was obtained in less than 32 h of cultivation in a fed-batch fermentation employing intermittent feeding with 12% glycerol. The GAP promoter-based HSA expression system showed a higher specific production rate and required a much simpler fermentation process than the MOX promoter-based system, demonstrating that P(GAP) can be a practical alternative of the MOX promoter in the large-scale production of HSA from H. polymorpha.     

Binding to human serum albumin of zidovudine (AZT) and novel AZT derivatives. Experimental and theoretical analyses

This work presents the binding of AZT and nine novel AZT derivatives to human serum albumin (HSA), both defatted (HSA(D)) and complexed with fatty acids (HSA(FA)). The bound fractions and binding site were determined by applying an ultrafiltration procedure, with an increased affinity for the majority of these derivatives to HSA(D) being found with respect to that of AZT, while only one derivative exhibited an increased affinity for HSA(FA). By means of computational methods, we observed that specific electrostatic interactions are responsible for the increased affinity for HSA(D), while the presence of fatty acids complexed to HSA caused an intense electrostatic repulsion with negatively charged ligands located in Sudlow site I, thus diminishing their bound fractions. A strong relationship between the calculated energetic components and the observed experimental affinity was identified.     

Release of the recombinant proteins, human serum albumin, beta-glucuronidase, glycoprotein B from human cytomegalovirus, and green fluorescent protein, in root exudates from transgenic tobacco and their effects on microbes and enzymatic activities in soil.

We determined the release in root exudates of human serum albumin (HSA), beta-glucuronidase (GUS), glycoprotein B (gB) from human cytomegalovirus, and green fluorescent protein (GFP) from genetically modified transgenic tobacco expressing the genes for these proteins in hydroponic culture and non-sterile soil. GUS, gB, and GFP were expressed in the plant but were not released in root exudates, whereas HSA was both expressed in the plant and released in root exudates, as shown by a 66.5-kDa band on SDS-PAGE and Western blot and confirmed by ELISA. Root exudates from GUS and gB plants showed no bands that could be attributed to these proteins on SDS-PAGE, and root exudates from GFP plants showed no fluorescence. The concentration of HSA in root exudates was estimated to be 0.021 ng ml(-1), whereas that in the plant biomass was estimated to be 0.087 ng ml(-1). The concentration of HSA in soil was estimated to be 0.049 ng g(-1). No significant differences in the number of microorganisms and the activity of selected enzymes were observed between rhizosphere soil of non-modified and HSA tobacco.     

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 O(2)(?-), 68% and 61% against H(2)O(2), 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.     

Polarographic direct determination and homogeneous immunoassay of anti-human serum albumin (HSA) by parallel catalytic hydrogen wave of anti-HSA or HSA

Human serum albumin (HSA) or anti-human serum albumin (anti-HSA) yields a catalytic hydrogen wave at about -1.85V (vs Ag/AgCl) in 0.25M NH(3).H(2)O-NH(4)Cl (pH 8.58) buffer. When 1.0 x 10(-2)M K(2)S(2)O(8) is present, the catalytic hydrogen wave is further catalyzed, producing a parallel catalytic wave of hydrogen as catalyst in nature, termed the parallel catalytic hydrogen wave. The sensitivity of the parallel catalytic hydrogen wave is higher by two orders of magnitude than that of the catalytic hydrogen wave. Using the parallel catalytic hydrogen wave of anti-HSA or HSA in the presence of K(2)S(2)O(8), two sensitive methods for the determination of anti-HSA were developed. One is a direct determination based on the parallel catalytic hydrogen wave of anti-HAS itself, and the other is a homogeneous immunoassay based on measuring the decrease of the peak current of the parallel catalytic hydrogen wave of HSA after homogeneous immunoreaction of HSA with anti-HSA. In the direct determination, the second-order derivative peak current of the parallel catalytic hydrogen wave of anti-HSA itself is rectilinear to its titer in the range from 1:1.0 x 10(7) to 1:8.4 x 10(6). In the homogeneous immunoassay, the decrease in the second-order derivative peak current of the parallel catalytic hydrogen wave of HSA is linearly related to the added anti-HSA in the titer range from 1:3.0 x 10(7) to 1:6.0 x 10(6). These assays are highly sensitive and rapid in operation and can be used to evaluate such antigens and their antibodies as those that could yield the parallel catalytic hydrogen wave.