Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments

The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity. Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion). Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase. In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates. Consequently two variants were designed and constructed using site-directed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones. Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability. After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank. A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.     

125I labelling of human serum albumin and fibrinogen and a study of protein adsorption properties on the surface of titanium oxide film

In order to detect the surface concentration of proteins adsorbed on a solid surface for selecting blood compatible materials, a gentle iodination reagent, Iodogen, was used to label human serum albumin and fibrinogen, and has been applied to the study of protein adsorption properties on a plate of titanium oxide film. The yields of the labelled albumin and fibrinogen are 69.7% and 49.6%. The results of adsorption show that [125I]HSA and [125I]HFG are efficacious at the surface concentration detection and can be used to investigate the protein adsorption properties of a solid material.     

Rigidity of human alpha-fetoprotein tertiary structure is under ligand control

Comparative study of the natural ligand effect on structural properties and conformational stability of human alpha-fetoprotein (AFP) and its homologue, human serum albumin (HSA), was performed using several approaches, including circular dichroism, fluorescence spectroscopy, and scanning microcalorimetry. Here we show that denaturation of AFP, induced by the increase of temperature or urea concentration, is irreversible. We have established the fact that this irreversibility is caused by ligand release from the AFP molecule. Interestingly, the ligand-free form of AFP has no rigid tertiary structure but exhibits substantial secondary structure and high compactness. This means that the rigid tertiary structure of AFP is controlled by interaction with ligands, while their release results in transition of a protein molecule into a molten globule-like intermediate. In contrast, processes of HSA denaturation and unfolding are completely reversible. Release of ligands from HSA results only in a small decrease in stability but not transformation into the molten globule state.     

Stereoselective irreversible binding of ketoprofen glucuronides to albumin. Characterization of the site and the mechanism

We have previously shown that the acyl glucuronide of racemic ketoprofen can irreversibly bind in vitro to plasma proteins (Dubois, N., et ai., Drug Metab. Dispos. 21, 617-623, 1993), but the mechanism of the reaction has not been characterized. In the present study, the reactivity toward albumin of the glucuronide of both ketoprofen enantiomers was investigated. The extent of binding increased with the concentration of both protein and glucuronide. However, the two diastereoisomers showed different reactivities toward human serum albumin (HSA): the maximum yield of adducts with the glucuronide of the S-enantiomer was twice that obtained with the glucuronide of its antipode. The maximum extent of irreversible binding was at 4 hr for the R-ketoprofen conjugate, but was later for the S-form. Chemical modifications of albumin indicated that the glucuronide of the S-isomer reacted only with lysine residues, whereas the R-form linked covalently mainly with tyrosine residues and secondarily with lysine residues. A competition study using specific binding probes and fatty acids showed that the conjugates of S- and R-ketoprofen reacted with amino acids located in sites I and II of HSA, respectively. Taken together, these findings suggest that the irreversible binding of ketoprofen to albumin depends on the stereochemistry of the aglycon: the R-enantiomer binds to site II of the protein probably by a nucleophilic attack by tyrosine and/or lysine residues, whereas adduct formation via the conjugate of the S-enantiomer could occur at site I of HSA by the Schiff base mechanism. This irreversible binding at sites I and II may affect the major function of albumin (i.e. the transport of drugs and endogenous compounds).     

Reproductive hormonal responses to ergotamine and ergonovine in cows during the luteal phase of the estrous cycle

We report the abnormal albumin in members of a Thai family that presented with high serum total T3 but not T4 when measured by radioimmunoassay. In contrast, total T3 values were very low when measured by ELISA and chemiluminescence. The subjects have no goiter, and clinically euthyroid. Their serum free T4, free T3, and TSH were normal. Spiking of T3 to affected serum showed good recovery by radioimmunoassay, but very poor recovery by ELISA and by chemiluminescence. The immunoprecipitation with labeled T3 bound to albumin showed high percent precipitation in affected serum. T3-binding studies showed that the association constant of serum albumin in affected subjects was 1.5 x 10(6) M-1 or 40-fold that of unaffected relatives of 3.9 x 10(4) M-1. In contrast, the Ka of HSA for T4 in an affected subject was only 1.5-fold that of a normal. Albumin complementary DNA from leukocytes of affected member was amplified and sequenced. We found the second nucleotide of normal codon 66 (CTT), a thymine, was substituted by a cytosine (CCT), resulting in the replacement of the normal leucine by proline. This is the first report of variant albumin causing familial dysalbuminemic hypertriiodothyroninemia.     

Expression of recombinant psoriasis-associated fatty acid binding protein in Escherichia coli: gel electrophoretic characterization, analysis of binding properties and comparison with human serum albumin

The psoriasis-associated fatty acid binding protein (PA-FABP, also known as FABP5) is a novel keratinocyte protein that is highly up-regulated in psoriatic plaques (P. Madsen, H. H. Rasmussen, H. Leffers, B. Honoré and J. E. Celis, J. Invest. Dermatol. 1992, 99, 299-305). Here we have expressed PA-FABP in Escherichia coli as a fusion protein containing an NH2-terminal hexa-His tag followed by a factor Xa cleavage site. The recombinant protein was expressed at a level of about 30% of the soluble proteins and was purified to homogeneity using a simple two-step protocol consisting of affinity chromatography on Ni2+-nitrilotriacetic acid agarose followed by gel filtration. The recombinant protein was then digested with factor Xa and characterized by two-dimensional gel electrophoresis. The ability of PA-FABP to bind saturated fatty acids ranging from 6 to 16 carbons was determined directly by dialysis and compared to human serum albumin (HSA). The results showed that PA-FABP binds multiple molecules of the fatty acids hexanoate (C6:0), octanoate (C8:0), decanoate (C10:0) and laurate (C12:0), all with a K1 of about 10(4) M(-l), and myristate (C14:0) with a K1 of 4.4 X 10(5) M(-l). Palmitate (C16:0) also bound strongly with multiple molecules. Due to the very low solubility of palmitate its affinity to PA-FABP was measured relatively to HSA and found to be 8.1 times lower. At ligand/protein ratios below 1, all fatty acids bound to PA-FABP with about one to three orders of magnitude lower affinity than to HSA. The difference in the fatty acid binding properties of the two proteins may reflect differences in their three-dimensional structures, which in the case of PA-FABP consists mainly of beta-sheets while HSA contains predominantly alpha-helices.     

Paradox as symptom in the borderline patient''s struggle for self-differentiation

A dramatic uncoupling of the expression of chimaeric beta-lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation, both HSA RNA and protein were expressed at high levels. However, when the explants were grown in hormone-free medium. HSA RNA continued to accumulate, whereas the synthesis of the corresponding protein was dependent on the presence of insulin and prolactin with a minor contribution of hydrocortisone. The untranslated HSA RNA was indistinguishable from its translatable counterpart in its mobility on agarose gels, was transported normally from the nucleus to the cytoplasm and was translated efficiently in rabbit reticulocyte lysate. In the presence of cycloheximide, HSA RNA rapidly disappeared suggesting a dependency on ongoing protein synthesis. Its estimated half-life of 5-6 h in hormone-free medium increased significantly in the presence of insulin, hydrocortisone and prolactin and was comparable to that of beta-casein RNA. The uncoupling of the expression of the BLG/HSA transgenes at the RNA and protein levels was also confirmed by in situ hybridization and immunohystochemistry on sections from virgin mammary explants. HSA synthesis was initiated within 13 h of the addition of insulin and prolactin in explants that had accumulated untranslated HSA RNA and was fourfold higher than that observed with insulin alone. Addition of hydrocortisone contributed to an additional 20% in HSA synthesis. We believe this is the first demonstration of translational control of exogenous milk protein gene expression in the mammary gland of transgenic animals.     

Localization of bacterial DNA polymerase: evidence for a factory model of replication

Two general models have been proposed for DNA replication. In one model, DNA polymerase moves along the DNA (like a train on a track); in the other model, the polymerase is stationary (like a factory), and DNA is pulled through. To distinguish between these models, we visualized DNA polymerase of the bacterium Bacillus subtilis in living cells by the creation of a fusion protein containing the catalytic subunit (PolC) and green fluorescent protein (GFP). PolC-GFP was localized at discrete intracellular positions, predominantly at or near midcell, rather than being distributed randomly. These results suggest that the polymerase is anchored in place and thus support the model in which the DNA template moves through the polymerase.     

H-NMR and CD studies on the structural transition of serum albumin in the acidic region--the N-->F transition

Helical content (f(alpha)) of bovine mercaptalbumin (BMA) showed the characteristic two-step decrease in the acidic region, one corresponding to the N-->F transition (pH 4.40-->3.75; f(alpha), 0.68-->0.58) and the other to the F-->E transition (the acid-expansion) (pH 3.60-->2.90; falpha, 0.58-->0.48). However, falpha of human serum albumin (HSA) mainly decreased in the N-->F transition (N-->F, pH 4.6-->3.4; falpha, 0.70-->-0.55 and F-->E, below pH 3.0; falpha, 0.55-->0.52). The difference in pH-profile of f(alpha) between BMA and HSA might be due to the microheterogeneity. The 1H-NMR spectra and cross-relaxation times (T(IS)) from irradiated to observed protein protons, which reflect the structural fluctuation and/or mobilability in proteins, were measured on the N-, F-, E-forms of HSA and BMA, and the N*-form (8.23 M urea, neutral pD) of iodoacetamide-blocked HSA (IA-HSA) and bovine serum albumin (IA-BSA). The 1H-NMR spectra and elongations of T(IS) values for the F- and E-forms of HSA and the E-form of BMA were quite similar to those for the N*-form of IA-HSA and IA-BSA, indicating the liberation of the intramolecular motion in the F- and E-forms. Those for the F-form of BMA were intermediate between the N- and E-form. The present results together with the reported data on hydrodynamic radii and D-H exchange reaction, indicate that the F-form of HSA and presumably BMA has a native-like globule form with a highly helical state and fluctuating tertiary structure. Thus, all of the present findings on the F-form of serum albumin seem to be in accord with the structural features for the F-form suggested by Foster's group (1-3, 19, 20, 22, 23) and the molten globule state demonstrated by Dolgikh et al. (40), and Ohgushi and Wada (36, 37).     

Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin

In this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 +/- 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.     

Reversal of stereoselectivity in the hepatic availability of verapamil in isolated perfused rat livers. Role of protein binding

The effects of serum proteins on the stereoselective kinetics of the high clearance drug verapamil (VER) and its metabolite, norverapamil (NOR), were studied in isolated perfused rat livers (IPRLs). Livers were perfused, in a recirculating manner, with a solution containing human serum albumin (HSA), bovine serum albumin (BSA), or no serum albumin (N = 5 for each group). After presystemic administration of a single dose of racemic VER (2 mg), the concentrations of VER and NOR enantiomers in the perfusate were measured over 90 min. In addition, the fraction of the enantiomers bound to the plasma of perfusate was determined. Perfusate concentrations of both VER and NOR were stereoselective in all of the perfusates studied. However, the direction of stereoselectivity in the concentrations of VER enantiomers in the BSA perfusate (S-VER > R-VER) was opposite that in the HSA and albumin-free perfusates (R-VER > S-VER); this was associated with an opposite stereoselectivity in the concentrations of NOR in the BSA perfusate was higher than that in the HSA and albumin-free perfusates, an observation in agreement with the higher stereoselectivity in the binding of NOR to BSA. These data, along with other kinetic parameters such as apparent hepatic availability and intrinsic clearance, suggest that the apparent stereoselectivity in the presystemic elimination of VER by IPRLs is significantly influenced by the stereoselectivity in the protein binding of the drug.     

Structural specificity requirements in the binding of beta lactam antibiotics to human serum albumin

The binding of some cephalosporins of pharmacological interest, to human serum albumin was studied using ultrafiltration method. The identification of the binding sites in albumin was also performed using probes for the so-called sites I, II, bilirubin and fatty acids binding sites. Cephalosporins were classified into three groups according to their affinity for albumin: low affinity (K = 10-10(2) M-1), medium affinity (K = 10(3) M-1) and high affinity (K = 10(4) M-1). Cephalosporin binding to albumin produced a perturbation of several basic amino acids of the protein such as histidine and lysine. It was found that only cefuroxime, ceftazidime and cefoperazone interact slightly with site I on serum albumin, while site II possesses capacity to bind: cephradine, cephalexin, ceftazidime, ceftriaxone, cefoperazone, cefaclor and cefsulodin. The bilirubin binding site showed capacity to interact with a great number of cephalosporins: ceftriaxone, cefazolin, cephaloglycin, cefamandole, cefotaxime, cefoxitin, cefuroxime, cefoperazone and cefadroxil. Ceftriaxone showed capacity to bind to the fatty acid binding site on HSA. No relation was found between the displacement of the marker and the chemical nature of the substituents at R1 and R2. Cephalosporins interact with HSA at the binding region that involves: tyrosyl 411, histidyl 146 and lysyls 195, 199, 225, 240 and 525 residues. The chemical modification of specific amino acids showed that the interaction of these amino acids with beta lactam antibiotics is not carried out to the same extent for all the cephalosporins tested. The results obtained revealed that the binding sites for cephalosporins on albumin are structurally heterogeneous, having different amino acids in the vicinity of the ligand molecule.     

Human serum albumin as a probe for protein adsorption to nanoparticles: relevance to biodistribution

A range of poloxamers and poloxamines were adsorbed to biodegradable poly(lactide-co-glycolide) (PLGA) and non-biodegradable polystyrene (PS) particulate systems in order to alter their surface characteristics and produce potential drug targeting systems. Human serum albumin (HSA) was chosen as a model protein to investigate protein adsorption to the above systems and was quantified by two techniques. I125 radiolabelled HSA proved to be a useful probe for determining protein adsorption but was limited by a modification that occurred on storage. Also, HSA eluted from the particle surface was quantified by densitometry following it's development on an SDS-PAGE gel. Both techniques produced similar results. For cleaned coated PS particles it was found that the PEO chain length and the molecular structure of the block copolymer were important in preventing protein adsorption. The presence of excess block copolymer in the uncleaned preparations resulted in further suppression of HSA adsorption, which was thought to be due to their detergent properties. Due to the different results obtained with similarly coated PLGA particles, it was concluded that the block copolymers adsorb onto the surface of the PLGA particles in a different conformation to those adsorbed onto PS particles. Correlating in vivo biodistribution in terms of the prevention of protein (opsonin) adsorption was of only limited success and it was concluded that adsorption data for a single model protein can only be used with caution to predict the in vivo behaviour of colloidal targeting systems.     

Interactions between dansyl amino acids and human serum albumin using high-performance liquid chromatography: mobile-phase pH and temperature considerations

The reversed-phase liquid chromatography (RPLC) retention mechanism of a series of dansyl amino acids was investigated over a wide range of mobile-phase pH and column temperatures using human serum albumin (HSA) as a chiral stationary phase. Thermodynamic constants for the transfer of a solute from the mobile to the HSA stationary phases were determined. Different van't Hoff plot shapes were observed with different mobile-phase pH values, indicating a change in the retention mechanism. Enthalpy-entropy compensation revealed that the solute retention mechanism was independent of the compound molecular structure, the same at four pH values (5.5, 6, 6.5, and 8), but changed at pH = 7 and 7.5. Differential scanning calorimetry was used to show phase transition in the HSA stationary phase at pH = 7 and 7.5. A new theory was presented to explain that the HSA protein structure balance between a disordered and an ordered solid-like state. Variations of column temperature and mobile-phase pH tend to cause this phase transition between these two states, explaining the observed thermodynamic constant variations with pH and temperature.     

The persistence of leptospiral agglutinins titers in human sera diagnosed by the microscopic agglutination test

Human serum albumin (HSA) is used in large amounts as an excipient in many biopharmaceutical formulation to prevent loss of the active ingredient through adsorption and/or degradation. Traditionally, iso-electric focusing has been used to demonstrate charge heterogeneity in HSA preparations. In an effort to develop new methods for the analysis of formulation components, a capillary zone electrophoresis method was developed for the analysis of HSA. Under initial separation conditions using untreated silica capillaries and 20 mM sodium phosphate, pH 6.0 as electrophoretic buffer, HSA migrated as a single peak. Addition of 1,4-diaminobutane allowed separation of several components which could be further resolved by varying the buffer pH. Optimal separation conditions were attained at 5 mM 1,4-diaminobutane and pH 8.5. The reproducibility of the separation conditions was verified by using capillaries from a different manufacturer. A comparative analysis of HSA preparations from different manufacturers provided evidence that the method may be used to qualitatively differentiate individual preparations. The analysis of rhEPO formulations, composed largely of HSA, showed levels of heterogeneity comparable to that of HSA preparations. Electrospray ionisation mass spectrometry (ESA-MS) was used as an independent method to confirm the heterogeneous nature of HSA.     

Significant prognostic factors for 5-year survival after curative resection of renal cell carcinoma

The aim of this study was, (1) to characterize the serum protein binding of lerisetron, a new 5-hydroxytryptamine (5-HT3) receptor antagonist under investigation as an antiemetic agent, and (2) to measure the percentage of unbound lerisetron in cancer patients. The binding parameters were determined in human serum albumin (HSA), alpha1-acid glycoprotein (AAG) and in pooled serum from six healthy volunteers. Concentrations of lerisetron ranging from 50 ng/ml to 2 microg/ml were used. The serum protein binding of 14C-lerisetron (2 microg/ml) was determined by ultrafiltration in three groups of individuals. Group I comprised healthy subjects (n = 11), group II comprised cancer patients undergoing radiotherapy (n = 9), and group III comprised cancer patients receiving chemotherapy (n = 18). The unbound concentration of lerisetron was measured in all samples by liquid scintillation counting. Concentrations of both AAG and HSA were also measured in all serum samples. The drug was extensively bound in pooled serum, involving a nonsaturated process. In HSA, lerisetron was also highly bound (4.04+/-0.8% unbound) and the protein binding was essentially unchanged within the studied concentration range of lerisetron. The extent of binding to AAG was high but significantly lower than in serum and in HSA and was also independent of lerisetron concentration. The unbound lerisetron was significantly decreased in group II cancer patients when compared with group I subjects (2.38+/-0.64% vs 3.70+/-0.70%; P < 0.001). No significant changes in lerisetron binding were observed in group III patients. HSA was diminished in both groups of patients and AAG was only significantly increased in group II. Unbound lerisetron was correlated with AAG in group II and with HSA in group III.     

Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.     

Steroid binding to human serum albumin and fragments thereof. Role of protein conformation and fatty acid content

The binding properties of the steroids testosterone and pregnenolone to human serum albumin (HSA) and derived fragments of albumin have been investigated by means of equilibrium dialysis and circular dichroism. The 46 kDa peptic fragment (P46) of HSA comprises domains one and two of the HSA structure, whereas the other fragment, the 45 kDa tryptic fragment (T45) is composed of domains two and three. A comparison of the binding behaviour of the steroid ligands to HSA and its fragments showed that the single primary testosterone binding site in all probability is located in the second domain of the HSA molecule. For pregnenolone it was found that at least two primary binding sites are present, also located in domain two. Both steroids show pH dependent binding profiles in the case of HSA and the P46 fragment. The binding of the steroids to the T45 fragment seems to be pH independent. The same phenomenon was observed with the circular dichroism experiments, indicating a link between the steroid binding properties and the structural behaviour of the proteins. In fact, the binding properties of the steroids can be assigned to the neutral-to-base (N-B) transition. The possible role of fatty acids as modulators in the transport processes of steroids in the body is discussed.