Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods

Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.     

Interaction of phosphatidylcholine with bovine serum albumin. Specificity and properties of the complexes

Phosphatidylcholine dispersed on Celite was rapidly solubilized by neutral bovine serum albumin solutions. Stable protein-lipid complexes were isolated by Agrose gel filtration or by ultracentrifugal flotation in high density solvents, and the physicochemical properties of the complexes were investigated in terms of the stoichiometry of binding, effect of fatty acid ligands on phosphatidylcholine binding, effect of high ionic strength on the stability of the complexes, intrinsic fluorescence and circular dichroism spectra, and sedimentation velocity coefficients. Complexes containing from 2 to 30 phosphatidylcholine molecules per protein molecule were observed; however, no saturation of binding sites could be detected in this range of molar ratios. Oleic acid binding by serum albumin prevents interaction of the protein with phosphatidylcholine, indicating possible competition of these ligands at low contents of the phospholipid. For molar ratios of up to 10 phosphatidylcholine molecules per serum albumin, binding is primarily due to hydrophobic interactions that have no effect on the overall shape and secondary structure of the native protein except for local modifications at tryptophan residues, whose fluorescence becomes quenched and blue shifted on phosphatidylcholine binding. Similar phosphatidylcholine uptake experiments performed with a series of globular proteins indicated that the lipid extraction from Celite surfaces is a non-specific process, accelerated by several other proteins (e.g. aldolase, egg albumin, chymotrypsinogen, soybean trypsin inhibitor, and the major apolipoprotein from bovine serum high density lipoprotein). Formation of stable protein-lipid complexes, however, was only observed with bovine serum albumin, which in contrast to the other proteins is known to have affinity binding sites for anions with hydrophobic side chains.     

Kinetics of peroxynitrite reaction with amino acids and human serum albumin

An initial rate approach was used to study the reaction of peroxynitrite with human serum albumin (HSA) through stopped-flow spectrophotometry. At pH 7.4 and 37 degreesC, the second order rate constant for peroxynitrite reaction with HSA was 9.7 +/- 1.1 x 10(3) M-1 s-1. The rate constants for sulfhydryl-blocked HSA and for the single sulfhydryl were 5.9 +/- 0.3 and 3.8 +/- 0.8 x 10(3) M-1 s-1, respectively. The corresponding values for bovine serum albumin were also determined. The reactivity of sulfhydryl-blocked HSA increased at acidic pH, whereas plots of the rate constant with the sulfhydryl versus pH were bell-shaped. The kinetics of peroxynitrite reaction with all free L-amino acids were determined under pseudo-first order conditions. The most reactive amino acids were cysteine, methionine, and tryptophan. Histidine, leucine, and phenylalanine (and by extension tyrosine) did not affect peroxynitrite decay rate, whereas for the remaining amino acids plots of kobs versus concentration were hyperbolic. The sum of the contributions of the constituent amino acids of the protein to HSA reactivity was comparable to the experimentally determined rate constant, where cysteine and methionine (seven residues in 585) accounted for an estimated 65% of the reactivity. Nitration of aromatic amino acids occurred in HSA following peroxynitrite reaction, with nitration of sulfhydryl-blocked HSA 2-fold higher than native HSA. Carbon dioxide accelerated peroxynitrite decomposition, enhanced aromatic amino acid nitration, and partially inhibited sulfhydryl oxidation of HSA. Nitration in the presence of carbon dioxide increased when the sulfhydryl was blocked. Thus, cysteine 34 was a preferential target of peroxynitrite both in the presence and in the absence of carbon dioxide.     

Effect of temperature on binding of warfarin by human serum albumin

The in vitro binding of warfarin by human serum albumin was studied at various temperatures and at pH 7.4 by a frontal gel filtration technique. The results can be best described in terms of a two class-of-binding site model, in which the numbers of primary and secondary sites are constrained to the average values for all experiments (n1 = 1.38 and n2 = 3.73). Analysis of the temperature dependence of the binding yielded the following thermodynamic parameters: deltaH1 =-2.55 kcal/mole, deltaS1=16.1 eu, and deltaF1=-7.34 kcal/mole for the primary binding and deltaH2=-5.08 kcal/mole, deltaS2=-1.10 eu, and deltaF2=4.72 kcal/mole for the secondary binding. Calculations based on these results showed that, for the therapeutic concentration range, warfarin was over 99% bound to albumin present in physiological concentration. These findings are compared and contrasted to binding data in the literature for warfarin and salicylate.     

Structural characterization with circular dichroism of native and modified human serum albumin

OBJECTIVE: Extragondal germ cell tumors are rare and have a high affinity for the midline structures. Herein we describe a young, male patient with a retroperitoneal primary tumor. METHODS/RESULTS: The unorthodox form of presentation of germ cell tumors corroborates their embryologic gonadal origin which is distant from the site of presentation. CONCLUSION: The diagnostic algorithm, chemotherapeutic and surgical treatment guidelines utilized in the management of primary gonadal germ cell tumors with retroperitoneal involvement remain applicable in this rare form of presentation.     

Interactions of prostaglandin H2 and thromboxane A2 with human serum albumin

The present report describes the interactions of human plasma proteins with the unstable endoperoxide, prostaglandin H2 and thromboxane A2, generated by incubation of platelets with prostaglandin H2 or arachidonic acid. It was found that both compounds reacted very rapidly with plasma proteins to form covalently bound derivatives. The major reacting plasma protein was human serum albumin. Depending on conditions, 20-40% of added prostaglandin H2 and 50-80% of generated thromboxane were bound to proteins. This reaction of both prostaglandin H2 and thromboxane A2 prevents their detection by classical analytical methods. The protein binding of thromboxane was more pH-sensitive than the binding of prostaglandin H2. The reactions cause reduced levels of both endoperoxide and thromboxane B2 in suspensions of washed platelets using human serum albumin as compared to buffer. It was also shown that the half-life of prostaglandin H2 was considerably reduced in the presence of albumin.     

The fluorescence decay of human serum albumin and its subfractions

Human serum albumin does not decay monoexponentially although it contains a single tryptophan residue per molecule. The molecular population is thus heterogeneous with respect to the tryptophan emission. The separated monomeric and dimeric molecules of this protein, as well as various fractions isolated by the procedures of Foster and his coworkers, exhibit deviations from monoexponential decay which are comparable to those of the unfractionated protein; thus, the heterogeneity in molecular population of human serum albumin persists in the various fractions. By comparing the fluorescence decay data of this protein in the presence of thyroxine with the corresponding quenching data it was found that the fluorescence of the protein does not respond uniformly to the binding for all protein molecules. Qualitatively similar behavior was found for bovine serum albumin. In view of the above, binding studies followed by fluorescence should be viewed as averages over a heterogeneous population of the molecules of the serum albumin.     

Dynamic dialysis method. III. Effect of sulfonamides on the binding of warfarin with bovine serum albumin

Enflurane, a fluorinated methylethyl ether, is metabolized, in part, to inorganic fluoride. Methoxyflurane has similar metabolism, and cases of fluoride ion-induced renal failure have been reported after its use. This prospective study was initiated to determine fluoride ion kinetics after enflurance anesthesia in 16 healthy patients, 18 anephric patients, and 6 patients each having a creatinine clearance of less than 5 ml/min (on dialysis). Serum and urine inorganic fluoride levels were determined. There was no clinical or statistical significance difference among the 3 groups with respect to maximum inorganic fluoride ion concentration or the time to reach it. The fluoride ion values were never above the 50 muM level that has been reported to cause subclinical renal toxicity. The fluoride ion concentration in serum fell rapidly after termination of anesthesia even in the anephric patients. This is presumed to be due to uptake of the ion by bone. Patients with low creatinine clearance also have low fluoride ion clearance. Statistical but not clinical significance was found in the comparison between pre-enflurane and the 24-hr fluoride ion values in the anephric and low creatine clearance patients, but this did not persist after one dialysis.     

Solvation of human serum albumin in aqueous alkylurea solutions

In order to list the negative emergency laparotomies, the records of 24,494 laparotomies performed from 1950 to 1989 were examined. 211 negative laparotomies were performed over this 40 years period: 49 for abdominal trauma, 42 for supposed intestinal obstruction, 44 for supposed peritonitis or visceral infection, 46 for presumed early post-operative abdominal complications and 30 for gastrointestinal bleeding. Over these 4 decades, the emergency laparotomy rate and negative laparotomy rate remained stable despite changes in the diagnostic tools, in the age of the patients and the frequency of their diseases.     

The decline in serum albumin after conversion to high flux, high efficiency dialysis

In previous works using cytofluorometry, we demonstrated a broad range of IgE and IgE-receptor levels within individual mast cell populations with a 60 to 80% occupancy of the IgE receptors on mast cells by native IgE. This study was performed in order to confirm our previous findings using an independent method and to visualize the distribution of IgE-receptor complexes on mast cells at an ultrastructural level. For this purpose an indirect immunocolloidal gold-labelling technique has been applied. By counting the number of labelled gold particles, a relative measure of IgE-receptor surface expression and IgE occupancy of the receptors could be obtained. With respect to mast cell morphology and anti-IgE binding specificity criteria, 1% glutaraldehyde + 4% paraformaldehyde (1:1, vol/vol) was found to be the best of the seven fixatives applied in this study. This technique revealed numerous gold particles on the surface of mast cells from barrier-maintained rats (26 +/- 11 per mast cell section, mean +/- SD). Increased numbers of gold particles were counted if the mast cells were incubated with rat myeloma IgE (20 micrograms/ml) (46 +/- 33 per mast cell section, mean +/- SD). There were significantly increased numbers of gold particles on the mast cells of rats infected with N. brasiliensis (126 +/- 30 per mast cell section, mean +/- SD). This indicates that some of the IgE receptors (about 50% of the total number of IgE receptors in this case) on mast cells were occupied by native IgE and that parasite infection significantly increased the number of IgE molecules on the surface of the mast cells. These results correspond with the findings we have made using the cytofluorometric technique and confirm the large individual variations in the density of IgE receptors and IgE among the mast cells of a given cell population. Macrophages, lymphocytes and eosinophils, carrying the low-affinity IgE receptors (Fc epsilon RII), contained less than 5 (normal rats after incubation in rat IgE) or 10 (nematode-infected rats) gold particles per cell section. We also observed some non-granulated lymphocyte-like cells which bound a large number of gold particles after incubation with rat myeloma IgE (20 micrograms/ml), indicating that they contained IgE receptors Fc epsilon RI). They were interpreted as mast cell precursors which have previously been shown to exist in the peritoneal cavity.     

Calcium ion binding of three different types of oligo/polysialic acids as studied by equilibrium dialysis and circular dichroic methods

Ca2+ binding properties of three different types of oligo/polysialic acid chains, i.e., oligo/poly(Neu5Ac), oligo/poly(Neu5Gc), and oligo/poly(KDN), were studied by equilibrium dialysis and circular dichroism. Colominic acid, high molecular weight polysialoglycoprotein (H-PSGP), low molecular weight polysialoglycoprotein (L-PSGP), and 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN) glycoprotein were found to bind calcium ions with about 8-100 times the affinity of sialic acid monomer. Analysis by equilibrium dialysis of the binding of Ca2+ to colominic acid was biphasic, and the high-affinity interaction was shown to change with the degree of polymerization. Specific binding of Ca2+ to polysialic acid (polySia) caused characteristic effects in the circular dichroism spectrum. A pronounced decrease in the circular dichroism of polySia at 205 nm was observed upon addition of calcium. H-PSGP was found to bind calcium ions with 3-fold higher affinity than L-PSGP.     

Interaction of NADPH-adrenodoxin reductase with NADP+ as studied by pulse radiolysis

The reduction of flavin in NADH--adrenodoxin reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced directly the flavin of the reductase to form a blue semiquinone of the enzyme. Subsequently, the semiquinone decayed by dismutation to form the oxidized and fully reduced forms of the enzyme with a second-order rate constant of 4.4 x 10(4) M-1 s-1. In the presence of equimolar NADP+, the decay of eaq- accompanied an absorption increase at 400 nm, the spectrum of which, formed transiently, is identical to that of NADP radical (NADP.). Subsequently, the transient species decayed concomitantly with the formation of the semiquinone. The rate constant in the formation of the semiquinone was independent of the concentration of the enzyme (6.1 x 10(4) s-1 at pH 7.5). From these results, it is concluded that eaq- reacts with NADP+ bound to the enzyme to form NADP. initially, and subsequently, an electron flows from the NADP. to the flavin by an intracomplex electron transfer. A similar result was obtained in the reaction of CO2- or N-methylnicotinamide radical with the NADP(+)-adrenodoxin reductase complex. These results suggest that the nicotinamide moiety of NADP+ bound to the enzyme is accessible to the solvent and masks the flavin completely.     

Evidence of a strong interaction of 2,4-dichlorophenoxyacetic acid herbicide with human serum albumin

The interaction of 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) with human serum albumin (HSA) was studied using fluorescence and differential scanning calorimetry (DSC). Fluorescence displacement of 1-anilino-8-naphtalenesulfonate (ANS) bound to HSA was used to evaluate the binding affinity of 2,4-D to HSA. The binding is associated to a high affinity site of HSA located in the IIIA subdomain. The association constant (Kass) of the herbicide was about 5 microM(-1), several times higher than the affinity found for pharmaceutical compounds. This relatively strong interaction with HSA was evidenced by the increase in HSA protein thermostability induced as consequence of herbicide interaction. 2,4-D induces an increase in the midpoint of thermal denaturation temperature from 60.1 degrees C in herbicide free solution to 75.6 degrees C in full ligand saturating condition. The calorimetric enthalpy and the excess heat capacity also increased upon 2,4-D binding. To investigate the possibility of other/s system/s of 2,4-D transport in blood, besides of HSA, the interaction of the herbicide with lipid monolayers was explored. No interaction was detected with any of the lipids tested. The overall results provided evidence that high affinity 2,4-D-HSA complex exhibits enhanced thermal stability and that HSA is the unique transport system for 2,4-D in blood.     

Effect of chain length on interactions of aliphatic alcohols with suspended human serum albumin

Enthalpy changes on the immersion of human serum albumin (HSA) into n-butanol, n-propanol, ethanol and methanol containing different amounts of water have been measured calorimetrically at 25 degrees C. Water sorption isotherms on HSA were also determined in water-n-butanol and water-ethanol mixtures. From comparison of the calorimetric and sorption data, it was concluded that there is a significant enthalpy change on the HSA immersion into methanol and ethanol even under conditions where there is no change in the quantity of adsorbed water. No similar contribution was found in the n-butanol based suspensions. Water monolayer capacity evaluated from the Langmuir model decreases also significantly when going from ethanol to n-butanol. Considering this non water sorption contribution, values of the monolayer capacity and the shape of the experimental dependences, it was inferred that a relatively small change of the solvent molecule structure (from n-propanol to ethanol) affects strongly the interactions of the protein with the solvent.     

Association equilibrium of d-methamphetamine and l-methamphetamine with serum albumin

The association equilibria for complex formation between serum albumin (bovine, rat) and the optical isomers of methamphetamine (MAMP) was determined using an ultrafiltration method. It was found that serum albumin/d-MAMP and serum albumin/l-MAMP complexes had distinctly different Scatchard plots with bovine and rat albumin. The binding parameters of each association equilibrium were estimated from the Scatchard plots by Rosenthal's graphic method. This distinguished two kinds of specific binding sites in terms of the association equilibrium between bovine serum albumin and d-MAMP, and one binding site for rat serum albumin and d-MAMP. One specific binding site was found between serum albumin and l-MAMP in both bovine and rat. Molar ellipticities, [theta], of peaks were decreased in the CD spectra of the complexes formed between bovine serum albumin and d-MAMP or l-MAMP when compared with the CD spectrum of bovine serum albumin alone. However, no difference in [theta] was found between the CD spectra of the enantiomers of MAMP in the measured wavelength range. The non-specific binding site was distinct from the specific binding site and resulting from altered tertiary structure of the albumin molecule.