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.     

Relationship between alcohol intake and immunoglobulin a immunoreactivity with acetaldehyde-modified bovine serum albumin

Acetaldehyde, the main metabolite of ethanol, is a highly reactive species that reacts with macromolecules to produce unstable and stable adducts. Acetaldehyde-modified proteins are immunogenic and have been detected in the liver and blood of alcoholics. Furthermore, antibodies reactive with acetaldehyde-modified proteins have been detected in the plasma of social drinkers and alcoholics. However, the class distribution of immunoglobulins reactive with modified proteins was different in the two groups, being predominantly immunoglobulin (Ig)M in social drinkers, but IgM and IgA in alcoholics. In this study, we demonstrate that heavy drinkers (alcohol intake > 130 g/week for females and 150 g/week for males) also exhibit IgA reactivity with acetaldehyde-modified proteins. The IgA adduct-specific reactivity (IgA reactivity with acetaldehyde-modified bovine serum albumin-reactivity with native bovine serum albumin) showed a moderate correlation with self-reported alcohol intake, but did not correlate with markers such as plasma transaminase, gamma-glutamyltransferase activity, or mean corpuscular volume. IgA adduct-specific reactivity had similar specificity to the conventional tests of alcohol abuse, but had higher sensitivity than the other tests, especially with heavy drinkers. Data presented herein demonstrate that elevated IgA reactivity with acetaldehyde-modified epitopes is associated with heavy drinking and is a potential marker for high alcohol intake.     

Heat treatment modifies the allergenicity of beef and bovine serum albumin

The effect of heat on the allergenicity of beef and bovine serum albumin was investigated among 10 toddlers skin prick test (SPT)-positive to raw and cooked beef. The meat-allergy diagnosis was confirmed during double-blind, placebo-controlled food challenge (DBPCFC) with 180 g of beef cooked for 5 min at 100 degrees C. SPT with homogenized and freeze-dried beef, and heated and unheated bovine serum albumin were performed. Both heated and unheated bovine serum albumin, homogenized beef, and freeze-dried beef were used in trial DBPCFC. All children were SPT-positive to unheated bovine serum albumin. Seven were positive to heated bovine serum albumin, one to freeze-dried beef, and none to homogenized beef. DBPCFCs were negative for homogenized beef and freeze-dried beef, positive for unheated bovine serum albumin in five patients, and positive for heated albumin in four children. We conclude that heating reduces sensitization to beef and bovine serum albumin but does not abolish reactivity to albumin under home conditions. However, industrially heat-treated and sterilized homogenized beef and freeze-dried beef may be suitable substitutes in beef-allergic children's diets.     

In-situ photochemical spectrofluorimetric detection of 9,10-anthraquinone-labeled bovine serum albumin

Bovine serum albumin (BSA) labeled with 9,10-anthraquinone (AQ) shows a greatly enhanced photochemical fluorometric activity compared with that of free AQ. The spectral characteristics of the photoreduction product of conjugated AQ was investigated and large blue shifts in the excitation and emission bands compared with those of free AQ were observed. The enhancement in the photochemical reactivity can be employed for sensitive detection of labeled BSA by a simple in-situ photochemical kinetic fluorimetric method. The kinetic behavior of the photochemical reaction and the effects of some experimental conditions were investigated. The calibration graph was linear over the range 0-1.8 x 10(-7) M BSA. The detection limit was 1.2 x 10(-10) M BSA and the relative standard deviation was 2.24% for the 1.42 x 10(-8) M BSA (n = 7).     

Pharmacokinetic analysis of hepatic uptake of galactosylated bovine serum albumin in a perfused rat liver

Hepatic uptake of 111In-labelled galactosylated bovine serum albumin (Gal-BSA) with different number of galactose residues per BSA were studied in rat liver perfusion experiments. During a single-pass constant infusion mode, [111In]Gal-BSAs (0.1-2.0 micrograms/ml) were continuously extracted by the liver and its extraction ratio at steady-state (Ess) was lowered as the inflow concentration increased. Hepatic clearance of [111In]Gal-BSAs increased significantly according to the increase in the number of galactose residues per BSA at an inflow concentration of 0.7 micrograms/ml. The outflow patterns of [111In]Gal-BSAs at various inflow concentrations were simultaneously fitted to a one-organ pharmacokinetic model, by which we can characterize their binding to the cell surface and internalization processes separately. The parameters obtained were varied significantly among [111In]Gal-BSAs depending on the number of galactose residues and indicate that not only the binding to the receptors but also the internalization after the binding are regulated by the number of galactose residues per BSA during hepatic uptake.     

NMR relaxation investigation of water mobility in aqueous bovine serum albumin solutions

T1 and T2 dispersion measurements of proton spin relaxation on aqueous solutions of bovine serum albumin as a function of both protein concentration and temperature, covering Larmor frequencies from 3 kHz to 75 MHz, will be presented as a most conclusive test for a three-phase fast-exchange relaxation model. By means of a computer curve fitting of this model to the experimental data, we were able to separate 3 distinct water environments and, in addition, the rotational tumbling of the bovine serum albumin molecules.     

SS-interchanged and oxidized isomers of bovine serum albumin separated by isoelectric focusing

Column isoelectric focusing separates commercial bovine serum albumin in 5 fractions with isoionic points in the vicinity of that of mercaptalbumin (pI 5.24). About 20% of the bovine albumin have isoionic points higher than mercaptalbumin and are split into two fractions, both recognized as SS-interchanges isomers: (1) pI 5.39 is the "aged" albumin described by Nikkel and Foster (1971, Biochemistry 10, 4479); (2) pI 5.45 represents a further degree of SS-interchange, catalyzed by small amounts of cysteine in the solution ('cysteine-aged' albumin). In 6 M urea the "cysteine-aged" albumin is electrofocused to the same pH value as mercaptalbumin. In 6 M urea 40% of commercial albumin focuses in 3 fractions with isoionic points lower than mercaptalbumin. This percentage will increase during incubation at oxidizing conditions ("oxidized" albumin). Electrofocused in water the oxidized fractions have isoionic points at pI 5.28, 5.18 and 5.12, respectively. The shifts in isoionic point of the "oxidized" albumins are caused by irreversible changes in the primary structure. Although the free SH group of albumin is oxidized during the oxidation reaction, the observed changes in isoionic points are caused by modifications of some other amino acid residues. Both "cysteine-ageing" and "oxidation" are inhibited by alkylation of the SH group. "Cysteine-ageing" is furthermore inhibited when the bovine albumin is "oxidized".     

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.     

Zinc(II) and copper(II) binding to serum albumin. A comparative study of dog, bovine, and human albumin

Metal binding strategies employing low molecular weight chelators and equilibrium dialysis were used to investigate several unresolved aspects of zinc and copper binding to serum albumin. Direct measurement of histidine binding to bovine serum albumin when the histidine is presented either as a metal-chelate or alone provides no evidence for an albumin-metal-histidine ternary complex. Using previously determined intrinsic constants for Zn(II) and Cu(II), we have measured zinc binding to bovine serum albumin in the presence of saturating amounts of copper. The results of these experiments unambiguously show that zinc and copper bind at separate noninteracting sites on this protein. The intrinsic constants for zinc and copper binding to dog serum albumin have been determined. Contrary to previous reports, we find that dog serum albumin has a specific high affinity site for copper, log10K 10.17 for Cu(II) compared to 6.85 for Zn(II) at the separate site.     

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.     

Genetic variants of human serum albumin in Italy: point mutants and a carboxyl-terminal variant

Of the > 50 different genetic variants of human serum albumin (alloalbumins) that have been characterized by amino acid or DNA sequence analysis, almost half have been identified in Italy through a long-term electrophoretic survey of serum. Previously we have reported structural studies of 11 Italian alloalbumins with point mutations, 2 different carboxyl-terminal variants, and 1 case of analbuminemia in an Italian family. This article describes confirmation by DNA sequencing of mutations previously inferred from protein sequencing of 4 of the above alloalbumins; it also reports the mutations identified by protein and DNA sequence analysis of 4 other Italian alloalbumins not previously recorded: albumin Larino, His3-->Tyr; Tradate-2 (protein sequencing only), Lys225-->Gln; Caserta, Lys276-->Asn; and Bazzano, a carboxyl-terminal variant. The first 3 have point mutations that produce a single amino acid substitution, but a nucleotide deletion causes a frameshift and an altered and truncated carboxyl-terminal sequence in albumin Bazzano. In these 4 instances the expression of the alloalbumin is variable, ranging from 10% to 70% of the total albumiN, in contrast to the usual 50% each for the normal and mutant albumin. The distribution of point mutations in the albumin gene is nonrandom; most of the 47 reported point substitutions involve charged amino acid residues on the surface of the molecule that are not concerned with ligand-binding sites.     

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.     

T cells from children with IDDM are sensitized to bovine serum albumin

Epidemiological and experimental evidence suggested that denial of dietary cow milk protein early in life protects genetically susceptible children and animals from insulin-dependent diabetes (IDDM). Bovine serum albumin (BSA) was proposed as a candidate milk-borne mimicry antigen responsible for the diabetogenic cow milk effect. Elevated anti-BSA antibodies have been observed in patients and diabetic rodents, and these antibodies precipitate p69 from islet cell lysates. IDDM is a T cell mediated disorder but efforts to detect BSA-specific T cells in diabetic children have so far failed. We describe here a culture system which allowed the detection of BSA-specific T cells and we mapped this response to the ABBOS peptide (pre-BSA position 152-169) previously identified as a possible mimicry epitope. ABBOS-sensitized T cells were found in 28/31 children with recent onset IDDM but not in non-diabetic controls nor in children with SLE or JRA. T cell proliferative responses declined within the first few years of diabetes diagnosis. Although no effector cell role for BSA/ABBOS specific T lymphocytes has been demonstrated, the presence of BSA peptide-specific T cells strengthens the postulated link between a cow milk protein and IDDM.     

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.     

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.     

Characterization of phospholipase A inhibition of stereospecific opiate binding and its reversal by bovine serum albumin

Opiate receptor binding is inhibited by phospholipase A from some sources, whereas the enzyme from other sources is inactive even at much higher concentrations. Evidence is presented that an active enzymatic site is required for inhibition and that the degree of inhibition seems to correlate with the extent of phospholipolysis. The inhibition is reversed almost completely by treatment with 0.5 to 1% bovine serum albumin, even up to 90% inhibition by phospholipase. As more enzyme is added or incubation time is increased, the extent of reversal diminishes. Based on our evidence, the most likely explanation of these results is that the inhibition of opiate binding activity by phospholipase A is due to the toxicity of the products of phospholipolysis and that bovine serum albumin reverses the inhibition by removing these products from the membranes, thereby restoring the active conformation of the receptors.     

Somatic cell count, electrical conductivity, and serum albumin concentration for detecting bovine mastitis

Cell concentration, electrical conductivity, and serum albumin concentration of milk were evaluated for predicting infection status of quarters in three herds. Probability of misclassifying quarters was lowest for cell concentration. For discriminating quarters infected with Staphylococcus aureus from quarters free from infection, probability of misclassification for cell concentration ranged from 8 to 20% among herds. For electrical conductivity probability of misclassification ranged between 22 and 32%, whereas for serum albumin the range among herds was from 15 to 48%. Among herds there was also considerable variation of the trait, which minimized the probability of misclassification. Cell concentrations for the three herds were 158, 200, and 251 x 10(3) cells/ml whereas for electrical conductivity counts were 4.6, 5.8, and 6.5 mS/cm. For electrical conductivity and serum albumin comparisons within cow by differences and ratios from different quarters yielded little advantage over absolute estimations. For a single sampling, cell concentration provided the most accurate prediction of quarter infection status. We suggest that 200 x 10(3) cells/ml should be the threshold above which quarters are considered likely to be infected with a major pathogen.