Characterization of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.     

Use of human albumin as an additional cryoprotective agent in freeze preservation of hematopoietic stem cells

14 bone-marrow samples of healthy donors were cryopreserved with 5% of DMSO in combination with different volume percentages of 20% human albumin, with the protective impact of the respective freezing mixture on the proliferating capacity of early hemotopoietic precursor cells being determined by means of CFU-c-technique. A combination of 5% of DMSO with 20% of human albumin with an addition of 20% of autologous serum proved to be the most favourable freezing protection. CFU-c-recovery which was achieved in this way amounted to 78 +/- 7%. A protective impact of human albumin for CFU-c during incubation with the freezing medium at room temperature was statistically not significant. Thus, the method presented is suitable for cryopreservation of bone marrow for autologous transplantation. The advantages over the traditional procedure results from the slight degree of toxicity by reducing the DMSO percentage from the common 10% to 5% as well as from the fact that the thawed cell suspension may be directly infused with the substance for freezing protection, thus avoiding an additional loss of proliferative material. The procedure may be used especially for autologous transplantation in childhood.     

Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin

Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.     

Interaction of an organic selenium compound with human serum albumin

The interaction between 4,4'-diselenadibenzoic acid and human serum albumin (HSA) was investigated by fluorescence and absorption spectroscopy. The quenching mechanism of fluorescence of HSA by 4,4'-diselenadibenzoic acid was discussed. It is proved that the fluorescence quenching of HSA by 4,4'-diselenadibenzoic acid is a result of the formation of the HSA-4,4'-diselenadibenzoic acid complex. The binding sites number n, apparent corporation constant K, and corresponding thermodynamic parameters, deltaH(theta), deltaG(theta), and deltaS(theta) were calculated. Results indicate that the electrostatic interactions forces played major role in the reaction.     

Partially folded state of the disulfide-reduced form of human serum albumin as an intermediate for reversible denaturation.

The conformation of the fully disulfide-reduced state of human serum albumin was investigated by tryptophan fluorescence spectrum, CD analyses, and size-exclusion chromatography. Both the reduction of the native disulfide-bonded form under nondenaturing conditions and the refolding of the urea-denatured disulfide-reduced form under reduced conditions yielded almost exactly the same disulfide-reduced state with partially folded unique conformation that was clearly distinguished from either the native or fully denatured state. In addition, the interconversion between the urea-denatured reduced form and the partially folded reduced form was reversible with each other; by reoxidation, the partially folded reduced form was converted to the disulfide-bonded form. The conformation of disulfide-reduced serum albumin was highly variable depending on pH and ionic strength conditions. Thus, we concluded that the disulfide-reduced state with partially folded variable conformation is involved in the reversible interconversion between the denatured reduced form and the native disulfide-bonded form of human serum albumin.     

Anion-binding centers of human serum albumin during an N-F conformation transition and in isolated albumin and blood serum

Fluorescent probe N-phenyl-1-amino-8-sulfonaphthalene (ANS) was used for studying pH-dependent structural N-F-transition in human serum albumin of two kinds: in commercial albumin and in natural blood serum. The kinetics of ANS fluorescence decay in albumin solutions was measured. There were found two types of the sites occupied by ANS in albumin under physiological conditions (pH 7.4). In the first binding site ANS fluorescence decay time was 16.6 +/- 0.3 nsec and it was not significantly changed at N-F transition (pH 4.0). In the second binding site the decay time was dependent on pH in commercial albumin and was not significantly changed in serum. In the second binding site there were individual differences of ANS decay time (4.3 +/- 0.6 nsec). The observed ANS fluorescence intensity enhancing (about 40-50%) in N-F transition may be explained by an increase of albumin binding sites capacity for ANS.     

Magnetic dye-affinity beads for human serum albumin purification

Cibacron Blue F3GA was covalently attached onto magnetic poly(vinyl alcohol) (mPVAL) beads (100-150 μm in diameter) for human serum albumin (HSA) adsorption from human plasma. Despite low nonspecific adsorption of HSA on mPVAL beads, Cibacron Blue F3GA attachment significantly increased the HSA adsorption. The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma. Desorption of HSA from mPVAL beads was achieved by medium containing 1.0 M KSCN at pH 8.0. To test the efficiency of albumin adsorption from human serum, before and after albumin adsorption was demonstrated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses. HSA molecules could be reversibly adsorbed and desorbed 10 times with the magnetic beads without noticeable loss in their HSA adsorption capacity.     

A high-capacity hydrophobic adsorbent for human serum albumin

A simple method, based on salting out hydrophobic interaction chromatography, for the efficient removal of trace amounts of serum albumin from partially purified protein preparations is described. The method is also successfully applied for the purification of albumin from Cohn fraction IV, a by-product obtained from the commercial fractionation of human serum proteins by the ethanol precipitation procedure. About 70% of the adsorbed albumin can be eluted by buffer of low ionic strength and can thus be lyophilized directly, if required. The adsorbent can be used for several cycles of adsorption and desorption without affecting its selectivity or capacity. Its adsorption properties and capacity for serum albumin are compared with those of the commercially available adsorbent Blue Sepharose CL-6B.     

Antireticular cytotoxic serum as an agent for stimulating the immunity system]

The changes in the immunological indices characterizing the T and B immunity system and also in biological resistance of atherosclerosis patients were studied under conditons of the use of the antireticular cytotoxic serum microdoses. A possibility of employing the ACS for stimulation of the immune system was demonstrated.     

Resveratrol binding to human serum albumin

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419-426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclooxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents (microM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K(Res) = 2.56 x 10(5) M(-1). The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of alpha-helix from 57% (free HSA) to 62% and a decrease of beta-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

Sulfenic acid in human serum albumin

Summary. Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.     

Polyamine analogues bind human serum albumin

Polyamine analogues show antitumor activity in experimental models, and their ability to alter activity of cytotoxic chemotherapeutic agents in breast cancer is well documented. Association of polyamines with nucleic acids and protein is included in their mechanism of action. The aim of this study was to examine the interaction of human serum albumin (HSA) with several polyamine analogues, such as 1,11-diamino-4,8-diazaundecane (333), 3,7,11,15-tetrazaheptadecane.4HCl (BE-333), and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333), in aqueous solution at physiological conditions using a constant protein concentration and various polyamine contents (microM to mM). FTIR, UV-visible, and CD spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind nonspecifically (H-bonding) via polypeptide polar groups with binding constants of K333 = 9.30 x 10(3) M(-1), KBE-333 = 5.63 x 10(2) M(-1), and KBE-3333 = 3.66 x 10(2) M(-1). The protein secondary structure showed major alterations with a reduction of alpha-helix from 55% (free protein) to 43-50% and an increase of beta-sheet from 17% (free protein) to 29-36% in the 333, BE-333, and BE-3333 complexes, indicating partial protein unfolding upon polyamine interaction. HSA structure was less perturbed by polyamine analogues compared to those of the biogenic polyamines.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

Flavonoid binding to human serum albumin

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10⁻⁶ and 3.9 × 10⁻⁵ M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.     

Stereoselective binding of human serum albumin

Stereoselectivity in binding can have a significant effect on the drug disposition such as first-pass metabolism, metabolic clearance, renal clearance, and protein and tissue binding. Human serum albumin (HSA) is able to stereoselectively bind a great number of various endogenous and exogenous compounds. Various experimental data suggested that the two major drug-binding cavities, namely, site I and site II, do not seem to be the stereoselective binding sites of HSA. Stereoselective binding of HSA under disease conditions such as renal and hepatic diseases was found to be enhanced. In addition, site-to-site displacement of a site II-specific drug by another site II-specific drug was found to be stereoselective, too. Endogenous compounds such as long-chain fatty acids and uremic toxins are likely to cause combined direct and cascade effects that contribute to the preferential binding of a particular drug enantiomer. Taking together the findings of other studies, it is highly possible that the stereoselective binding site exists at the interface of the subdomains.