Intraspecific variability in fractional composition of the serum proteins of the sturgeon Acipenser stellatus]

Comparative studies on fractional composition of the blood serum proteins in two sympatric populations of the sturgeon A. stellatus (South-Caspian and North-Caspian) have been made by means of polyacrylamide gel disc-electrophoresis. Serum proteins are fractionated into 13-18 electrophoretic components, the heterogeneity of proteins being somewhat higher in the North-Caspian population than in the South-Caspian one. Most pronounced differences were found in the relative content of albumins and beta-globulins. Special interest is attracted to different heterogeneity of albumins and beta-globulins (transferrins) in the two populations of the Caspian sturgeon.     

Structure of fish serum albumins

Data are presented about the presence of serum albumins in fish of different classes and orders inhabiting different ecological conditions, about structure of typical albumins and albumin-like proteins, and about the degree of homology of these proteins to mammalian albumins. There is shown a wide spectrum of structural diversity of albumins in Pisces due to their participation in osmotic, plastic, and transport functions under conditions of environment and the organism internal media. Detection of similar motifs in the fish and mammalian albumin genes allows uniting these genes into one superfamily and considering vertebrate albumins the homologous proteins.     

Peculiarities of secondary structure of serum albumin of some representatives of the animal kingdom

Methods of infrared (IR) spectroscopy and circular dichroism (CD) are suitable techniques for detection of proteins structural changes. These methods were used for determinating peculiarities of the secondary structure of serum albumins in some representatives of two classes of reptiles: Horsfield's tortoise (Testudo horsfieldi), water snake (Natrix tessellata) and grass snake (Natrix natrix) and birds: domestic goose (Anser anser), domestic chicken (Gallus domesticus), domestic duck (Anas platyrhyncha) and dove colored (Columba livia). An analysis of IR spectra and spectra obtained by the method of CD of serum albumins of both classes representatives revealed that beta-folding structure and alpha-helical sections that form the alpha-conformation play an important role in conformational structure formation of polypeptide chain and also disordered sites of molecules of these proteins. It was observed that certain redistribution depending on animals species exists, in the formation of secondary structure of serum albumins of the investigated representatives of reptiles and birds classes between the content of beta-folding structure, alpha-helical sections and disordered sites in molecules of these proteins.     

Studies on the mechanism of binding of serum albumins to immobilized cibacron blue F3G A.

The interaction of Cibacron Blue F3G A-Sepharose 4B with several serum albumins was studied. Although all albumins used were fond to bind to this adsorbent, human serum albumin was bound to a far greater extent than were the others. From the results of competition experiments and n.m.r. studies of Cibacron Blue and/or bilirubin binding to human serum albumin it is proposed that the mechanism of the interaction between human serum albumin and cibacron Blue is consistent wit Cibacron Blue binding to bilirubin-binding sites. In contrast with these findings with human serum albumin, there is little or no interaction of Cibacron Blue and the bilirubin-binding sites of albumins from rabbit, horse, bovine or sheep sera, although some interaction occurs between Cibacron Blue and the fatty acid-binding sites of these proteins. Structural analogues of Cibacron Blue have been used to investigate the binding of albumins to these ligands.     

Investigations of interaction mechanism and conformational variation of serum albumin affected by artemisinin and dihydroartemisinin

In this work, binding interactions of artemisinin (ART) and dihydroartemisinin (DHA) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated thoroughly to illustrate the conformational variation of serum albumin. Experimental results indicated that ART and DHA bound strongly with the site I of serum albumins via hydrogen bond (H-bond) and van der Waals force and subsequently statically quenched the intrinsic fluorescence of serum albumins through concentration-dependent manner. The quenching abilities of two drugs on the intrinsic fluorescence of HSA were much higher than the quenching abilities of two drugs on the intrinsic fluorescence of BSA. Both ART and DHA, especially DHA, caused the conformational variation of serum albumins and reduced the α-helix structure content of serum albumins. DHA with hydrophilic hydroxyl group bound with HSA more strongly, suggesting the important roles of the chemical polarity and the hydrophilicity during the binding interactions of two drugs with serum albumins. These results reveal the molecular understanding of binding interactions between ART derivatives and serum albumins, providing vital information for the future application of ART derivatives in biological and clinical areas.     

Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin

Quaternary amine of diethylaminoethyl rosin ester (QRMAE), chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5), and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC). It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s) responsible for the aggregate formation, in addition to various other industrial applications.     

Metals in biochemistry : P.M. Harrison and R.J. Hoare,Chapman & Hall,New York, 1980, 80 pp. $5.95 (paper)

An ultrafiltration technique was used to study stripping by glycine of the first copper and zinc ion equivalents bound by bovine, dog, and rat serum albumins at pH 7.5. Affinity of dog serum albumin for copper was poorer than for the other albumins, consistent with the absence in the former albumin of the copper binding site present at the amino terminus of the latter albumins. Affinities of all three proteins for zinc were similar, suggesting that the albumin amino terminus is not the primary zinc ion binding site.     

Photo-induced riboflavin binding to the tryptophan residues of bovine and human serum albumins

Summary The photobinding between riboflavin and the Trp residues from human and bovine serum albumins at two pH-dependent protein conformations was studied. At pH 7.0 both proteins showed photo-adduct formation with hyperbolic kinetics. In the bovine serum albumin this is attributed to the different locations of the two Trp residues. In the case of the human serum albumin, which has only one Trp residue, this behaviour may be related to different molecular conformations of the protein, as is also manifest in the iodide quenching experiments. At pH 3.5, the kinetics of the photo-adduct formation were found to be slower and showed a monophasic behaviour. These results are due to the conformational change of these proteins at acidic pH; the Trp residues of both proteins being now located in a more hydrophobic environment. When bovine serum albumin was anaerobically irradiated at pH 7.0 in the presence of¹⁴C-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of¹⁴C-riboflavin in these samples was significantly higher at the level of the peptide containing the Trp-134 residue. Furthermore, it was demonstrated, that the energy transfer from enzymatically generated triplet acetone to riboflavin can also promote the binding of this vitamin to the Trp residues of human and bovine serum albumins.     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

Complexation with albumins of chiral aromatic substrates and their chemistry in ground and excited states. Catalytic and chirality recognition properties of the protein in the cases of binaphthol, its photoisomers, and ketoprofen.

The reactivity of organic molecules can be modified upon complexation with proteins: these changes can be different and more significant when the substrate is in an electronically excited state. Here we review UV, CD, and fluorescence spectroscopy studies on the photochemistry and on the chemistry of atropisomeric binaphthols and of ketoprofen, complexed to serum albumins. The chemical and photochemical properties of the organic substrates, complexed to the albumins or free in common solvents, are different. The role of the protein complexation is also evidenced in photoresolution processes of racemate-protein complexes. Catalytic effects due to serum albumins are also reported. In particular, the Arrhenius parameters for the rate of thermal isomerization of a metastable photoproduct of binaphthol in common solvents are compared with those of the bovine serum albumin catalyzed isomerization.     

Divergence of the two albumins of X. laevis. Evidence for the glycosylation of the major 74K albumin

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis, differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

Divergence of the Two Albumins of X. laevis

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis , differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

The effect of commercial bovine serum albumin and other proteins on the activity of bovine milk galactosyltransferase

The widespread use of bovine serum albumin preparations for the stabilization of purified glycosyltransferases has prompted us to study the effects of different preparations of albumins on the galactosyltransferase activity of bovine milk. For comparison, several other proteins were tested as well. The albumins caused a large stimulation of transferase activity (400-700%) which varied depending on the source of the albumin and the treatment to which it had been subjected. Several other unrelated proteins were tested for their effects on transferase activity. Some proteins stimulated, while others had little effect. Lysozyme stimulated the activity by 178% and poly-L-lysine had little effect. Other proteins stimulated to variable extents. The stimulations obtained with albumin and myelin basic protein were noteworthy. The stimulation was considerably less marked when the enzyme was incorporated into lipid vesicles. These results emphasize the need for caution when adding proteins such as bovine serum albumin to purified enzymes for the purpose of stabilizing the activity of the enzyme.     

Age-related variability of serum protein antigens in sturgeons (Acipenseridae, Acipenseriformes)

We studied the variability of antigenic properties of proteins in two sturgeon species at different stages of postembryonic development. The deepest changes occurred in individual components of albumins and beta-globulins (transferrins) and were mostly related to an increased proportion of the protein accounting for these antigens. Transformation of the main component of albumins A1 into adult antigens was completed in 5-month old fry. The main component of beta-globulins A (component of transferrins) appeared in the blood flow much later than other proteins and could retain the fry features until the age of 3-4 years. Other antigens belonging to alpha1- and alpha2-globulins and the second component of transferrins were more stable and did not undergo substantial changes. The direction of ontogenetic variability of serum antigens in sturgeon fry did not depend on the habitat of adult fish in fresh or sea water.     

Comparison of Methods for Separating Proteins Using Bisalbuminemia as a Model

Sera from bisalbuminemic chicken-turkey hybrids contain two albumins in equal amounts. These are observed as inherited electrophoretic variants and originate from the respective chicken and turkey parents. Sera from the hybrid birds served as a model system by which fractionating and identification procedures for evaluating serum albumin variants were compared.

The two albumins in the hybrid were isolated with preparative polyacrylamide gel electrophoresis (PAGE) and starch block preparative electrophoresis. Isoelectric focusing of the hybrid albumins resulted in the isolation of the turkey albumin. Interference of ampholinea prevented the complete isolation of the chicken albumin.

The two albumins in the hybrid have identical molecular weights and cannot be identified by sedimentation coefficient, gel filtration behavior, or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because of the close relatedness the chicken and turkey albumins in the hybrid cross reacted with rabbit anti-hybrid serum as well as with rabbit anti-chicken and anti-turkey sera.     

Studies on the antigenic properties of serum albumins

Summary Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well known and its antigenic properties have been described in animal models. The aim of our work was to evaluate the role of conformation on antigenicity of serum albumins. This study was performed using electrophoresis associated with the immunoblotting technique, where sera from children allergic to BSA were used. Data obtained in this research indicate that serum albumin antigenicity is only partially correlated to its native three-dimensional structure. Heat treatment and chemical denaturation (SDS treatment) are not able to significantly decrease its capability to bind circulating IgEs. Only the reducing treatment is able to modify the antigenicity of this protein. Moreover, a direct correlation between the cross-reactivity observed in immunoblotting between different serum albumins and the percentage of their sequence identity (phylogenetic similarity of the species) was shown.     

Isolation and characterization of denatured serum albumin from rats with endotoxicosis

Due to its rapid breakdown in the body, denatured serum albumin has not been identified in biological samples. In this study we attempted to determine whether denatured albumin could be identified in rats with endotoxicosis. Male Wistar rats were injected with lipopolysaccharide (LPS; 5 mg/kg body weight). Plasma albumin concentration decreased to one-third the normal level at 2 days after the injection. By using the purified IgG against the specific epitope of chemically denatured albumin, two immunoreactive plasma proteins (bands D2 and D3) were identified by native PAGE followed by Western blot analysis. The plasma concentration of these two proteins increased significantly at 1 and 1.5 days after LPS injection. Peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI/TOF-MS) identified these two proteins as serum albumin. In order to characterize their conformational nature, ion-exchange chromatography was used to isolate D2 and D3 albumins from rats injected with LPS. Far- and near-UV circular dichroism (CD), tryptophan and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence, and proteolytic susceptibility showed conformational alterations in the D2 and D3 albumins as compared with native albumin. These data indicate the presence of denatured albumin in circulating rat plasma, and this fact may contribute to a further understanding of the molecular mechanisms of albumin breakdown in physiological and pathophysiological conditions.     

Atlantic salmon (Salmo salar) serum albumin: cDNA sequence, evolution, and tissue expression

Atlantic salmon serum albumin is one of the most abundant proteins in salmon liver, representing 1.6% of all clones in a cDNA library made from salmon liver RNA. The DNA from a number of clones was sequenced to reveal an open reading frame of 1,827 bases encoding a 608-amino-acid protein. The sequenced 5' untranslated region is 69 bases long and the 3' untranslated region contains two putative polyadenylation signals and poly(A) tail. Sequence analysis of different clones indicates the presence of a second cDNA for salmon serum albumin. Multiple alignments of salmon serum albumin deduced amino acid sequence with Xenopus laevis, rat, bovine, and human serum albumins shows significant conservation of cysteine residues. The triple domain structure of serum albumin proteins is maintained. Unlike mammalian systems where serum albumin expression appears to be specific to liver only, salmon serum albumin is expressed in muscle also.     

Stable Mammalian Serum Albumins Designed for Bacterial Expression

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.