Cleavage Of Bovine Serum Albumin Research Articles

Effects of Maillard reaction products on the oxidative cleavage and polymerization of protein under ascorbic acid-transition metal system.

This study investigated the effects of Maillard reaction products (MRPs) on the oxidative cleavage and polymerization of BSA (bovine serum albumin) in an aqueous system. In L-ascorbic acid (AsA) and Cu(II) or Fe(III) reaction system, 50-60% of BSA was cleaved under physiological conditions (37 degrees C, pH 7.2). The oxidative cleavage induced by AsA-Cu(II) system was suppressed to the extent of 32-86% by model melanoidins or brown pigments from amino acids and foodstuffs. In the AsA-Fe(III) system, the oxidative cleavage was inhibited to the extent of 45-93% by melanoidins and brown pigments. However, this cleavage was promoted by amino acid Amadori rearrangement products and brown pigment from soy paste. Therefore, MRPs show both suppression and promotion activity on oxidative cleavage of BSA in the system of AsA and a transition metal. The quantity of Amadori rearrangement moiety (ARM) in melanoidins from Lysine and brown pigments molecules from foods was also measured. From these data, it was estimated that the suppression and/or promotion of oxidative cleavage of BSA did not only depend on the quantity of ARM, but also depended on the chemical structure of ARM in melanoidins or brown pigments.

Functional molecules based on polyazometals. (1) artificial metalloproteinases prepared by conjugation of polyazometals with poly(allylamine)

Polyazometals (PAMs) conjugated with poly(allylamine) manifested high catalytic activity in the hydrolytic cleavage of bovine serum albumin. Based on cleavage sites, the mechanism for the PAM-catalyzed peptide hydrolysis is suggested.

Photochemical protease: site-specific photocleavage of hen egg lysozyme and bovine serum albumin.

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 +/- 0.3 x 10(5) M-1 and 6.5 +/- 0.4 x 10(7) M-1, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at approximately 460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

Bilirubin attenuates radical-mediated damage to serum albumin

Oxidative damage to biological macromolecules has been implicated in a number of diseases. Much interest has focused on how non-proteinaceous, low-molecular weight antioxidants prevent oxidative damage to lipids, while comparatively little is known about protein antioxidation. Here we show that bilirubin (BR), the end-product of heme catabolism, when bound to bovine serum albumin (BSA), is oxidised by hydroxyl ('OH), hydroperoxyl (HO 2), and Superoxide anion (O xxx 2) radicals to so far mostly uncharacterised products. The initial oxidation rates of BSA-bound BR decreased in the order OH ⪢ HO 2 ⪢ O −. 2 BR protected its carrier protein from oxidative damage inflicted by OH radicals. This protective action included a reduction in the OH-mediated cleavage of BSA, conversion of Trp into kynurenine and formation of ‘bityrosine-specific’ fluorescence. BR also strongly inhibited OH-mediated formation of protein carbonyls, whereas ascorbate and Trolox (a water-soluble analogue of vitamin E) were much less effective. These results support an antioxidant-protective function of BR and point towards significant differences in the efficacies of various antioxidants in the prevention of oxidative damage to lipids and proteins.

Direct liquid chromatographic separation of enantiomers on immobilized protein stationary phases : VIII. A comparison of a series of sorbents based on bovine serum albumin and its fragments

A mixture of three peptides ( M r 38 000), obtained by enzymatic cleavage of bovine serum albumin (BSA) was isolated, immobilized to silica and used as a chiral stationary phase in liquid chromatography. A comparison of sorbents containing these fragments and intact BSA, showed that the enantioselectivity is preserved for only a limited number of compounds. Under identical mobile phase conditions, retention on the columns based on BSA fragments was also much lower than on those containing intact BSA. The method used for immobilization has a great influence on the retentive and enantioselective properties of the sorbent obtained. When sorbents based on BSA entrapped in silica and 3-aminopropylsilica, by cross-linking with glutaraldehyde were compared under identical mobile phase conditions, the latter were generally found to give larger capacity factors and often also larger α values. These results indicate that the increased hydrophobicity, primarily caused by the aminopropyl groups, partly contributes to the overall retention and chiral discrimination process and that the situation may be different from that in a solution of the free protein.

Effect of oxidative sulfitolysis of disulfide bonds of bovine serum albumin on its structural properties: a physiochemical study.

The effect of S-S bond cleavage of bovine serum albumin (BSA) on some of its structural properties, including solubility, viscosity, and conformation, were investigated. Cleavage of S-S bonds decreased the solubility of serum albumin and also shifted its isoelectric point to lower pH values. S-S bond cleavage resulted in changes in shape and hydrodynamic volume of the protein, increasing the specific viscosity, with cleavage of up to 14 S-S bonds, followed by a decrease with further cleavage. Both UV difference and fluorescence spectral measurements indicated that conformational flexibility increases with S-S bond cleavage. Secondary structure estimations by far UV-CD suggested a gradual decrease in alpha-helical content of the protein with progressive cleavage of its S-S bonds. However, fully S-S bond cleaved protein maintained some alpha-helical structure. Sulfitolysis of the protein also decreased its 1,8-anilino-naphthalene sulfonate-binding ability.

Optical detection of triplet-state magnetic resonance studies on individual tryptophan residues of serum albumin: correlation between phosphorescence and zero-field splittings.

Cyanogen bromide cleavage of bovine serum albumin (BSA) yields two fragments, N (1-183) and C (184-582), containing 183 and 399 amino acid residues, respectively. Each in each fragment are characterized in this study by phosphorescence and optically detected magnetic resonance spectroscopy, and the results are compared with those of the intact albumin. Trp-134 in fragment N is located in a hydrophobic environment in the interior of the protein, as reflected by its red-shifted phosphorescence and characteristic zero-field splittings. The spectral properties of Trp-212 in fragment C suggest its location in a partially buried, inhomogeneous environment. They show great similarity to those of human serum albumin, which contains a single Trp at position 214. The Trp phosphorescence 0,0-bands of fragments C and N are fitted with Gaussian functions by computer, and their relative contributions to the phosphoresence 0,0-band of BSA are adjusted to fit the observed BSA 0,0-band. The wavelength dependence of the [D[-[E[ transition frequencies of fragments N and C is then weighted by their 0,0-band intensity, taking into account differences in spin alignment, and summed to predict the peak frequency of the [D[-[E[ band profile as a function of phosphorescence wavelength for the intact BSA. Good agreement between predicted and observed behavior of [D[-[E[ vs. wavelength for the intact protein provides strong evidence for the additivity of the phosphorescence and ODMR spectra of the individual Trp sites in BSA. We find that Trp-134 and Trp-212 have wavelength-independent and wavelength-dependent zero-field splittings, respectively.

Cleavage of bovine serum albumin with cyanogen bromide and alignment of the fragments

Bovine serum albumin was split by cyanogen bromide treatment in two main fragments which were characterized by their amino-acid composition, N-terminal amino acids and thiol content. The five expected polypeptides were liberated on cleavage of the disulfide bonds of the two fragments. They were characterized by their amino-acid composition and N- and C-terminal amino acids. The isolation and characterization of two intermediates after reduction allowed an unequivocal alignment of the five polypeptides in the sequence. The amino acids surrounding the methionine residues were determined and the two tryptophan residues and the single thiol group were localized in the fragments. La sérumalbumine bovine a été scindée par traitement au bromure de cyanogène en deux fragments principaux, qui ont été caractérisés par leur composition en acides aminés, par leurs groupes N-terminaux et par leur teneur en thiol. Par rupture des ponts disulfure les deux fragments ont libéré les cinq polypeptides escomptés, qui ont été caractérisés par leur composition en acides aminés et par leurs groupes N-et C-terminaux. La séparation et la caractérisation de deux intermédiaires lors de cette réduction a permis d'aligner sans équivoque les cinq polypeptides dans la séquence. Les acides aminés près des résidus méthionine ont été identifiés et les deux résidus tryptophane et le groupe thiol unique localisés dans les fragments.