Lysine Residues Of Albumin Research Articles

Development of Albumin‐closo‐Dodecaborate Conjugates as Boron Carriers for Neutron‐Capture Therapy by Ru(bpy)3‐Photocatalyzed Modification of Tyrosine

Serum albumin has attracted significant attention as a drug‐delivery carrier to tumor tissue. We previously reported boron neutron capture therapy (BNCT), an effective method that uses maleimide‐functionalized closo‐dodecaborate albumin conjugates (MID‐ACs). MID can form covalent bonds with the free thiol group of Cys34 and with lysine residues in albumin. In this study, we synthesized tyrosyl‐radical‐trapping‐moiety‐functionalized closo‐dodecaborates (TRT‐DBs), which were expected to form covalent bonds with tyrosine residues. N′‐Acetyl‐N,N‐dimethylphenylenediamine was chosen as the TRT moiety to bind to closo‐dodecaborate through an amide linker (TRT‐DB 1) and an ammonium linker (TRT‐DB 2). TRT‐DB 1 more efficiently conjugated to bovine serum albumin (BSA) than 2: approximately 2.4 molecules of 1 were bound to each BSA if BSA (10 µm) was treated with 1 (1 mm) in the presence of Ru(bpy)3 (1 mm; bpy = 2,2′‐bipyridyl) and ammonium persulfate (1 mm) under light irradiation for 1 min. Furthermore, double modification of BSA with 1 and MID increased the boron density in BSA for efficient boron delivery to tumors. Western blot analysis using the anti‐B12 cluster antibody revealed that closo‐dodecaborate‐conjugated BSAs were taken up by colon 26 cells.

Mass Spectrometric Characterization of Circulating and Functional Antigens Derived from Piperacillin in Patients with Cystic Fibrosis

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.

Calcium ion binding to clinically relevant chemical modifications of human serum albumin

Calcium binding to glycated, penicilloylated, acetylated, and normal defatted human serum albumin as well as to mercapt- and nonmercaptalbumin was studied by equilibrium dialysis of radioactive Ca2+. Binding was quantified by five Scatchard constants [ni = 1, (i = 1-4) and n5 = 10]. Glycation resulted in increased k1- and k2-values and unchanged k3-k5-values, whereas penicilloylation increased all five association constants. The increments were greater the more pronounced the modification, and the enhancements caused by penicilloylation were, for the same degree of modification, greater than those produced by glycation. In contrast, acetylation by acetylsalicylate did not affect calcium binding. Likewise, binding to mercapt- and nonmercaptalbumin was the same, a finding showing that the thiol group of cysteine 34 is not important for calcium binding. D-Glucose and penicillin G are known to react with lysine residues of albumin, and the enhancement of binding resulting from glycation or penicilloylation is probably brought about by unspecific electrostatic effects, possibly supplemented by conformational changes of the protein molecule. The relative importance of the three domains of human serum albumin for calcium binding is discussed.

Studies on the interaction between human serum protein fractions and 18O-labeled oxosteroids.

The nature of the interaction between progesterone or testosterone and human albumin as well as the interaction between progesterone and partially purified human transcortin has been studied. Modification of lysine residues of albumin with maleic anhydride resulted in a decreased binding of the steroid as judged from equilibrium dialysis experiments. This suggested that lysine residues in albumin interact with the oxosteroids. In order to check this hypothesis, steroids labeled with 18O in their oxo function (testosterone and progesterone) were synthesized for use as probes of the interactions. However, no loss of label was noted when testosterone or progesterone specifically 18O-labeled in their oxo functions were incubated with albumin. This suggested that no covalent interaction between the steroidal oxo group and albumin took place. This was in contrast to the results obtained with 3,20-18O-labeled progesterone and partially purified transcortin, where a complete loss of 18O label in the protein-bound steroid was found. The nonbound steroid showed an almost complete retention of label. These results indicate a participation of steroid oxo groups in the binding of progesterone to transcortin. Of the possible mechanisms discussed, imine bonds between the steroid and transcortin seem most likely although other types of interactions cannot be ruled out.

Receptor-mediated endocytosis of aldehyde-modified proteins by sinusoidal liver cells

Formaldehyde-treated serum albumin (f-Alb) is known to be endocytosed by sinusoidal lever cells via a receptor-mediated mechanism. The receptor purified from rat livers exhibited a molecular weight of 125,000, consisting of two glycoprotein components with molecular weights of 53,000 and 30,000, respectively. Experiments using antireceptor antibody demonstrated that the f-Alb receptor is distinct from the receptor that mediates endocytotic uptake of acetylated low-density lipoprotein, but they share a common property of being inhibited by several polyanions, suggesting that polyanion-sensitivity might play an important role in the scavenger function of simusoidal liver cells. Studies on the ligand specificity of this receptor revealed that a covalent modification by formaldehyde of a limited number of lysine residues in albumin has led to the formation of a receptor-recognition domain(s). Furthermore, in addition to formaldehyde, the ligand activity was also generated with albumin modified by other aliphatic aldehydes, such as glycoaldehyde and glyceraldehyde. This phenomenon was extended to several proteins other than albumin. These data suggest therefore that the f-Alb receptor originally described as being specific for albumin modified by formaldehyde may play a general role as a scavenger receptor for aldehyde-modified proteins.

The relative extent of glycation of haemoglobin and albumin

The level of non-enzymatic glycation of a protein is thought to depend on the number of sites available for reaction, the half-life of the protein and the ambient concentration of glucose. Accordingly, the modification of two blood proteins with a similar number of potential sites but different survival times was examined in non-diabetic patients by periodate oxidation and by reduction with [ 3H]borohydride. The amount of glycation of haemoglobin and its sub-fractions HbA 1 and HbA 1c were determined to be 0.44, 2.42 and 2.24 mol/mol respectively and the corresponding value for albumin was 0.37 mol/mol protein. Amino acid analysis showed that the ϵ amino groups of albumin were more extensively modified than they were in haemoglobin and thus it is concluded that the average rate of reaction of the lysine residues in albumin is markedly faster than in haemoglobin.

8] Nonenzymatic glucosylation of lysine residues in albumin

Publisher Summary This chapter describes the preparation and characterization of radiolabeled glycosylamine (GA) and N-substituted-l-amino-l-deoxyketopyranose (KP) derivatives of albumin. It also illustrates procedures for determining kinetic constants involved in their formation and dissociation and for detecting glucose adducts to lysine residues in human serum albumin (HSA) and other proteins. Nonenzymatic glucosylation is a common posttranslational modification of body protein, in which glucose reacts directly with primary amino groups on protein to yield stable covalent adducts. The sugar reacts primarily with the ɛ-amino groups of lysine residues in albumin and other proteins, although reaction at the α-amino terminus also occurs. Since, the rate of hydrolysis of glycosylamines is relatively slow at neutral pH and low temperature, the kinetics of dissociation of GA adducts can be measured by Sephadex G-25 chromatography. The Amadori adduct to the protein (after acid discharge of GA) is stable to extended incubation of the protein at 37° and has an estimated minimum half-life of about 3 weeks.

PREPARATION OF AN ESTRONE–PROTEIN CONJUGATE

A procedure is described for the preparation of an estrone–protein conjugate. The 17-amino derivative of estrone was prepared and converted by phosgenation to estrone-17-isocyanate. The latter was coupled to rabbit serum albumin. Ultraviolet analysis and determination of free amino groups of both albumin and estrone–albumin conjugate showed that the latter contained about 25 estrone residues per albumin molecule. Electrophoretic analysis provided additional evidence that coupling had occurred at the ε-amino groups of the lysine residues of albumin.