Residues Of Bovine Serum Albumin Research Articles

Study of the interaction of kaempferol with bovine serum albumin

The binding of kaempferol with bovine serum albumin (BSA) was investigated at three temperatures, 296, 310 and 318 K, by the fluorescence, circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR) at pH 7.40. The CD and FT-IR studies indicate that kaempferol binds strongly to BSA. The association constant K was determined by Stern–Volmer equation based on the quenching of the fluorescence BSA in the presence of kaempferol. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: Δ H 0 and Δ S 0 possess small negative (−1.694 kJ/mol) and positive values (88.814 J/mol K), respectively. According to the displacement experimental and the thermodynamic results, it is considered that kaempferol binding site II (subdomain III) mainly by hydrophobic interaction. The results studied by FT-IR and CD experiments indicate that the secondary structures of the protein have been changed by the interaction of kaempferol with BSA. The distance between the tryptophan residues in BSA and kaempferol bound to site II was estimated to be 2.78 nm using Foster's equation on the basis of fluorescence energy transfer.

Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin

AbstractThe reaction mechanism of chitosan, bovine serum albumin (BSA), and gelatin with genipin (a natural crosslinking reagent) was examined with infrared, ultraviolet–visible, and 13C NMR spectroscopies; protein‐transfer reaction mass spectrometry; photon correlation spectroscopy; and dynamic oscillatory rheometry. Two reactions that proceeded at different rates led to the formation of crosslinks between primary amine groups. The fastest reaction to occur was a nucleophilic attack on genipin by a primary amine group that led to the formation of a heterocyclic compound of genipin linked to the glucosamine residue in chitosan and the basic residues in BSA and gelatin. The second, slower, reaction was the nucleophilic substitution of the ester group possessed by genipin to form a secondary amide link with chitosan, BSA, or gelatin. A decreased crosslinking rate in the presence of deuterium oxide rather than water suggested that acid catalysis was necessary for one or both of the reactions to proceed. The behavior of the gel time with polymer concentration was consistent with second‐order gelation kinetics resulting from an irreversible crosslinking process, but was complicated by the oxygen radical‐induced polymerization of genipin that caused the gels to assume a blue color in the presence of air. The lower elastic modulus attained after a given time during crosslinking of the globular protein BSA as compared to the coiled protein gelatin, despite possessing more crosslinkable basic residues, demonstrated the importance of protein secondary and tertiary structures in determining the availability of sites for crosslinking with genipin in protein systems. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3941–3953, 2003

Mimosine prevents the death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite.

Immunostimulated astrocytes become highly vulnerable to glucose deprivation (Choi and Kim: J Neurosci Res 54:870-875, 1998a). The increased vulnerability is caused by the enhanced level of peroxynitrite endogenously produced in glucose-deprived immunostimulated astrocytes. In the present study, we report that the plant amino acid mimosine can attenuate the increased death by scavenging peroxynitrite. Treatment with mimosine blocked the increase of nitrotyrosine immunoreactivity, a marker of peroxynitrite, in glucose-deprived immunostimulated astrocytes. Furthermore, mimosine directly inhibited the nitration of tyrosine residues of bovine serum albumin and the oxidation of dihydrorhodamine-123 to rhodamine-123 by peroxynitrite. Mimosine has been used experimentally as a cell cycle G1/S phase transition blocker (Lalande: Exp Cell Res 186:332-339, 1990; Hoffman et al.: Cytometry 12:26-32, 1991). Flow cytometry analysis, however, showed that the cytoprotective effect of mimosine was not attributed to its inhibition of cell cycle progression. Furthermore, under our experimental conditions, mimosine did not alter the levels of cell cycle regulatory proteins, including p21(WAF1/CIP1), cyclins D1 and E, and proliferating cell nuclear antigen. In addition, cyclin-dependent kinase inhibitors olomoucine and roscovitine did not block the increased death. These results indicate that mimosine inhibits the augmented death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite rather than suppressing the cell cycle progression.

15N Chemically Induced Dynamic Nuclear Polarization during Reaction of N-Acetyl-[formula omitted]-tyrosine with the Nitrating Systems Nitrite/Hydrogen Peroxide/Horseradish Peroxidase and Nitrite/Hypochloric Acid

During reaction of N-acetyl-l-tyrosine with the nitrating system 15NO−2/H2O2/HRP at pH 7.5, emission is observed in the 15N NMR signals of 3-nitro-N-acetyl-l-tyrosine and the 1-nitrocyclohexa-2,5-dien-4-one derivative of N-acetyl-l-tyrosine. The occurrence of 15N CIDNP proves the formation of radical pairs. The nitration products are formed by recombination of 15NO•2 and N-acetyl-l-tyrosinyl radicals Tyrac•. The 15N NMR signals of 15NO−2 and of 15NO−3 show enhanced absorption. The 15N CIDNP effects are built up in radical pairs [15NO•2, Tyrac•]F formed by diffusive encounters of the radicals. Nuclear polarization is transferred into 15NO−2 and 15NO−3 by disproportionation reactions of 15NO•2 and by H abstraction of 15NO•2 from N-acetyl-l-tyrosine. If N-acetyl-l-tyrosine is not present, 15NO−3 does not show 15N CIDNP, indicating that peroxynitrite is not formed as an intermediate during the reaction. Peroxynitrate is not formed either, which is indicated by the absence of 15N CIDNP during reaction or a 15N NMR signal after reaction. Emission also appears in nitrated tyrosine residues of bovine serum albumin during reaction of albumin with the 15NO−2/H2O2/HRP system. Analogous but smaller effects are observed during reaction of the nitrating system 15NO−2/HOCl with N-acetyl-l-tyrosine at pH 7.5, proving the radical character of this reaction, too. The 15N CIDNP effects are weak because of the low yield of nitration products.

Endogenous Formation of Protein Adducts with Carcinogenic Aldehydes: IMPLICATIONS FOR OXIDATIVE STRESS

In the present study, we characterize the covalent modification of a protein by crotonaldehyde, a representative carcinogenic aldehyde, and describe the endogenous production of this aldehyde in vivo. The crotonaldehyde preferentially reacted with the lysine and histidine residues of bovine serum albumin and generated a protein-linked carbonyl derivative. Upon incubation with the histidine and lysine derivatives, crotonaldehyde predominantly generated beta-substituted butanal adducts of histidine and lysine and N(epsilon)-(2,5-dimethyl-3-formyl-3,4-dehydropiperidino)lysine (dimethyl-FDP-lysine) as the putative carbonyl derivatives generated in the crotonaldehyde-modified protein. To verify the endogenous formation of crotonaldehyde in vivo, we raised the monoclonal antibody (mAb82D3) against the crotonaldehyde-modified protein and found that it cross-reacted with the protein-bound 2-alkenals, such as crotonaldehyde, 2-pentenal, and 2-hexenal. The anti-2-alkenal antibody recognized multiple crotonaldehyde-lysine adducts, including dimethyl-FDP-lysine and an unknown product, which showed the greatest immunoreactivity with the antibody. On the basis of the chemical and spectroscopic evidence, the major antigenic product was determined to be a novel Schiff base-derived crotonaldehyde-lysine adduct, N(epsilon)-(5-ethyl-2-methylpyridinium)lysine (EMP-lysine). It was found that the lysine residues that had disappeared in the protein treated with crotonaldehyde were partially recovered by EMP-lysine. The presence of immunoreactive materials with mAb82D3 in vivo was demonstrated in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate. In addition, the observations that the metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of proteins resulted in an increase in the antigenicity of the protein indicated that lipid peroxidation represents a potential pathway for the formation of crotonaldehyde/2-alkenals in vivo. These data suggest that the formation of carcinogenic aldehydes during lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.

Oxidative Modification of Tryptophan Residues Exposed to Peroxynitrite

The aim of this study was to clarify the mechanism of loss of Trp residues in proteins exposed to peroxynitrite. The Trp residues in bovine serum albumin and collagen IV were decreased by peroxynitrite treatment. To identify the degradation products of the Trp residue by peroxynitrite,tert-butoxycarbonyl-l-tryptophan (Boc-Trp) was used as a model of the Trp residue in proteins, and the products formed from peroxynitrite-treated Boc-Trp were then isolated. Boc-Trp decreased with an increase in peroxynitrite concentration. N-Formylkynurenine, oxindole, and hydropyrroloindole were identified as major products. The formation of these products may account for the losses of Trp residues in proteins by peroxynitrite.

Formation of Nitrating and Chlorinating Species by Reaction of Nitrite with Hypochlorous Acid: A NOVEL MECHANISM FOR NITRIC OXIDE-MEDIATED PROTEIN MODIFICATION

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO-). We show here that reaction of nitrite (NO2-), the autoxidation product of nitric oxide (.NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO2- with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO2-/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO2-/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO2-/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO2-/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (.NO2) and tyrosyl radical. We propose that NO2- reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl. and .NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO-.

Comparison of the Effects of Ozone on the Modification of Amino Acid Residues in Glutamine Synthetase and Bovine Serum Albumin

During exposure to ozone, the methionine and aromatic amino acid residues of Escherichia coli glutamine synthetase (GS) and bovine serum albumin (BSA) are oxidized rapidly in the order Met > Trp > Tyr approximately His > Phe. The loss of His is matched by a nearly equivalent formation of aspartate or of a derivative that is converted to aspartic acid upon acid hydrolysis. Conversion of His to aspartate was confirmed by showing that the oxidation of E. coli protein in which all His residues were uniformly labeled with 14C gave rise to 14C-labeled aspartic acid in 80% yield and also by the demonstration that His residues in the tripeptides Ala-His-Ala or Ala-Ala-His gave rise to nearly stoichiometric amounts of aspartic acid whereas oxidation of His-Ala-Ala yielded only 36% aspartate. The oxidation of BSA and GS led to formation, respectively, of 11 and 3.3 eq of carbonyl groups and 0.5 and 0.3 eq of quinoprotein per subunit. Although BSA and GS contain nearly identical amounts of each kind of aromatic amino acid residues, oxidation of these residues in BSA was about 1.5-2.0 times faster than in GS indicating that the susceptibility to oxidation is dependent on the primary, secondary, tertiary, and quaternary structure of the protein.

Protective effects of β-blockers against 2,2′-azobis(2-amidinopropane)-dihydrochlorideinduced damage

The protective effects of β-blockers against 2,2′-azobis(2-amidinopropane)-dihydrochloride (AAPH)-induced damage were investigated. With the exception of pindolol, none of the β-blockers tested inhibited arachidonate peroxidation induced by AAPH in the absence of iron. In contrast, ADP-Fe 3+- and NADPH-dependent microsomal lipid peroxidation was inhibited by all the β-blockers tested, although the inhibitory effects of atenolol and metoprolol were very slight. Oxidation of tryptophan residues in bovine serum albumin (BSA) induced by AAPH was strongly inhibited by pindolol and propranolol but not by atenolol or metoprolol. All the β-blockers tested, however, inhibited AAPH-induced carbonyl formation of BSA. Furthermore, all the β-blockers tested also strongly inhibited the deoxyribose degradation induced by AAPH, suggesting that these agents act as hydroxyl radical scavengers to inhibit carbonyl formation. DNA strand scission was induced by AAPH in the absence or presence of O 2. Only pindolol strongly inhibited the DNA damage in the absence of O 2. In the presence of O 2, however, all the β-blockers tested effectively prevented the DNA damage. These results suggested that the hydroxyl radicals produced from AAPH damaged DNA and, that β-blockers might act as hydroxyl radical scavengers to protect DNA against the AAPH-induced oxidative damage.

Receptor-mediated endocytic uptake of methylglyoxal-modified serum albumin. Competition with advanced glycation end product-modified serum albumin at the advanced glycation end product receptor.

Methylglyoxal binds and irreversibly modifies arginine and lysine residues in bovine serum albumin (BSA) under physiological conditions, producing a protein with an increased net negative charge at physiological pH. At 4 degrees C, methylglyoxal-modified BSA (MG-BSA) was bound by cell surface receptors on murine P388D1 macrophages. The apparent dissociation constant KD value was 435 +/- 2 nM, and there were 8.89 +/- 0.02 x 10(5) receptors/cell (n = 6), compare with an apparent KD value of 263 +/- 52 nM and 10.17 +/- 0.93 x 10(5) receptors/cell (n = 11) for advanced glycation end product-modified BSA (AGE-BSA). AGE-BSA competed with MG-BSA for binding to a common receptor; however, a component of AGE-BSA receptor binding could not be displaced by MG-BSA, and a component of MG-BSA receptor binding could not be displaced by AGE-BSA, suggesting that there are binding sites for both AGE-BSA and MG-BSA, competitive and noncompetitive, to MG-BSA and AGE-BSA on P388D1 cells at 4 degrees C. At 37 degrees C, receptor binding of AGE-BSA and MG-BSA was followed by endocytosis and lysosomal degradation of the modified protein. Methylglyoxal-modified proteins are ligands for the AGE receptor, and their formation and metabolism may be linked to the development of diabetic complications.

Modification of Histidine Residues in Bovine Serum Albumin by Reaction with (E)-2-Octenal

Reaction of bovine serum albumin (BSA) with the lipid peroxidation product (E)-2-octenal leads to the generation of (E)-2-octenal-BSA adducts. Amino acid analysis of the modified protein (after reduction with NaBH4) showed that histidine residues were major targets of the reaction and showed the formation of new amino acid derivatives. The same products were detected in acid hydrolysates of poly(L-histidine) and N-(carbobenzoxy)-L-histidine after their reactions with (E)-2-octenal and NaBH4. The reaction of N-(carbobenzoxy)-L-histidine with (E)-2-octenal led to the production of isomeric forms of N-(carbobenzoxy)-1(3)-[1' (formylmethyl)heyl]-L-histidine dihydrate. Upon acid hydrolysis, these compounds yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of 1(3)-[1'-(hydroyethyl)hexyl]-L-histidine] that were indistinguishable from those obtained from BSA, poly(L-histidine), and N-(carbobenzoxy)-L-histidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in BSA by (E)-2-octenal involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha,beta-unsaturated bond of (E)-2-octenal. The reaction of histidine residues with (E)-2-octenal provides the basis for methods by which the contributions of (E)-2-octenal in the modification of proteins can be determined.

Synthesis of Neoglycoproteins as Artificial Antigens

Abstract The synthesis of various allyl glycosides of L-rhamnose containing ether and/or ester substituents are reported. Ozonolysis of allyl glycosides followed by reductive amination with ∊-amino groups of lysine residues in bovine serum albumin using sodium cyanoborohydride at pH 7.8 provided different structural neoglycoproteins as artificial antigens.

Thyroid peroxidase: kinetics, pH optima and substrate dependency.

The oxidation of iodide, guaiacol and 2,2'-azino-di[3-ethyl-benzthiazoline-(6)-sulphonic acid] and the iodination of tyrosyl residues in bovine serum albumin, catalysed by partly purified thyroid peroxidase, were studied. The enzyme showed pH optima with all electron donors. With the exception of guaiacol, the position of the pH optima depended upon both the electron donor and hydrogen peroxide concentrations. With increased hydrogen peroxide concentrations the optima shifted to lower pH, and with increased iodide concentration to higher pH. For monoiodotyrosine (MIT) formation in bovine serum albumin the position of the pH optimum was also dependent on the hydrogen peroxide concentrations. The position of the pH optimum of the oxidation of guaiacol was pH 9 and independent of substrate and hydrogen peroxide concentrations. It is obvious from these findings that iodination reactions must be studied under well-defined conditions.

A specific radioimmunoassay for the measurement of ICI 200,880, an elastase inhibitor, in human serum

ICI 200,880, 4-(4-chlorophenylsulphonylcarbamoyl)benzoyl- l-valyl- l-proline-1( RS)-(1-trifluoroacetyl-2-methylpropyl)amide ( I), is a human neutrophil elastase inhibitor in development by ICI Pharmaceuticals Group. A specific and sensitive radioimmunoassay has been developed for this compound in human serum. An hydroxysuccinimide ester analogue of ICI 200,880 was coupled to the lysine residues of bovine serum albumin, characterized by gel electrophoresis and injected into rabbits to stimulate antibody formation; a highly specific, high titre antibody was obtained. An 125I-diiodinated 4-hydroxy-phenyl derivative of ICI 200,880 was utilized as the radiolabelled antigen. Satisfactory zero and non-specific binding were achieved with a 2-h pre-incubation of ICI 200,880 and antiserum at 37°C, followed by the addition of radiolabelled antigen and a second 2-h incubation. Separation of bound and free radiolabel was achieved by employing PEG-goat anti-rabbit IgG separant. Sensitivity of 25 pg ml −1 ICI 200,880 in human serum was achieved ( n = 12), with quantitative recovery of 106%, inter-assay precision of 6.3% and an average relative binding statistically different than that of pooled human serum. Serum quality control samples spiked at 600 and 100 pg ml −1 ICI 200,880 averaged 104% recovery over 6 validation days, with intra-assay precision of 12.8% RSD and inter-assay precision of 9.2% RSD ( n = 24). Cross-reactivity of the ICI 200,880 antibody to three known metabolites and several analogues was negligible. Over 1200 clinical samples following aerosol administration of ICI 200,880 have been analysed by this procedure. After maximum exposure ranging from 0.3 to 32 mg ICI 200,880, mean peak levels from 0.08 to 20.5 ng ml −1 were measured, respectively, with compound detected generally up to 16 h post-dose.

Synthesis of allyl glycosides for conversion into neoglycoproteins bearing epitopes of mycobacterial glycolipid antigens

Neoglycoproteins bearing key glycosyl substituents of several glycopeptidolipid antigens of pathogenic Mycobacterium species have been synthesized. Allyl glycosides of the terminal 6-deoxyhexose-containing units of the antigens were prepared, with appropriate ether and ester substituents in place. Ozonolysis of the allyl glycosides was then followed by reductive coupling with ε-amino groups of lysine residues in bovine serum albumin, using sodium cyanoborohydride at pH 7.8. The resulting neoglycoproteins emulated the antigenicity of the native molecule in several serological tests.

Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding.

Using a double modification technique about 20% of the lysine residues of bovine serum albumin (BSA) which are not easily accessible in the native protein have been modified. The technique involved approximately 80% modification of lysine residues of BSA with citraconic anhydride followed by chemical modification of the remaining lysine residues with acetic anhydride, succinic anhydride, potassium cyanate, or O-methylisourea. Finally, these preparations were decitraconylated under mild acidic conditions to yield acetylated, succinylated, carbomylated or guanidinated BSA. All of these preparations were found to be homogeneous with respect to charge and size. The spectral, hydrodynamic and bilirubin binding properties of these preparations are described. In contrast to most of the highly modified proteins these preparations with the exception of succinylated BSA are very similar to native BSA in their spectral and hydrodynamic properties. However, the equilibrium association constant (Ka) with bilirubin measured by fluorescence quenching was decreased by about 100-fold in acetylated, carbamylated and succinylated BSA, but only 3-fold in guanidinated BSA. Since conformationally acetylated and carbamylated BSAs are identical to guanidinated BSA we conclude that the decrease in Ka in these preparations is solely due to loss of positive charge on 'critical' lysine residues. The results support a binding model for BSA in which bilirubin binding site is buried and the protein undergoes a series of relaxational changes in conformation upon interaction with bilirubin.

TB(III) as a fluorescent probe for the structure of bovine serum albumin

Tb(III) was used as a fluorescent probe in the study of the calcium-binding sites on Bovine Serum Albumin(BSA). The fluorescence of Tb(III) is enhanced markedly when bound to BSA and nonradiative energy transfer between two fluorescent tryptophan(Trp) residues and Tb(III) bound to calcium-binding sites on BSA occurred. Experimental results show that the major groups in BSA bound to metal ion are the carboxyl side groups of glutamic acid(Glu) and aspartic acid(Asp). The average distance between the bound Tb(III) and the two tryptophan residues in BSA calculated by a Föster dipole-dipole nonradiative energy transfer mechanism is 1.48 nm.

Expedient syntheses of neoglycoproteins using phase transfer catalysis and reductive amination as key reactions

Starting from peracetylated chloro- or bromo-glycosyl donors of N-acetylneuraminic acid, N-acetylglucosamine, glucose and lactose, the corresponding p-formylphenyl glycosides were synthesized stereospecifically under phase transfer catalysed conditions at room temperature in yields of 38-67%. After Zemplén de-O-acetylation, the formyl groups were directly and chemoselectively coupled to the lysine residues of bovine serum albumin by reductive amination using sodium cyanoborohydride. The conjugation reactions were followed as a function of time and under a series of different molar ratios of the reactants to provide glycoconjugates of varying degree of antigenicities. Thus, carbohydrate protein conjugates were made readily available using essentially two key reactions.

Heterotropic binding of alclofenac and dansylsarcosine to bovine serum albumin

The binding data for the interaction of alclofenac (AF) and dansylsarcosine (DS) to bovine serum albumin (BSA) have respectively yielded nonlinear Scatchard plots. The plots have been subjected to Rosenthal’s method of analysis and thus the ligands have been found to possess two different kinds of sites in BSA. The binding capacities of these sites have been evaluated. The fluorescence competition studies have revealed that the binding of DS to BSA is noncompetitively inhibited by AF. Therefore, the presence of distinct binding sites for AF and DS in BSA could be inferred. The fluorescence quenching studies have also been able to demonstrate this aforesaid fact. The analysis of the quenching data by the modified Stern-Volmer plot has indicated that both the tryptophan (Trp) residues of BSA are accessible to DS for the quenching in absence of AF, but only one of them is accessible in presence of AF. This has led to suggest that the binding site of DS has been in the vicinity of loop 3–4, involving Trp-134 and Trp-212. The binding of AF at a distinct site from that of DS has exerted heterotropic interactions at the DS binding site and thereby inhibited the binding of DS to BSA.

Binding of bromophenol blue to bovine serum albumin and its succinylated forms

Interaction of bromophenol blue with bovine serum albumin and its five succinylated forms was studied spectrophotometrically at three different ionic strengths, i.e. 0.04, 0.15 and 1.0 and at two different pH values, namely pH 7.0 and pH 5.0 respectively. Results showed a decrease in bromophenol blue binding on increasing succinylation at low ionic strengths. This decrease was more marked at pH 7.0 than pH 5.0. However, at both the pH values binding returned to a significant degree on increasing the ionic strength to 1.0. Succinylation also caused marked conformation changes at pH 7.0 and ionic strength 0.15 as evidenced by changes in hydrodynamic properties and reduction in antigen-antibody precipitin reaction. However, an increase in ionic strength to 1.0 or decrease in pH to 5.0 caused signifciant reversal in hydrodynamic parameters. These studies show that lysine residues of bovine serum albumin are not important in bromophenol blue binding.