Protein Cysteinyl-S-Nitrosylation: Analysis and Quantification.

The thiol moiety of cysteine residues can undergo a number of biologic modifications including oxidation, sulfenylation, nitrosylation, persulfidation, metalation, and other modifications. These modifications can control biological function, including gain as well as loss of function. Herein, we focus attention on the proteomic analysis of S-nitrosylation in health and disease. We describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. We present several studies where suitability of this approach for investigating endogenous S-nitrosylation is addressed.

Automated Ferguson analysis of glycoproteins by capillary electrophoresis using a replaceable sieving matrix.

Obtaining accurate molecular weight estimates for glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moieties of the proteins. This leads to lower charge-to-mass ratios for SDS-glycoprotein complexes, resulting in over-estimation of molecular weights by SDS-PAGE. In order to minimize these inaccuracies for proteins with abnormal charge-to-mass ratios, a Ferguson plot may be employed. This application requires the determination of relative mobilities for standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular because it requires time-consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates all of the data required for a Ferguson plot using a replaceable sieving matrix (thereby eliminating gel polymerization) in a capillary format. In addition, this technique possesses the advantages inherent to capillary electrophoresis, namely, very fast separation times, and on-line monitoring which allows quantitation and precludes post-separation staining and destaining of gels.

Size-dependent separation of proteins denatured in SDS by capillary electrophoresis using a replaceable sieving matrix.

The determination of molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most powerful electrophoretic techniques for protein characterization. A separation media has been developed which allows this type of analysis to be performed in the capillary format. A replaceable polymeric solution rather than a polymerized gel was used as the sieving matrix. This solution allowed for the separation of proteins denatured in SDS in a size-dependent manner as demonstrated by the linear correlation between the proteins' relative migration and the concentration of the sieving matrix (Ferguson plot). The logarithm of the molecular weight of protein standards correlated linearly with the relative mobility of the denatured proteins over the molecular weight range of 14,000 to 205,000. The calculated resolution at half-peak height was such that proteins that differed by as little as 4% in molecular weight would be resolved. Finally, the integrated peak areas at 215 nm were linearly proportional to the mass of the protein injected.

Isoelectric focusing by free solution capillary electrophoresis.

A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient was developed. RNase T1 and RNase ba are two proteins that have isoelectric points (pI's) at the two extremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and pI's determined in the same experiment. The pI's of RNase T1 wild-type, its three mutants, and RNase ba were determined for the first time as 2.9, 3.1, 3.1, 3.3, and 9.0, respectively. The paper describes the protocol for isoelectric focusing by capillary electrophoresis, as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.

Automated capillary electrophoresis applied to therapeutic drug monitoring.

Chromatographic separations in conjunction with physical chemical detection methods can provide extremely accurate and highly precise measurements of drugs in biological matrices. Immunochemical methods for drug analyses, on the other hand, although usually fully automated, often lack accuracy, may be subject to matrix interference effects, and frequently exhibit nonlinearities that contribute to suboptimal analytical results. Automated capillary electrophoresis (CE) promises the high accuracy and precision of physical methods with no operator intervention once the sample is loaded into the sample cup. A clean-up step is necessary for the analysis of drugs in biological matrices, but that could also be fully automated. Reagent costs per test with CE approach zero, because of the use of extremely small volumes of buffers, standards, and controls. We present data supporting CE as an alternative separation strategy for 28 commonly used therapeutic drugs. These can be resolved in less than 16 min with an optimized micelle-based buffer system.

Separation of cationic proteins via charge reversal in capillary electrophoresis.

Analysis of proteins by capillary electrophoresis requires strategies which minimize coulombic interactions with the capillary surface. Thus buffers with pH's above the isoelectric points (pI) of proteins, or near the pI of silanol are required for efficient separation. Covalent modification of the capillary surface is also effective; however, this strategy is technically difficult, abolishes endosmotic flow and suffers from the inherent lability of the siloxane bond. Finally, "dynamic coating" agents, which interact weakly with the capillary surface and therefore, must be included in the separation buffer, suffer from the potential interaction of coating agent with analytes, altering the selectivity of the system. In the following paper, we describe another approach which overcomes all of these difficulties, and demonstrate the ease of use, nondenaturing property, stability and selectivity of the coating strategy with several model protein systems.

Studies on histone acetyltransferase. Partial purification and basic properties.

A rapid and reproducible method for the purification of rat liver histone acetyltransferase is presented. Extraction of nuclei in low salt, followed by phenyl-Sepharose hydrophobic affinity chromatography, G-200 gel filtration in the presence of 1 M urea, CM-cellulose ion exchange and acetyllysine affinity chromatography minimize exposure of the enzyme to high salt. Evidence is provided which indicates that the instability of the enzyme activity is due in part to hydrophobic interactions. The molecular weight of the enzyme is 96,000 as judged by gel filtration. In agreement with others, the enzyme is unstable in the presence of divalent cations, although a requirement for low concentration of Mg2+ or Ca2+ was observed. The enzyme is also sensitive to sulfhydryl blocking agents and is susceptible to rapid thermal denaturation at 37 and 45 degrees C (t1/2 = 22.2 and 9.54 min, respectively). The optimum pH and the energy of activation for the reaction were pH 7.5 and 5230 +/- 378 cal/mol, respectively. In the presence of all five histones, the enzyme catalyzes the acetylation in the order of H3 greater than H4 greater than H2b greater than H2a greater than H1 and appears to operate in a nonprocessive manner. While no other isozymic forms of nuclear acetyltransferase were detected, the enzyme exhibits the properties of both nuclear isozymic forms which have been reported, histone acetyltransferase A and DB, observed in calf thymus and bovine lymphocytes, respectively.

Substrate and product inhibition initial rate kinetics of histone acetyltransferase.

Initial velocity and product inhibition kinetics of the histone acetyltransferase (EC 2.3.1.48) reaction indicate that the rat liver nuclear enzyme operates under a rapid equilibrium ordered bireactant mechanism. Histone adds first to the enzyme, and under the conditions of the experiment Ka = 0 as acetyl coenzyme A (CoA) concentration approaches saturating conditions. The Km for acetyl-CoA was 2.10 +/- 0.48 micrometer. Inhibition with acetyllysine resulted in a Kiq for the enzyme-acetyllysine complex of 1.96 +/- 0.30 mM. Inhibition with CoA yielded Kip for the ternary complex of 3.19 +/- 0.48 micrometer. These results indicate that the enzyme activity is comparatively independent of histone concentration, and, since the enzyme is sensitive only to acetyl-CoA and CoA concentrations, the enzyme will tend to maintain histones in the acetylated state.

Demonstration of cross-reacting material in Tay-Sachs disease.

Antibodies against placental hexosaminidase A and kidney alpha-subunits were raised in rabbits after cross-linking the antigens with glutaraldehyde. Anti-(alpha(n)-subunit) antiserum (anti-alpha(n)) precipitated hexosaminidase A but not hexosaminidase B, whereas anti-(hexosaminidase A) antiserum precipitated both hexosaminidases A and B. Specific anti-(hexosaminidase A) antiserum was prepared by absorbing antiserum with hexosaminidase B. Both anti-alpha(n) and anti-(hexosaminidase A) antisera precipitated the CR (cross-reacting) material from eight unrelated patients with Tay-Sachs disease. Immunotitration, immunoelectrophoresis, double-immunodiffusion and radial-immunodiffusion techniques were used to demonstrate the presence of CR material. The CR-material-antibody complex was enzymically inactive. Antiserum raised against kidney or placental hexosaminidase A, without cross-linking with glutaraldehyde, failed to precipitate the CR material, implying that treatment of the protein with glutaraldehyde exposes antigenic determinants that are hidden in the native protein. Since anti-(hexosaminidase B) antiserum did not precipitate the CR material during the immunoelectrophoresis of Tay-Sachs liver extracts, it is suggested that altered alpha-subunits do not combine with beta-subunits. By using immunotitration we have demonstrated the competition between the hexosaminidase B-free Tay-Sachs liver extract and hexosaminidase A for the common binding sites on monospecific anti-(cross-linked hexosaminidase A) antiserum. The amount of CR material in the liver samples from seven cases of Tay-Sachs desease was found to be in the same range as theoretically calculated alpha-subunits in normal liver samples. Similar results were obtained by the radial-immunodiffusion studies. The present studies therefore suggest that Tay-Sachs disease is caused by a structural-gene mutation.

Purification and properties of human kidney-cortex hexosaminidases A and B.

Hexosaminidases (EC 3.2.1.30) A and B from human kidney cortex were purified to homogeneity by using concanavalin A affinity chromatography, ion-exchange chromatography and gel filtration. The yield of homogeneous isoenzymes improved approx. 20-fold, giving preparations of hexosaminidases A and B with specific activities of about 200 and 325 units/mg of protein respectively. The kinetic and structural properties of kidney hexosaminidase isoenzymes were studied and compared with the hexosaminidase isoenzymes from human placenta. The amino acid composition of hexosaminidase A was significantly different from that of hexosaminidase B. In the event of success in developing enzyme-replacement therapy for Tay-Sachs and Sandhoff's diseases, this modified procedure can furnish larger amounts of homogeneous isoenzymes.

Interrelationship of hexosaminidases A and B: conformation of the common and the unique subunit theory.

Human kidney hexosaminidase A (beta-N-acetylglucosaminidase; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) is a heteropolymer of two immunologically distinct subunits designated as alpha and beta. Hexosaminidase B, however, is a homopolymer comprised entirely of beta subunits. When human kidney hexosaminidase A was dissociated into its subunits by p-hydroxymercuribenzoate, three distinct proteins having isoelectric points of pH 7.2.5.4, and 4.3 were isolated. The fraction having an isoelectric point of pH 7.2, designated as beta fraction, was electrophoretically and immunologically identical to hexosaminidase B and was enzymatically active. The proteins having isoelectric points of pH 5.4 and 4.3, designated as hexosaminidase Ai and alpha fractions, respectively, were enzymatically inactive and crossreacted with antiserum against hexosaminidase A and not with antiserum against hexosaminidase B. Upon incubation of p-hydroxymercuribenzoate-treated hexosaminidase A with dithiothreitol,, hexosaminidase A activity, as well as antigenicity, was regenerated, indicating that alpha and beta subunits hybridize to form hexosaminidase A. Antibodies raised in rabbits against beta fractions reacted with both hexosaminidase A and B, whereas the antibodies against alpha and hexosaminidase Ai fractions reacted only against hexosaminidase A. This would indicate that both fractions are composed only of subunits unique to hexosaminidase A. The molecular weights of alpha,beta, and hexosaminidase Ai fractions were estimated to be 47,000, 120,000, and 180,000 respectively, by Sephadex gel filtration. Upon urea-sodium dodecyl sulfate polyacrylamide electrophoresis, each of the three fractions dissociated into a single polypeptide having a molecular weight of approximately 18,000. It is concluded that p-hydroxymercuribenzoate dissociates hexosaminidase A, (alphabeta)3, into its subunits, and the beta subunits can reassociate to form relatively stable hexosaminidase B, (betabeta)3, while the alpha subunits reassociate in both the dimeric state, alpha2, and a polymeric state, alpha8.