sepharose and benzeneboronic-acid

The complexity of the human plasma proteome is greatly increased by post-translational modifications. Besides physiological modifications, pathological conditions such as diabetes are responsible for adding to this complexity by producing advanced glycation endproducts (AGEs). When searching for specific biomarkers it is a prerequisite to reduce this complexity prior to analysis. To do this, agarose hydrogel was used to create a high-capacity affinity layer on the modified aluminum surface of MALDI (matrix-assisted laser desorption/ionization) targets. 3-Aminophenylboronic acid was immobilized via cyanogen bromide activation as a ligand for affinity sorption of glycated proteins, followed by their direct detection by MALDI. High protein capacity of prepared MALDI chips, efficient separation and low non-specific protein binding were demonstrated. The results show that phenylboronic acid modified hydrogels are very suitable for creating affinity surfaces for the high-throughput analysis of AGEs.

Topics: Aluminum; Boronic Acids; Cyanogen Bromide; Fluorescence; Glycation End Products, Advanced; Glycoproteins; Hydrogels; Indicators and Reagents; Ligands; Mass Spectrometry; Protein Array Analysis; Proteomics; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrophotometry, Ultraviolet

Aminophenylboronate-substituted agarose in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, pH 8.5, selectively adsorbs immunoglobulins and complement factors C3 and C4 from human serum. The selectivity of binding is strongly influenced by the presence of magnesium chloride in the sample buffer. Adsorbed immunoglobulins are quantitatively eluted by sorbitol, but only partially by ethylene glycol or methylcellosolve. Aniline-agarose of a similar degree of substitution shows only weak adsorption of serum proteins under similar experimental conditions, thus indicating the important contribution of the boronate moiety to this interaction. Immunoglobulin adsorption seems not to be due to the cis-diol complexation used extensively for the chromatographic determination of non-enzymatically glucosylated proteins. Hydrophobic and pi-pi interactions with the aromatic structure of the ligand seem also to contribute to protein binding. The behaviour of aminophenylboronate-liganded agarose is, in some respects, rather similar to that of the so-called "thiophilic adsorbents".

Topics: Adsorption; Blood Proteins; Boronic Acids; Chromatography, Liquid; Complement C3; Complement C4; Electrophoresis, Polyacrylamide Gel; Glucose; Humans; Hydrogen-Ion Concentration; Immunoglobulins; Magnesium Chloride; Sepharose; Serum Albumin

Boronic acids are active-site inhibitors of serine beta-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify beta-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purification of Staphylococcus aureus beta-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal beta-lactamase exhibited non-active-site-mediated adsorption to the agarose used as a support for the meta-aminophenylborate ligand, preventing the recovery of beta-lactamase from the column. Second, the staphylococcal beta-lactamases exhibited low affinity for meta-aminophenylborate with inhibition constants (Kis) ranging from 8.0 x 10(-3) to 20.0 x 10(-3) M. These problems were resolved by modifying the buffers utilized during chromatography and increasing the dimensions of the affinity column, and a two-stage procedure consisting of cation-exchange chromatography followed by affinity chromatography was used to purify each of the four variants of staphylococcal beta-lactamase. The mean specific activities of the purified type A, B, C, and D beta-lactamases were 44.6, 12.2, 10.6, and 30.8 mumol of nitrocefin hydrolyzed per min/mg of protein, respectively. Dimer formation, presumably from intramolecular cysteine-cysteine cross-linking, was observed with the type D beta-lactamase but not with the type A, B, or C enzyme.

Topics: beta-Lactamase Inhibitors; beta-Lactamases; Boronic Acids; Chromatography, Affinity; Chromatography, Ion Exchange; Culture Media; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; Sepharose; Staphylococcus aureus

Detergent-solubilized plasma-membrane proteins from lymphocytes of patients with chronic lymphocytic leukaemia were applied to phenylboronic acid-agarose. Some of the polypeptides were bound specifically and subsequently eluted with sorbitol. Immunoprecipitation analyses showed that no surface immunoglobulin M was bound, but that most of the histocompatibility antigens HLA-A, HLA-B, HLA-C and HLA-DR were.

Topics: alpha-Fetoproteins; Boronic Acids; Chromatography, Agarose; Chromatography, Gel; Deoxycholic Acid; HLA Antigens; Humans; Leukemia, Lymphoid; Lymphocytes; Membrane Proteins; Octoxynol; Polyethylene Glycols; Receptors, Antigen, B-Cell; Sepharose