sepharose and sodium-borohydride

Cationized agaroses with different degrees of substitution (0.04-0.77) were synthesized, employing 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC). The influence of different reaction parameters on the substitution degree and molecular weight was evaluated. The investigated parameters were concentration of reagents, temperature, time, and addition of NaBH(4). The products were characterized by means of scanning electronic microscopy, infrared spectroscopy, viscosimetry, and NMR spectroscopy. Methanolysis products were studied by electrospray ionization mass spectrometry. The higher the concentration of CHPTAC employed, a higher degree of substitution was obtained, if the optimum concentration of NaOH in each case was employed. Insufficient quantities of NaOH reduced epoxide formation and the reacting alkoxides of the polysaccharide, whereas an excess of NaOH favored degradation of the epoxide and decrease in the molecular weight of the product. A reaction time of 2h was sufficient to obtain products with the maximum degree of substitution for each case. The addition of NaBH(4) gave products with a slightly higher molecular weight, but the extra cost involved should not justify its use for large-scale application.

Topics: Borohydrides; Cations; Infrared Rays; Magnetic Resonance Spectroscopy; Microscopy, Electron, Scanning; Propanols; Quaternary Ammonium Compounds; Sepharose; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Infrared; Viscosity

Pig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate in which the active site glutamate (E344) of both subunits each forms thiolester links with coenzyme A. Reaction of this dimeric enzyme-coenzyme A species with sodium borohydride leads to inactivation of the enzyme and reduction of the thiolester on both subunits to the corresponding enzyme alcohol, as judged by electrospray mass spectrometry. Reaction of the dimeric enzyme-coenzyme A intermediate with either succinate or acetoacetate, however, results in only one-half of the coenzyme A being transferred to the acceptor carboxylate to form either succinyl-coenzyme A or acetoacetyl-coenzyme A. Reaction of this latter enzyme species with borohydride caused no loss of enzyme activity despite the reduction of the remaining half of the enzyme-coenzyme A thiolester to the enzyme alcohol. That this catalytic asymmetry existed between subunits within the same enzyme dimer was demonstrated by showing that the enzyme species, created by successive reaction with acetoacetyl-coenzyme A and succinate, bound to Blue MX-2G Sepharose through the remaining available active site and could be eluted as a single chromatographic species by succinyl-coenzyme A. It is concluded that while both of the subunits of the succinate-coenzyme A transferase dimer are able to form enzyme-coenzyme A thiolester intermediates, only one subunit is competent to transfer the coenzyme A moiety to a carboxylic acid acceptor to form the new acyl-coenzyme A product. The possible structural basis for this catalytic asymmetry and its mechanistic implications are discussed.

Topics: Acyl Coenzyme A; Animals; Borohydrides; Carboxylic Acids; Catalysis; Chromatography, Agarose; Coenzyme A-Transferases; Dimerization; Esters; Mitochondria, Heart; Myocardium; Peptide Fragments; Sepharose; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity; Sulfhydryl Compounds; Swine

The glycolipid profiles of two Leishmania major strains which differ in their expression of the major glycoconjugate, lipophosphoglycan (LPG), have been compared. All the glycolipids in these strains belong to a class of glycoinositolphospholipids (GIPLs) which can be metabolically labelled with [3H]inositol and are sensitive to phosphatidylinositol-specific phospholipase C. The major glycolipids in the LPG-producing L. major strain V121 are tetraglycosyl phosphatidylinositol (GIPL-1), pentaglycosyl phosphatidylinositol (GIPL-2), hexaglycosyl phosphatidylinositol (GIPL-3) and lyso-GIPL-3. These were identified by their sensitivity to lipases, and by BioGel P4 chromatography of the glycan fragments released after nitrous acid deamination. Similar glycolipids are also present in the LPG-deficient L. major strain LRC-L119. However, this strain also produces several highly polar GIPLs (GIPL-4, 5 and 6) which are absent from V121 and which may represent truncated forms of LPG. Evidence is presented showing that the LPG and GIPLs from both strains contain galactofuranose, based on identification of labelled arabinose after mild periodate oxidation and reduction with NaB [3H]4. Furthermore, analysis of the deaminated glycan moieties after mild acid hydrolysis suggests that the GIPLs and the glycolipid anchor of LPG contain a common glycan core, which includes the galactofuranose. Finally, radiolabelling of intact cells indicates that there is restricted expression of some of the GIPLs in the plasma membrane and that the GIPL-2 is the predominant cell surface glycolipid. These data are consistent with some, but not all, of the GIPLs in V121 having a major role as precursors to the glycolipid core of LPG.

Topics: Animals; Borohydrides; Glycolipids; Glycosphingolipids; Glycosylphosphatidylinositols; Inositol; Isotope Labeling; Leishmania tropica; Membrane Lipids; Periodic Acid; Phosphatidylinositols; Sepharose

A novel approach to the preparation of immunopeptide-carrier protein conjugates of improved chemical definition based on a solid-phase synthetic protocol combining incorporation of a Cys(Npys) N-terminal residue with systematic acetylation after every coupling, is described. The potential of the method is demonstrated in a synthesis of the tridecapeptide (Npys)-Cys-Val-Asn-Tyr-Ile-Arg-Lys-Arg-Ser-Leu-Gln-Thr-Val-OH in which the main product is purposely contaminated by a number of shorter truncated sequences resulting from intentionally defective couplings. From this peptide crude, and rather independently of its complexity, the target sequence can be selectively recovered and attached to a carrier molecule through a disulfide bond formed by reaction of the Npys-protected cysteine residue and a thiol function in the carrier. The process can be properly named purification-by-conjugation.

Topics: Amino Acid Sequence; Antigens; Borohydrides; Carrier Proteins; Chromatography, High Pressure Liquid; Hemocyanins; Molecular Sequence Data; Peptide Fragments; Peptides; Protein Binding; Sepharose; Serum Albumin, Bovine

Glucosylated albumin of human serum isolated by dye-ligand chromatography on blue Sepharose, was not found to be completely reducible by sodium borohydride. The percentage reducible hexose as judged by phenol-sulphuric acid reaction was in the range of 49.7 +/- 12.8 in control subjects (n = 24) and 53.8 +/- 14.2 in diabetics (n = 50). Increase in the level of total hexose bound to albumin and reducible hexose were equally significant in diabetes (P less than 0.001). Sodium chloride gradient elution during chromatography on blue Sepharose showed that glucosylated albumin had lesser affinity than the native protein to the matrix. It is proposed that an addition product between hexose and albumin is formed during nonenzymatic reaction and this adduct is fairly stable and is not reducible by sodium borohydride.

Topics: Adult; Blood Glucose; Borohydrides; Chromatography, Agarose; Diabetes Mellitus; Hemoglobins; Hexoses; Humans; Middle Aged; Oxidation-Reduction; Sepharose; Serum Albumin