James H. Hageman

Boronic acids for affinity chromatography: Spectral methods for determinations of ionization and diol-binding constants

Arylboronic acids attached to solid matrices have proved useful for the diol-specific chromatography of biomolecules and affinity purification of enzymes by exchangeable-ligand chromatography. The latter use has been limited by the intrinsic ionization constant (pKa approximately 9) of the most common commercial products. The synthesis of several arylboronic acids with ionization constants near neutrality are described, and the application of a new general, spectral-difference method for determining acid ionization constants and formation constants with fructose is developed. In particular 4-(N-methyl) carboxamido-benzeneboronic acid was found to have a pKa of 7.86 and a formation constant with D-fructose of 8600. It was stable toward acid or base hydrolysis. We suggest that 4-carboxybenzeneboronic acid might be useful for preparing matrices for enzyme affinity chromatography.

Synthesis of a fluorescent boronic acid which reversibly binds to cell walls and a diboronic acid which agglutinates erythrocytes

Abstract N-(5-dimethylamino-1-napthalene sulfonyl)-3-aminobenzene boronic acid (Dns-PBA) and N,N′- bis -3(dihydroxylborylbenzene)adipamide (Bis-PBA) were synthesized. The former is found to reversibly associate with Bacillus subtilis , apparently through boronate diester linkages with carbohydrates on the cell surface. The latter displays the lectin-like property of agglutinating red blood cells.

Polyacrylamide-boronate beads saturated with biomolecules: A new general support for affinity chromatography of enzymes

Aminoethyl polyacrylamide beads (P-150) were quantitatively coupled to m-aminobenzeneboronic acid to yield a product containing 1.3 mmoles of boronic acid per gram (dry weight) of gel. The average pKa of the insolubilized boronic acid was determined to be 9.2. At pH 8.45 a number of enzyme substrates and cofactors, including NAD+, citric acid, pyridoxal, and epinephrine, were shown to bind to P-150-boronate beads packed in chromatography columns. At saturation, the P-150-boronate beads bound 2.5 to 80 μmoles of substrate or cofactor per ml of wet packed gel. A P-150-boronate column saturated with uridine 5′-triphosphate was used to achieve a 1000-fold purification of the enzyme uridine 5′-diphosphate glucose pyrophosphorylase (EC 2.7.7.9) from the slime mold, Physarum polycephalum.

Assay of adenylate cyclase by use of polyacrylamide--boronate gel columns.

A method is described for covalent attachment of up to 1.1 mmol of m-aminobenzeneboronic acid/g dry weight of polyacrylamide beads. Small (4 ml) columns of the derivatized beads have been used to separate quantitatively cyclic AMP from ATP in a single-step procedure. Columns of the polyacrylamide-boronate gel have been used as the basis of a convenient new assay of the soluble adenylate cyclase prepared from nuclei of Physarum polycephalum.

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Abstract Clarified cell-free extracts were prepared from rapidly dividing Bacillus subtilis cells and from rabbit liver cells. These extracts were treated with [ 3 H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the B. subtilis extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.

Photoredox reactions of metal ions for photochemical solar energy conversion

Abstract Solar energy conversion to chemical potential energy is thermodynamically feasible by many routes. One possible route is the photochemical reaction of metal ions in water to produce hydrogen and an oxidizer. The photooxidation of several low-valent transition metal ions, including europium(II), vanadium(II), and copper(I) complexes, proceeds in aqueous acidic media according to: Quantum yields in 1.0 M hydrochloric or perchloric acid at 313 nm are: Φ Eu(III) = 0.16, Φ V(III) = 0.15, and Φ Cu(II) = 0.34. This reaction proceeds in visible light with a minimum of photochemical complications for Eu(II) and Cu(I) salts, and since the oxidation of copper(I) halo-complexes is endergic and hence potentially useful for energy storage, the mechanism of photooxidation has been studied. The product quantum yield is strongly affected by the acidity, irradiation wavelength and H-atom scavengers. Photoredox reactions of a number of metal ions and the requirements for using such in a solar energy scheme are discussed.

Boronic Acid matrices for the affinity purification of glycoproteins and enzymes.

Boronic acids immobilized on insoluble matrices have been used for over a decade to purify proteins and enzymes (1,2). Boronic acid matrices have been used to purify: 1. Glycoproteins, 2. Enzymes for which a boronic acid moiety is an inhibitor, and 3. Enzymes that bind to specific dial-containing cofactors (such as UTP or NADP(+)).