Cyclic polyether-protonated organic amine binding: significance in enzymatic and ion transport processes.

The cyclic polyether, 18-crown-6, reacts with protonated amines in methanol to form complexes whose formation constants (log K) decrease in the order NH4+, RNH3+ greater than R2NH2+ greater than R3NH+. In the case of the organic amines, this stability order is identical to the earlier observed permeability sequence for protonated organic amines in glyceryl dioleate bilayers treated with valinomycin, nonactin, or gramicidin, and in bullfrog and rabbit gallbladder membranes. The decrease in log K values in the above series is primarily a result of decreased enthalpy change (deltaH) values, the entropy change (TdeltaS) term being essentially constant for the systems studied.

Binding of alkali metal ions by cyclic polyethers: significance in ion transport processes.

Values for the formation constant (log K), the change in enthalpy (triangle upH degrees ), and the change in entropy (triangle upS degrees ) have been determined for the interaction of lithium, sodium, potassium, rubidium, and cesium ions with the two isomers of the cyclic polyether, 2,5,8,15,18,21-hexaoxatricyclo[20.4.0.0(9,14)] hexacosane. The stability order of these metal ions with either isomer is identical to the permeability order for these same metal ions with the structurally related antibiotics, valinomycin and monactin.

Characterization of His-X3-His sites in alpha-helices of synthetic metal-binding bovine somatotropin.

Variants of bovine somatotropin have been engineered to contain synthetic metal-binding sites consisting of two solvent-exposed histidines separated by a single turn of an alpha-helix (His-X3-His variants). The affinities of these proteins for Cu(II) were characterized by measuring their partition coefficients in an aqueous two-phase polymer system. The partition coefficients were used to generate binding constants for formation of a complex between the engineered metal-binding site and Cu(II) chelated to an iminodiacetic acid derivative of polyethylene glycol. For three His-X3-His variants described here, these constants range from 2 x 10(4) to 1.6 x 10(6) M-1. The metal affinity of a His-X3-His site depends on the rigidity of the helix into which the site is engineered. The affinities of the His-X3-His sites for Cu(II) are large enough to dramatically increase not only the partitioning of these proteins in aqueous two-phase systems, but also their retention times on a metal-affinity chromatography column. Both these features can greatly facilitate the purification of engineered proteins. Criteria for choosing positions for incorporating metal-binding sites are discussed.

Capillary zone electrophoresis of histidine-containing compounds.

Capillary zone electrophoresis has been tested for the separation of angiotensins, cationic heptapeptides and model histidine derivatives. Good separation efficiencies are seen for peptides and model compounds with negative to small positive net charges. For net charge greater than +2, addition of putrescine to pH 6 buffer greatly suppresses ion exchange at anionic sites on fused silica. When operating at pH values where histidine groups are neutral, addition of Zn2+ allows separations based on metal, rather than proton, binding. Separation efficiencies and relative migration times are dependent on capillary length when ion-exchange behavior occurs.

Purification of glycogen phosphorylase isozymes by metal-affinity chromatography.

Mammalian phosphorylase isozymes from muscle, brain and liver were expressed in Escherichia coli and purified from the crude bacterial cell extracts in one step using a copper-loaded, metal-affinity matrix. Good chromatographic behavior, enzyme activity and protein stability were maintained by judicious choice of pH and buffer which contained 250 mM sodium chloride and 25 mM beta-glycerophosphate at pH 7.0. Small amounts of beta-mercaptoethanol and EDTA in the buffers further stabilized the enzymes, but stripped some of the metal from the column which, nonetheless, retained good chromatographic characteristics. Owing to the presence of multiple surface histidine residues in the phosphorylase dimers, good enzyme purities (90-98%) and recoveries (>90%) were routinely obtained from crude bacterial lysates after two passes through the copper column. Of the various metal ions which were investigated, Cu2+ gave the best chromatographic results. Imidazole gradients at constant pH were used to selectively desorb the phosphorylase from the metal column whose capacity for phosphorylase binding in the presence of bacterial proteins exceeded 30 mg enzyme per milliliter of matrix.

Regulation of serine protease activity by an engineered metal switch.

A recombinant trypsin was designed whose catalytic activity can be regulated by varying the concentration of Cu2+ in solution. Substitution of Arg-96 with a His in rat trypsin (trypsin R96H) places a new imidazole group on the surface of the enzyme near the essential active-site His-57. The unique spatial orientation of these His side chains results in the formation of a stable, metal-binding site that chelates divalent first-row transition-metal ions. Occupancy of this site by a metal ion prevents the imidazole group of His-57 from participating as a general base in catalysis. As a consequence, the primary effect of the transition metal ion is to inhibit the esterase and amidase activities of trypsin R96H. The apparent Ki for this inhibition is in the micromolar range for copper, nickel, and zinc, the tightest binding being to Cu2+ at 21 microM. Trypsin R96H activity can be fully restored by removing the bound Cu2+ ion with EDTA. Multiple cycles of inhibition by Cu2+ ions and reactivation by EDTA demonstrate that reversible regulatory control has been introduced into the enzyme. These results describe a novel mode of inhibition of serine protease activity that may also prove applicable to other proteins.

Structure and intramolecular hydrogen bonding of 1-phenazinecarboxylic acid.

C13H8N2O2, Mr = 224.22, monoclinic, P2(1)/n, a = 18.149 (2), b = 14.2768 (9), c = 3.8191 (3) A, beta = 92.029 (6) degrees, V = 988.95 A3, Z = 4, Dx = 1.51 g cm-3, lambda (Cu K alpha) = 1.54178 A, mu = 7.61 cm-1, F(000) = 464, T = 163 (1) K, R = 0.037 for 1479 data with I greater than or equal to 2 sigma (I). An intramolecular hydrogen bond is observed between the carboxyl hydrogen atom and the nearby nitrogen atom with the N...O distance being 2.67 A.

Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12.

The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid. The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose. The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue. All four cysteines were shown to be present as thiols. Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis. Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M. A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.

Cu(II)-binding properties of a cytochrome c with a synthetic metal-binding site: His-X3-His in an alpha-helix.

A metal-binding site consisting of two histidines positioned His-X3-His in an alpha-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c. The synthetic metal-binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography. The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 x 10(4) M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy. Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.

Structure of an engineered, metal-actuated switch in trypsin.

The X-ray crystal structure of the copper complex of the rat trypsin mutant Arg96 to His96 (trypsin R96H) has been determined in order to ascertain the nature of the engineered metal-binding site and to understand the structural basis for the metal-induced enzymatic inhibition. In the structure, the catalytically essential His57 residue is reoriented out of the active-site pocket and forms a chelating, metal-binding site with residue His96. The copper is bound to the N epsilon 2 atoms of both histidine residues with Cu-N epsilon 2 = 2.2 A and N epsilon 2-Cu-N epsilon 2 = 89 degrees. The metal is clearly bound to a third ligand leading to a distorted square planar geometry at Cu. The X-ray results do not unambiguously yield the identity of this third ligand, but chemical data suggest that it is a deprotonated, chelating Tris molecule which was used as a carrier to solubilize the copper in alkaline solution (pH 8.0). Upon reorientation of His57, a unique water molecule moves into the active site and engages in hydrogen-bonding with Asp102-O delta 2 and His57-N delta 1. Except for small movements of the peptide backbone near His96, the remainder of the trypsin molecule is isostructural with the native enzyme. These data support the notion that the effective inhibition of catalytic activity by metal ions observed in trypsin R96H is indeed caused by a specific and reversible reorganization of the active site in the enzyme.

Human vascular permeability factor. Isolation from U937 cells.

Human vascular permeability factor (hVPF) is a glycoprotein that promotes fluid and protein leakage from blood vessels. The function of hVPF is at present unknown, but the potent bioactivities of this protein suggest that it could act during inflammation, wound healing, and tumor angiogenesis. hVPF was purified from serum-free conditioned medium of the human histiocytic lymphoma cell line U937 as a disulfide-linked dimeric 40-kDa protein that promoted dermal blood vessel leakage in guinea pigs at a dose of 20 ng (3 x 10(-9) M) and promoted in vitro endothelial cell growth at concentrations as low as 50 PM. Multiple forms of hVPF with apparent pI values greater than 7.5 were resolved using pH gradient electrophoresis. Antibodies against guinea pig vascular permeability factor were found to cross-react with hVPF. The N-terminal amino acid sequence of hVPF was similar to, but not identical with, the N-terminal sequence of guinea pig vascular permeability factor.