Formation Of Stoichiometric Complex Research Articles

Etiohemin as a prosthetic group of myoglobin

Sperm whale myoglobin was reconstituted with etioheme and the stoichiometric complex formation was confirmed. The proton NMR spectrum of the deoxy myoglobin exhibits an NH signal from the proximal histidine at 78.6 ppm, indicating heme incorporation into the heme pocket to form the FeN(His-F8) bond. The appearance of a single set of the heme-methyl NMR signals shows that etioheme without acid side-chains specifically interacts with the surrounding globin. The visible spectral data suggest retention of a normal iron coordination structure. The functional and NMR spectral properties of etioheme myoglobin are similar to those of mesoheme myoglobin, reflecting the absence of the electron-withdrawing heme vinyl groups.

Expression of functionally active human antithrombin III.

Human antithrombin III cDNA was cloned into an expression vector suitable for transient expression in COS cells. Upon transfection COS cells secreted a single immunoreactive 58-kDa protein. Quantitation of secretion levels by ELISA indicated that at 44 hr posttransfection cells were secreting 48 +/- 5 ng of antithrombin III per 10(6) cells per 24 hr. Heparin-agarose chromatography resulted in the elution of the COS-derived protein as a broad band between 0.3 and 1.0 M NaCl. 35S-labeled medium from transfected cells reacted with human thrombin (1.5 ng/ml) in the absence of heparin. In 40 min, greater than 80% of the immunoreactive material was found as a higher molecular weight species, consistent with stoichiometric covalent complex formation. In a two-stage chromogenic thrombin inactivation assay, under pseudo-first-order conditions, at 16 nM antithrombin III the t1/2 was 74 min and 50 min for plasma and COS cell-derived antithrombin III, respectively, in the absence of heparin. In the presence of 17.4 nM high-affinity heparin, the t1/2 was 5.2 min and 2.2 min, respectively.

Proton NMR study of the cyanide metmyoglobin reconstituted with meso-tetraalkylhemins. Dynamic free rotation of the synthetic hemins in the heme pocket.

Incorporation of the three synthetic hemins, Fe(III) meso-tetraalkylporphyrins with the methyl, ethyl, or n-propyl groups, into apomyoglobin was followed by spectrophotometry, and the stoichiometric complex formation was confirmed. The reconstituted myoglobins bind with an equimolar amount of cyanide to exhibit visible absorption peaks at 419, 570, and 608 nm. The spectral feature was independent of the cyanide concentrations. Proton NMR spectra of the cyanide complexes resolved the pyrrole-proton signals of the hemins in a -5 to -15-ppm region, which is comparable with that of the corresponding signals of deuterohemin-containing low-spin methemoproteins. These spectral observations indicate the presence of the NC-Fe-N(His-F8) structure in the presently reconstituted cyanide metmyoglobins. The pyrrole-proton NMR signals of the hemins in cyanide metmyoglobins appeared as a singlet, doublet, or quartet for the methyl, ethyl, or n-propyl hemin complexes, respectively. The systematic NMR spectral changes suggest the dynamic free rotation of the alkylhemins about the Fe-N(His-F8) bond. Temperature-dependent NMR spectral transition of the meso-tetraethylhemin-reconstituted myoglobin was consistent with thermally regulated dynamic free rotation of the hemin in the myoglobin heme pocket.

Binding of benzo[a]pyrene by purified cytochrome P-450c.

Benzo[a]pyrene (BP) fluorescence-emission intensities in phospholipid micelles are quantitatively described over a broad range of lipid and BP concentrations by excitation that is linearly dependent upon BP concentration and an offsetting excimer quenching that is dependent upon the square of the BP concentration. The fluorescence of BP is quenched by the presence of cytochrome P-450c in proportion to the concentration of the cytochrome in the micelles and in accord with stoichiometric complex formation. Parallel optical titrations indicate a change in spin state of P-450c to a predominantly high-spin state that correlates directly with the percentage fluorescence quenching of complexed BP. Neither change occurs with five other purified forms of rat liver P-450 that have low activity in BP metabolism. N-Octylamine, a ligand that binds to the heme of P-450, competitively inhibits both the spin-state changes and the fluorescence quenching in equal proportion. The Kd for the interaction of BP with P-450c is exceptionally low (10 nM) relative to the Km for monooxygenation (ca. 1 microM). Decreasing the concentration of either dilauroylphosphatidylcholine or dioleoylphosphatidylcholine concomitantly increases the high-spin state (from 30% to 80%) of fully complexed P-450c and the fluorescence quenching (50-100%) of the complexed BP (half-maximal at 80 micrograms of lipid/mL). It is concluded that spin state and fluorescence quenching both reflect the same changes in the interaction of the BP with the P-450 heme.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific endogenous inhibitor of two forms of Ca++ activated neutral proteases in platelets.

An endogenous, specific inhibitor of high molecular weight has been isolated from bovine blood platelets, which inhibits the activity of the 2 forms of platelet Ca++ activated neutral proteases reported previously by us. The inhibition is not due to chelation of Ca++ but results from a stoichiometric complex formation.

Solubilities in binary solvent systems II. The importance of non-specific interactions

Solubilities have been determined at 25°C for p-benzoquinone in binary mixtures of cyclohexane with n-heptane, cyclooctane and isooctane, in mixtures of n-heptane with n-dodecane, and in binary mixtures of carbon tetrachloride with n-heptane and n-octane. The Nearly Ideal Binary Solvent (NIBS) model predicts these solubilities with a maximum deviation of 5% and an overall standard deviation of 1.6%. The NIBS model correctly predicts maxima for the mole fraction solubility of p-benzoquinone in both cyclohexane + n-heptane and cyclohexane + isooctane mixtures. The success of this simple model, based entirely on non-specific interactions, is significant considering the solubility of p-benzoquinone changes by a factor of 6 in the two binary solvent mixtures containing carbon tetrachloride. Solution models that attribute all solubility enhancement to the formation of stoichiometric complexes require several equilibrium constants to mathematically describe the 6-fold range of p-benzoquinone solubilities in these two solvent mixtures.

Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle.

An endogenous inhibitor of calcium-activated neutral protease was purified to homogeneity from rabbit skeletal muscle using ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex A-50 columns, chromatofocusing, and hydrophobic interaction chromatography on a phenyl-Sepharose CL-4B column. The purified inhibitor was shown to be a dimer of identical subunits and each subunit has a molecular weight of about 34,000. This inhibitor was remarkably thermo- and acid-stable. It was specific for calcium-activated neutral protease and had no effect on any other protease examined (trypsin, papain, alpha-chymotrypsin, bromelain, etc.). It is demonstrated that the inhibition is due to the formation of stoichiometric complex between two enzyme molecules and one inhibitor molecule.

Merocyanine 540 as a fluorescent probe of membranes: Staining of electrically excitable cells

With the exception of certain blood cells considered in the accompanying paper ( Valinsky, Easton and Reich, 1978), merocyanine 540 (MC 540), a fluorescent membrane probe, selectively stains the membranes of a wide variety of electrically excitable cells, but not those of nonexcitable cells. This reaction is Ca 2+-dependent when staining is performed in buffered iso-osmotic sucrose, Ca 2+-independent when staining proceeds at high ionic strength, inhibited by La 3+ and sodium Suramin, enhanced by controlled, low level photo-sensitization of cell-associated dye and essentially irreversible. These characteristics of the staining reaction depend upon the maintenance of both cell viability and a normal unperturbed membrane structure. Although the mechanisms involved in the staining specificity remain unknown, observation of MC 540 partitioning between benzene and water in model reactions indicates that dye transport into hydrophobic solvents is accompanied by the formation of stoichiometric complexes with cations and phospholipids. These results may suggest the existence of specific, possibly phospholipid-rich membrane domains that mediate complex formation with MC 540 in excitable cells; comparable domains either would not exist, or would be inaccessible at the external surfaces of nonexcitable cells.

Excited state solvation dynamics of 2-anilinonaphthalene

Nanosecond fluorescence spectroscopy has been used to study the interaction of 2-anilinonaphthalene with polar solvent molecules which is shown to result in stoichiometric complex formation at low polar solvent concentrations. This is followed by reorientation of the solvent cage when the concentration of polar solvent is high.

The interaction of amine-hydrocarbon exciplexes with small dipolar molecules - stoichiometric complex formation

The interaction of an intramolecular naphthalene-alkylamine exciplex with small dipolar molecules is shown to result in stoichiometric complex formation. The binding energy is suggested to be derived from dipole-diple and polarization interactions, a hypothesis which is applicable in general to the interaction of polar excited states with individual molecules of polar solvents.

A Study of Monolayer Penetration by the Radiotracer Method

Abstract The surface pressure - area isotherms of monolayers of cholesterol, hexadecyl alcohol and octadecyl amine on sodium alkyl sulfate solutions and the surface excess of alkyl sulfate have been measured by the film balance and the radiotracer methods respectively. These measurements were carried out with (1) the monolayers spread on the alkyl sulfate solutions, (2) those below which the detergent solutions were injected, and (3) those formed by the spreading of the insoluble speciesdetergent mixtures on water. All the surface pressure - area curves showed remarkable expansions; the results were essentially the same as those observed by Schulman et al. and by others. On the other hand, the surface excess of alkyl sulfates showed no appreciable increase in the case of cholesterol and hexadecyl alcohol, and it was too low to form a stoichiometric complex between the insoluble species and the alkyl sulfate; only in the presence of the octadecyl amine did it increase to an amount sufficient to form the 1 : 1 stoichiometric salt. Thus it has been concluded that the expansion of the surface pressure - area curve can not be an indication of the stoichiometric complex formation. It has been pointed out that the surface pressure of the detergent solution increases with the compression, even in the absence of a monolayer; this is the main cause for the expansion of the surface pressure - area curve of the penetrated monolayer. The phenomenon of monolayer penetration has been explained in terms of the formation of the adsorbed layer of alkyl sulfate, and the reactivity for penetration has been related to the surface activity of detergents.

Precipitation and inhibition of lysozyme by surface-active agents

Crystalline lysozyme has been interacted with an anionic, a cationic, and two nonionic surface-active agents (SAA). Quantitative precipitation of lysozyme by the ionic SAA used was obtained at ratios of the reactants consonant with the formation of stoichiometric complexes dependent upon salt linkages between the SAA and the oppositely charged groups in the enzyme. Neither of the nonionic SAA tested caused precipitation of the enzyme. The inactivation of lysozyme is shown to be constant over a 50-fold range of enzyme concentration when calculated on the basis of the ratio of SAA to enzyme. Inhibition of lysozyme activity as a result of interaction with ionic SAA was obtained only when the ionic SAA were present in substantial excess of the amount required for formation of stoichiometric complexes with oppositely charged groups in the enzyme. Neither of the two nonionic SAA studied altered the enzymatic activity of lysozyme.