Selective Release of Proteins from Spirillum itersonii by Tris(hydroxymethyl)aminomethane and Ethylenediaminetetraacetate

Treatment of Spirillum itersonii with tris(hydroxymethyl)aminomethane (Tris)-ethylenediaminetetraacetate (EDTA) results in the quantitative release of alkaline phosphatase and ribonuclease into the surrounding medium. At the same time, about 90% of the total cellular soluble cytochrome c is liberated. This process occurs within 1 min of treatment at both 24 and 4 C. Release of these proteins by Tris-EDTA treatment is highly selective, since only 9% of the total cell protein is liberated, concomitantly with less than 5% ribonucleic acid, deoxyribonucleic acid, and malate dehydrogenase. Different sigmoidal curves are obtained for release of proteins as a function of EDTA concentration. The order of liberation with increasing EDTA is as follows: alkaline phosphatase, protein, soluble cytochrome c, and ribonuclease. Treatment of cells with Tris-EDTA under conditions which cause extensive loss of alkaline phosphatase, soluble cytochrome c, and ribonuclease results in cell death, with cessation of protein and ribonucleic acid synthesis. Cells treated with EDTA in phosphate buffer (in the absence of Tris) liberate a large portion of their soluble cytochrome c, but negligible amounts of alkaline phosphatase and ribonuclease. Addition of Tris to cells pretreated with phosphate-buffered EDTA releases high levels of alkaline phosphatase, but not ribonuclease. These results suggest that a common surface alteration is not solely responsible for release of periplasmic proteins. More likely, each protein of the periplasm is bound in an independent and specific manner.     

Mechanism of irreversible inhibition of O2 evolution in photosystem II by Tris(hydroxymethyl)aminomethane.

The dark reaction of tris(hydroxymethyl)aminomethane (Tris) with the O2-evolving center of photosystem II (PSII) in the S1 state causes irreversible inhibition of O2 evolution. Similar inhibition is observed for several other amines: NH3, CH3NH2, (CH3)2NH, ethanolamine, and 2-amino-2-ethyl-1,3-propanediol. In PSII membranes, both depleted of the 17- and 23-kDa polypeptides and undepleted, the rate of reaction of Tris depends inversely upon the Cl- concentration. However, the rate of reaction of Tris is about 2-fold greater with PSII membranes depleted of the 17- and 23-kDa polypeptides than with undepleted PSII membranes. We have used low-temperature electron paramagnetic resonance (EPR) spectroscopy to study the effect of Tris on the oxidation state of the Mn complex in the O2-evolving center, to monitor the electron-donation reactions in Tris-treated samples, and to observe any loss of the Mn complex (forming Mn2+ ions) after Tris treatment. We find that Tris treatment causes loss of electron-donation ability from the Mn complex at the same rate as inhibition of O2 evolution and that Mn2+ ions are released. We conclude that Tris reduces the Mn complex to labile Mn2+ ions, without generating any kinetically stable, partially reduced intermediates, and that the reaction occurs at the Cl(-)-sensitive site previously characterized in studies of the reversible inhibition of O2 evolution by amines.     

Tris(hydroxymethyl)aminomethane inhibits calcium uptake in vascular smooth muscle.

This report demonstrates that the commonly used buffering agent Tris(hydroxymethyl)aminomethane (Tris) in concentrations of 5 and 30 mM inhibits calcium (Ca2+) uptake in both rat aortic and portal venous smooth muscle. The data indicates that total exchangeable Ca2+ in portal vein is reduced by about 35% in 5 or 30 mM Tris, while the intracellular exchangeable Ca2+ is not significantly altered. On the other hand, in aortic smooth muscle, while 30 mM Tris reduces total exchangeable Ca2+ by about 20%, intracellular Ca2+ is reduced by 44% in 5 mM Tris and by 55% in 30 mM Tris. The present studies, thus, reveal that Tris exerts significant inhibitory effects on exchangeability and transmembrane movement of Ca2+ in at least two different types of smooth muscle.     

Tris(hydroxymethyl)aminomethane buffer modification of Escherichia coli outer membrane permeability.

The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8. Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase. The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer. The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer. Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component. Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.     

Quantitation of the Action of Ethylenediaminetetraacetic Acid and Tris(hydroxymethyl)aminomethane on a Gram-Negative Bacterium by Vancomycin Adsorption

Changes in the cell wall of a species of Flavobacterium can be quantitated by measuring vancomycin adsorption. Treatment of the cells with ethylenediaminetetraacetic acid or tris(hydroxymethyl)aminomethane (or both) increased adsorption of the antibiotic.     

Resurveying the Tris buffer solution: the specific interaction between tris(hydroxymethyl)aminomethane and lysozyme

An unusual phenomenon, the specific interaction between tris(hydroxymethyl)aminomethane (Tris) and lysozyme (LZM), was demonstrated for the first time by rapid screen analysis of interactions using a quartz crystal microbalance (QCM) biosensor. This phenomenon was also observed in a surface plasmon resonance (SPR) system. Further study using high-performance affinity chromatography (HPAC) confirmed this specific interaction between LZM and immobilized Tris with an apparent dissociation constant (K_D) of 6.7 × 10⁻⁵ M. Molecular docking was carried out to identify possible modes of binding between LZM and Tris linked to a binding arm. The estimated binding free energy was −6.34 kcal mol⁻¹, corresponding to a K_D of 2.3 × 10⁻⁵ M, which correlated well with the experimental value. Based on the docking model, the three hydroxyl groups of Tris form intermolecular H bonds with Asp52, Glu35, and Ala107 in LZM. This study reinforces the importance of buffer selection in quantitative biochemical investigations. For a lysozyme ligand binding study, it is better to avoid using Tris when the ligands under study are weak binders.     

Catalysis of hydrolysis and aminolysis of benzylpenicillin mediated by a ternary complex with zinc ion and Tris(hydroxymethyl)aminomethane

It was discovered that the combination of zinc ion and Tris in the pH range 7.5–10 is a very effective true catalyst for hydrolysis and aminolysis (by Tris) of benzylpenicillin. Both Cu²⁺ and Ni²⁺ ions were nonreactive in this system. The rate of loss of penicillin from solution was found to be a linear function of zinc ion at concentrations up to 10^?5M, while the dependence upon Tris concentration is maximal at about 0.02–0.03 M at each pH studied. At high penicillin concentrations saturation kinetics was observed. Product assays showed the major product to be a penicilloic acid at low Tris concentrations and N(penicilloyl)-Tris at high Tris concentrations. A mechanism is proposed which suggests that the reaction is mediated by a ternary complex in which the metal ion acts to bring the reactants (penicillin and Tris) into close proximity and to lower the pK_a of a Tris hydroxyl, creating a strong nucleophile. This mechanism also explains the results of the product assays and the lack of reactivity of the other metal ions tested. This reaction may be related to the mechanism for a known zinc-dependent β-lactamase.     

Co2+, Cu2+, and Zn2+ accumulation by cyanobacterium Spirulina platensis

The Spirulina platensis biomass was characterized for its metal accumulation as a function of pH, external metal concentration, equilibrium isotherms, kinetics, effect of co-ions under free (living cells, lyophilized, and oven-dried) and immobilized (Ca-alginate and polyacrylamide gel) conditions. The maximum metal biosorption by S. platensis biomass was observed at pH 6.0 with free and immobilized biomass. The studies on equilibrium isotherm experiments showed highest maximum metal loading by living cells (181.0 +/- 13.1 mg Co(2+)/g, 272.1 +/- 29.4 mg Cu(2+)/g and 250.3 +/- 26.4 mg Zn(2+)/g) followed by lyophilized (79.7 +/- 9.6 mg Co(2+)/g, 250.0 +/- 22.4 mg Cu(2+)/g and 111.2 +/- 9.8 mg Zn(2+)/g) and oven-dried (25.9 +/- 1.9 mg Co(2+)/g, 160.0 +/- 14.2 mg Cu(2+)/g and 35.1 +/- 2.7 mg Zn(2+)/g) biomass of S. platensis on a dry weight basis. The polyacrylamide gel (PAG) immobilization of lyophilized biomass found to be superior over Ca-alginate (Ca-Alg) and did not interfere with the S. platensis biomass biosorption capacity, yielding 25% of metal loading after PAG entrapment. The time-dependent metal biosorption in both the free and immobilized form revealed existence of two phases involving an initial rapid phase (which lasted for 1-2 min) contributing 63-77% of total biosorption, followed by a slower phase that continued for 2 h. The metal elution studies conducted using various reagents showed more than 90% elution with mineral acids, calcium salts, and Na(2)EDTA with free (lyophilized or oven-dried) as well as immobilized biomass. The experiments conducted to examine the suitability of PAG-immobilized S. platensis biomass over multiple cycles of Co(2+), Cu(2+), and Zn(2+) sorption and elution showed that the same PAG cubes can be reused for at least seven cycles with high efficiency.     

Probing the coordination properties of glutathione with transition metal ions (Cr2+,Mn2+,Fe2+,Co2+, Ni2+,Cu2+,Zn2+,Cd2+,Hg2+) by density functional theory

Complexes formed by reduced glutathione (GSH) with metal cations (Cr²⁺, Mn²⁺,Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺,Hg²⁺) were systematically investigated by the density functional theory (DFT). The results showed that the interactions of the metal cations with GSH resulted in nine different stable complexes and many factors had an effect on the binding energy. Generally, for the same period of metal ions, the binding energies ranked in the order of Cu²⁺>Ni²⁺>Co²⁺>Fe²⁺>Cr²⁺>Zn²⁺>Mn²⁺; and for the same group of metal ions, the general trend of binding energies was Zn²⁺>Hg²⁺>Cd²⁺. Moreover, the amounts of charge transferred from S or N to transition metal cations are greater than that of O atoms. For Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺ and Hg²⁺ complexes, the values of the Wiberg bond indices (WBIs) of M-S (M denotes metal cations) were larger than that of M-N and M-O; for Cr²⁺ complexes, most of the WBIs of M-O in complexes were higher than that of M-S and M-N. Furthermore, the changes in the electron configuration of the metal cations before and after chelate reaction revealed that Cu²⁺, Ni²⁺,Co²⁺ and Hg²⁺ had obvious tendencies to be reduced to Cu⁺,Ni⁺,Co⁺ and Hg⁺ during the coordination process.     

Iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26.1: characterization and reconstitution with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+

Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.1 were depleted of Fe by a simple procedure involving reversible dissociation of the H subunit. The resulting intact Fe-depleted RCs contained 0.1-0.2 Fe per RC as determined from atomic absorption and electron paramagnetic resonance (EPR) spectroscopy. Fe-depleted RCs that have no metal ion occupying the Fe site differed from native RCs in the following respects: (1) the rate of electron transfer from QA- to QB exhibited nonexponential kinetics with the majority of RCs having a rate constant slower by only a factor of approximately 2, (2) the efficiency of light-induced charge separation (DQA----D+QA-) produced by a saturating flash decreased to 63%, and (3) QA appeared readily reducible to QA2-. Various divalent metal ions were subsequently incorporated into the Fe site. The electron transfer characteristics of Fe-depleted RCs reconstituted with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+ were essentially the same as those of native RCs. These results demonstrate that neither Fe2+ nor any divalent metal ion is required for rapid electron transfer from QA- to QB. However, the presence of a metal ion in the Fe site is necessary to establish the characteristic, native, electron-transfer properties of QA. The lack of a dominant role of Fe2+ or other divalent metals in the observed rate of electron transfer from QA- to QB suggests that a rate-limiting step (for example, a protonation event or a light-induced structural change) precedes electron transfer.     

Super-high-affinity binding site for [3H]diazepam in the presence of Co2+, Ni2+, Cu2+, OR Zn2+

Chloride salts of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, and Fe³⁺ had no effect on [³H]diazepam binding. Chloride salts of Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ increased [³H]diazepam binding by 34 to 68% in a concentration-dependent fashion. Since these divalent cations potentiated the GABA-enhanced [³H]diazepam binding and the effect of each divalent cation was nearly additive with GABA, these cations probably act at a site different from the GABA recognition site in the benzodiazepine-receptor complex. Scatchard plots of [³H]diazepam binding without an effective divalent cation showed a single class of binding, with a Kd value of 5.3 mM. In the presence of 1 mM Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺, two distinct binding sites were evident with apparent Kd values of 1.0 nM and 5.7 nM. The higher-affinity binding was not detected in the absence of an effective divalent cation and is probably a novel, super-high-affinity binding site.     

Density-gradient centrifugation in lithium metatungstate and tris-(hydroxymethyl)aminomethane phosphotungstate

New substances--lithium metatungstate (MTL) and tris-(hydroxymethyl)aminomethane phosphotungstate (PTT)--have been presented for density-gradient preparation. The buoyant densities of protein, RNA, DNA and some nucleoproteins were determined in solutions of these salts. Nucleic acids have been smaller buoyant density (1.1 g/cm3) than the proteins in contrast to CsCl-gradients. The protein in PTT solution have buoyant density 1.5 g/cm3 and in MTL solution 2.0-2,3 g/cm3. It was shown that MTL gradients allow to reach better resolution in nucleoprotein analysis than CsCl gradients.     

Inhibition of trout (Salmo gairdneri R.) PBG-synthase by some metal ions (Mg2+, Pb2+, Zn2+)

The effect of metal ions on the activity of trout kidney and liver PBG-synthase was investigated. Heavy metals inhibited the kidney enzyme in a complex manner. Kinetic analysis of the inhibition of liver activity by Pb2+ (Ki = 1.3 mM) was consistent with non-competitive inhibition, whereas Zn2+ (Ki = 1.3 mM) and Mg2+ (Ki = 3.5 mM) were competitive inhibitors.