Imidazole Buffer Research Articles

Fructose‐1,6‐bisphosphatase from a cold‐hardy insect: Control of cryoprotectant glycerol catabolism

AbstractFructose 1,6‐bisphosphatase (FBPase) from the larvae of the gall moth, Epiblema scudderiana, was purified to homogeneity with a final specific activity of 1.6 U/mg protein. The enzyme had a native molecular weight of 74.0 ± 6.5 kD and a subunit molecular weight of 37.6 ± 3.0 kD; the dimeric structure of the enzyme in this species is unusual. The pH optimum was 7.00 in imidazole buffer at 22°C and rose to 7.31 at 5°C. An Arrhenius plot of enzyme activity vs. temperature was linear with an activation energy of 91 ± 4.1 kJ/mol−1. Km values for FBPase decreased from 4.7 ± 0.34 μM at 22°C to 1.3 ± 0.05 μM at 5°C. No allosteric activators were identified, but the enzyme was inhibited by fructose 2,6‐bisphosphate (F2,6P2), AMP, ADP, dihydroxyacetonephosphate, glycerol, and KCI. Inhibition by AMP and F2,6P2 increased at low temperature, and effects of these compounds may be key to preventing futile cycling of carbon at the FBPase/phosphofructokinase loci during the biosynthesis of glycerol cryoprotectant. Oppositely, glycerol clearance in the spring and reconversion into glycogen is promoted by interactions of temperature, inhibitors, and glycerol that promote FBPase activity: I50 values for AMP and F2,6P2 increase at 22°C (compared with 5°C), high glycerol levels override F2,6P2 inhibition of the enzyme, and deinhibitors (ATP, citrate) partially reverse AMP inhibition of the enzyme. © 1995 Wiley‐Liss, Inc.

Sequencing of oligonucleotides using high performance liquid chromatography and electrospray mass spectrometry

We report the sequence analysis of an oligodeoxyribonucleotide by electrospray mass spectrometry (ES-MS). For full sequence analysis of the oligomer, d(CGTAGCTACG), an enzyme digestion step followed by off-line high performance liquid chromatography was used. For ES-MS analysis, introducing the sample in an imidazole buffer with 70% acetonitrile increased the electrospray ion currents and helped reduce sodium-adduct ion peaks.

Metal-ion-promoted hydrolysis of uridylyl(3′,5′)uridine: internal vs. external general base catalysis

The hydrolysis of uridylyl(3′,5′)uridine promoted by Mg2+, Zn2+ and Zn2+[12]aneN3([12]aneN3= 1,5,9-triazacyclododecane) has been studied in imidazole, HEPES and triethanolamine buffers and the origin of marked electrolyte effects observed with Zn2+[12]ane N3 has been examined. The results obtained suggest that the basic buffer constituent does not serve as an external general base, but the catalytic activity of the metal ion species is influenced by the coordination to the buffer base and other Lewis bases present in the solution. The data lend additional support to a bifunctional mechanism, which consist of coordination of the metal ion to the anionic phosphodiester and intracomplex general base (or nucleophilic) catalysis by its hydroxo ligand.

The Nijmegen Modification of the Bethesda Assay for Factor VIII:C Inhibitors: Improved Specificity and Reliability

Antibodies against factor VIII coagulant activity can appear in haemophiliacs who are treated with factor VIII preparations but also spontaneously in non-haemophiliacs. The Bethesda assay is the most commonly used method to detect these antibodies, but it lacks specificity especially in the lower range resulting in unreliable data. Two modifications are proposed and tested to resolve the imperfections: 1. Buffering the normal plasma used in the assay- and control mixture with 0.1 M imidazole to pH 7.4. 2. Replacing the imidazole buffer in the control mixture by immunodepleted factor VIII deficient plasma. These modifications allow better discrimination between positive and negative samples and improve reliability.

Capillary Electrophoresis of Alkali and Alkaline-Earth Cations with Imidazole or Benzylamine Buffers

Abstract The separation of alkali, alkaline earth, and ammonium cations in several samples of water was achieved by capillary electrophoresis with indirect UV detection. A solution of imidazole (10−2 M, pH 4.5) was used as buffer to resolve a mixture of six cations (K+, Na+, Ca2+, Ba2+, Li+ and Mg2+) by capillary electrophoresis at 214 nm in less than 10 min. The addition of potassium cation to the running buffer has an influence on the resolution of Ca2+/Na+ and Na+/Mg2+ peaks. A linear relationship between the corrected peak area and concentration was obtained in the 1–10 ppm range for these cations using a hydrodynamic injector. This electrophoretic system permitted the separation of these inorganic cations at a 50 ppb-level concentration with an hydrodynamic injection, thus making it possible to quantitatively determine their presence in mineral waters by capillary electrophoresis. At pH 4.5, potassium and ammonium unfortunately have identical ionic mobilities causing them to comigrate in an imidazole...

Effect of pH on the stability and surface composition of emulsions made with whey protein isolate

Emulsions (20 wt % soy oil) made with various concentrations (0.5-2.5 wt %) of whey protein isolate (WPI) were most stable at pH 7 and least stable at pH 5.5. Emulsions made with imidazole buffer at pH 6 were stable, but those made with citrate buffer at the same pH were unstable. Emulsions prepared at pH 3, using citrate buffer, were stable. At pH 7 β-lactoglobulin and α-lactalbumin adsorbed in proportion to their concentration, but at lower pH values α-lactalbumin was found to adsorb preferentially, depending on the protein concentration, pH, and buffer. When emulsions (2 wt % WPI) were acidified from pH 7 to 3 more α-lactalbumin became adsorbed and the emulsion was stable, but reducing the pH from 7 to 6 did not alter the interfacial composition of protein and the emulsion became unstable

Kinetic fluorimetric determination of Cu(II) and Zn(II) by their incorporation reactions into a water-soluble porphyrin

Kinetics of incorporation of Cu, Zn, Fe, Co, Ni and Mn divalent ions into coproporphyrin-I in imidazole buffer solution, pH 7.0, has been studied by monitoring the decrease in fluorescence intensity of the free base porphyrin. All reactions followed simple second-order rate law, the rate constants being decreased in the order Zn > Cu ⪢ Co > Fe ⪢ Mn, Ni. the kinetic fluorimetric method for the determination of Cu(II) and Zn(II) using their incorporation reactions into the porphyrin was developed. Initial rate and fixed-time methods were used to construct calibration graphs over the range 0–1.0 × 10 −5 M of both metals. The analytical characteristics of the method and effect of foreign ions were determined. In the presence of sodium thiosulphate as the masking reagent the determination of micromolar concentrations of Zn in the presence of a 10-fold excess of Cu is possible.

Yeast Mitochondrial F1-ATPase-Effects of Metal-Ions

The effects of a series of metal ions (M) on the velocity (V) of the reaction catalyzed by the yeast mitochondrial F1-ATPase were investigated in N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and Imidazole buffers (pH 7.45), with total concentrations of ATP ranging from 2 to 8 mM. The biphasic metal ion activation/inhibition effects, observed with a constant total amount of ATP, were found to be consistent with a simple empirical kinetic equation, V1/V = 1 + K1/[M] + [M]/K2, where V1, K1, and K2 are empirical parameters and [M] the concentrations of metal ions not complexed with the ATP. Three alternative kinetic equations, involving the concentrations of either ATP or its metal chelate, consistent with this empirical equation, were shown to be invalid since changing the total amount of ATP did not affect the empirical reaction parameters V1, K1, and K2. The fact that changing the total ATP concentration failed to change the empirical reaction parameters suggests that the inhibition by an excess of ATP, which is observed with a constant total metal ion concentration, is due to chelation of free metal ions that are required for the ATP hydrolysis. When the total amount of added ATP was in excess of that of added magnesium ions it was found that calcium, strontium, and aluminum ions, which liberate free magnesium from the Mg-ATP chelate by complexing the ATP, all activated. These activations apparently confirm the fact that the ATPase requires a free magnesium ion as a cofactor for the hydrolysis of ATP.

Comparison of intra- and intermolecular proton transfer in human carbonic anhydrase II.

The catalysis of the hydration of CO2 by human carbonic anhydrase II (HCA II) includes the transfer of a proton from zinc-bound water to histidine 64 utilizing a network of intervening hydrogen-bonded water molecules, then the proton is transferred to buffer in solution. We used stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry to compare catalytic constants dependent on proton transfer in HCA II and in the mutant H64A HCA II containing the replacement His64-->Ala. Maximal velocities and oxygen-18 exchange catalyzed by H64A HCA II showed that nearly all of the proton transfer with this mutant proceeded through the imidazole buffer. The following parameters were very similar or identical in catalysis by H64A HCA II compared with catalysis by wild-type HCA II both in the presence of large concentrations of imidazole (100 mM): the maximal rate of initial velocity and of exchange of 18O between CO2 and water, solvent hydrogen isotope effects on the maximal velocity, and the dependence of these isotope effects on the atom fraction of deuterium in solvent water. These results indicate that the proton transfer involving the zinc-bound water in catalysis is not significantly affected by the difference between the mobility of the free imidazole buffer and the side chain of His 64. Moreover, data for both the wild-type and mutant enzymes are consistent with proton transfer through intervening hydrogen-bonded water bridges in the active sites. These features of the proton transfer are discussed in terms of a model in which the first proton transfer from the zinc-bound water to an adjacent water is rate limiting.

Purification and characterization of aldolase from the cold hardy insect Epiblema scudderiana: Enzyme role in glycerol biosynthesis

Aldolase was purified to homogeneity from larvae of the freeze avoiding gall moth, Epiblema scudderiana to a final specific activity 16.5 U/mg protein. The tetrameric enzyme had a native molecular weight of 160 ± 11 kDa and a subunit molecular weight of 37.8 ± 1.0 kDa. Aldolase in both 15°C and −4°C acclimated larvae occured in a single enzyme form with an isoelectric point of 5.0–5.1. The pH optimum of the enzyme was 6.0 when assayed in imidazole buffer at 22°C and increased to 6.8 at 5°C. The Arrhenius plot was linear between 5 to 40°C with an activation energy of 68.9 ± 2.31 kJmol −1. K m values for fructose 1,6-bisphosphate increased 2-fold when assay temperature was decreased from 22 to 5°C. No allostetic activators of the enzyme were found but α-glycerophosphate, inorganic phosphate, and glycerol were effective inhibitors under the saturating substrate concentrations that exist for aldolase during glycerol biosynthesis. Inhibitory effects of α-glycerophosphate and inorganic phosphate increased when assayed at 5°C but the opposite was true of inhibition by glycerol. Inhibitory controls on aldolase may function to help bring about the cessation of cryoprotectant synthesis as well as maintain the cryoprotectant glycerol pool over the winter months.

Electron transfer catalyzed by manganese tetratolyl‐prophyrinate across poly(γ‐methyl L‐glutamate) membrane

AbstractA poly(γ‐methyl L‐glutamate) (PMLG) membrane containing manganese tetratolylporphyrinate (MnTTP) was prepared in order to examine the transmembrane electron transfer in a redox system constructed as S2O42−/membrane/Fe(CN)63−. Electron transfer was observed in 0,1 M pH 7,0 imidazole (Im) buffer but not in 0,1 M pH 7,0 phosphate buffer. The apparent transfer rate increased with MnTTP content and also with the Im concentration in the buffer, indicating that electron is transferred by MnTTP, which is coupled with proton transport by Im distributed in the membrane. The transfer catalyzed by MnTTP was more effectively accelerated with Im than with 2‐methylimidazole (2‐Melm) but not at all with 1‐methylimidazole (1‐Melm). This implies that the 1‐N site on the imidazoles as the axial ligand of MnTTP is a crucial position for the mediation of electron transfer between MnTTP's. In the pH range 6,0–8,5 of Im buffer, the electron transfer rate showed a maximum at pH 7,0. This pH dependence is discussed in terms of the concentration and mobility of proton in the membrane.

Flow Injection Determinations of Creatine and Creatinine Using Packed Bed Enzyme Reactors Under Equilibrium Conditions

Abstract A flow injection system with immobilized enzyme reactors is used for sequential determinations of creatinine and creatine. The first reactor, creatinine amidohydrolase (CAH), catalyzed the hydrolysis of creatinine to creatine which was later phosphorylated by adenosine-5′-triphosphate (ATP) in a second reactor containing creatine kinase (CK), pyruvate kinase (PK) and 1-lactate dehydrogenase (LDH). The intermediate product, adenosine-5′-diphosphate (ADP), dephosphory-lated phosphoenol pyruvate (PEP) to pyruvate by PK catalysis. The LDH reaction converted pyruvate to L-lactate, oxidizing an equivalent quantity of reduced nicotinamide adenine dinucleotide, NADH. The consumption of NADH was measured spectrophotometrically at 340 nm and it was linear in the range 5 – 400 μM creatinine or creatine. The sample throughput was 25 – 30 h−1. The CAH-reactor was stable for at least five months and the multienzyme reactor for three months when used daily and stored in imidazole buffer (pH 7.6) containing 2-mercaptoethanol and EDTA.

Determination of malonaldehyde by coupled high-performance liquid chromatography—spectrofluorimetry after derivatization with luminarin 3

A high-performance liquid chromatographic method with fluorescence detection is described for the specific determination of free malonaldehyde (MDA). Malonaldehyde was labelled with luminarin 3 in acetate buffer (pH 4.0) at room temperature in the dark. An aliquot of the reaction mixture was injected on to an octadecyl-bonded column using acetonitrile—imidazole buffer (30:70, v/v) as the mobile phase. The eluate was monitored with a fluorescence detector at 395 nm (excitation) and 500 nm (emission). A linear calibration graph was established between 7.2 and 90 ng/ml of MDA and the limit of quantification (LOQ) was lower than 7 ng/ml of MDA. The precision was characterized by R.S.D.s of 11% at 7.2 ng/ml and 2% at 90 ng/ml. The structure of the derivative was confirmed as the 5-hydroxy-2-pyrazoline form. UV absorbance and corrected fluorescence spectral data and quantum yields of the luminarin 3 derivative of malonaldehyde are presented.

Isolation and characterization of a 54-kilodalton precursor of caltrin, the calcium transport inhibitor protein from seminal vesicles of the rat.

A basic 54-kDa protein (pI approximately 8.8) that cross-reacts with anti-caltrin antisera has been detected and isolated by gel filtration and cation exchange chromatography from seminal vesicle content of the rat. The soluble protein spontaneously precipitated in NaHCO3-buffered solution at pH 7.8, but it was kept soluble in imidazole buffer containing EDTA and dithiothreitol at pH 7.0. In addition to the main band of 54 kDa, two faint immunoreactive fractions with molecular weights around 45,000 and 14,000 were also revealed by Western blotting. The presence of the rat caltrin sequence within the primary structure of the 54-kDa molecule has been investigated by sequencing the peptides generated by trypsin digestion. The sequence of the first 46 amino acid residues of rat caltrin has been found in one of the fragments produced by enzymatic cleavage. However, the exact location of the caltrin sequence in the whole 54-kDa protein has not been determined. The purified 54-kDa protein did not inhibit Ca2+ uptake by epididymal spermatozoa. Results indicated that this molecule represents an inactive precursor of caltrin and is enzymatically processed in the lumen of the seminal vesicle to the small and active calcium transport inhibitor protein. The immunoreactive proteins with intermediate molecular weights (45,000 and 14,000) could represent partially degraded products of the precursor. The lack of inhibitory activity of the precursor may be related to the molecule's having a size and conformation that would make it unable to interact with caltrin receptors on the sperm surface.

Synthesis of protected peptide fragments and release from a solid support under neutral conditions

A linker has been developed for use in solid-phase peptide synthesis which allows a protected peptide to be cleaved from the resin under neutral conditions (nBu 3P, DMF, imidazole buffer pH 7) whilst retaining tBuO-, Boc- and Fmoc-protecting groups. The linker comprises a 2-azidomethyl-4-hydroxy-6,N-dimethylbenzamide moiety, which is coupled to polystyrene resin as a phenol ether and to the peptide via a N-hydroxymethyl group. The protected peptide fragments so produced may be useful, for example, in subsequent fragment condensations using the Fmoc-chemistry.

Recognition and catalysis in nucleic acid chemistry.

The enzyme ribonuclease A catalyzes the cleavage of RNA, using the imidazole groups of histidine-12 and histidine-119 as its principal catalytic groups. Model studies show that RNA can be cleaved by imidazole buffer itself and that, as in the enzyme, a bell-shaped pH vs. rate profile is seen. This indicates that one imidazole functions as a base, while the other, as the imidazolium ion, functions as an acid. However, in contrast to the enzymatic case, the simple model uses the imidazoles in sequential, rather than simultaneous, bifunctional catalysis. Mechanistic studies on this reaction and on the reactions of simple dinucleotides catalyzed by imidazole and other buffers establish the details of the process. The results let us propose a mechanism for the enzymatic process different from the standard one; they also stimulated us to design an improved mimic of the enzyme that uses a mechanism like that proposed for the enzyme. Critical to the mechanistic studies is observation of the rearrangement of normal 3',5'' RNA nucleotides to the 2',5'' isomers. This led us to investigate the properties of DNA isomers in which a 2',5'' link also replaces the normal 3',5'' one. The results indicate that poor base stacking in a double helix with such links makes them less suitable as genetic units.

Kinetics and mechanism in RNA cleavage.

Experimental studies on the cleavage of various RNA molecules--poly(U), 3',5''-UpU, 2',5''-UpU, and 3',5''-ApA--catalyzed by imidazole buffers show that there is a sequential bifunctional mechanism. One catalyst species converts the substrate to an intermediate phosphorane, and the second catalyst converts the phosphorane to cleavage products. Detailed steady-state kinetics are presented to determine all mechanisms that are consistent with the findings. Choice among these possibilities can be made considering other experimental evidence about the catalysis of an isomerization reaction that accompanies cleavage. It is concluded that all acceptable mechanisms involve action of the second catalyst on a phosphorane monoanion; it can be formed directly from the substrate in the first step or by proton equilibrations involving either the substrate or the phosphorane intermediate. The relationship of these conclusions to the likely mechanism of action of the enzyme ribonuclease is briefly discussed.

Refolding of Recombinant Pneumocystis carinii Dihydrofolate Reductase and Characterization of the Enzyme

The isolation of dihydrofolate reductase (DHFR) cDNA sequences from the messenger RNA of Pneumocystis carinii using the polymerase chain reaction is described. The 206-amino acid P. carinii DHFR was expressed to high levels in Escherichia coli inclusion bodies using the T7 promoter expression system. Solubilization of the inclusion bodies in 4 M guanidine hydrochloride and refolding of the recombinant protein in the presence of 0.5% polyethylene glycol 1450 yielded correctly folded DHFR which was purified to homogeneity by methotrexate-Sepharose affinity chromatography. The refolded enzyme was readily crystallized as a ternary complex with NADPH and various inhibitors. The enzyme exhibited a sharp pH optimum with maximum activity at pH 7.0 (turnover number = 6500 min −1). K m values for dihydrofolate (DHF) and NADPH were 2.3 and 3.0 μM, respectively, in 0.1 M imidazole buffer, pH 7. Folate did not act as a substrate. Comparison of the kinetic properties of the refolded enzyme with soluble P. carinii DHFR expressed at low levels in the T7 expression system showed similar pH-activity profiles, K m , values for DHF and NADPH, and IC 50 values for several known antifolates which were tested as inhibitors of the enzyme.

Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone.

Calcium binding properties of bone acidic glycoprotein-75, osteopontin, and bone sialoprotein were determined in 10 mM imidazole buffer (pH 6.8), containing either 60 mM KCl or 150 mM NaCl. Proteins assayed were first bound to nitrocellulose to mimic substrate-bound forms in vivo; retention of phosphoproteins was determined through use of radioiodinated tracers. Binding studies were carried out both as a function of calcium concentration and the amount of phosphoprotein. In the presence of 60 mM KCl, bone acidic glycoprotein-75 exhibited the largest calcium binding capacity (139 atoms/molecule at saturation), with bone sialoprotein intermediary (83 atoms/molecule) and osteopontin lowest (50 atoms/molecule). Sites detected for each phosphoprotein exhibited overall binding constants in the 0.5-1.0 mM extracellular range. In 150 mM NaCl and 1-2 mM total calcium, phosphoproteins bound between 72 and 19 mol of calcium/mol with the same relative order. Binding was proportional to amount of phosphoprotein in either salt condition. The presence of 5 mM calcium had a different effect on concentration-dependent binding to type I collagen for each phosphoprotein. Bone acidic glycoprotein-75 alone was found to undergo an unusual calcium-enhanced polymerization reaction, confirmed by light scattering measurements, wherein collagen binding was greatest with polymeric forms. These findings demonstrate that acidic phosphoproteins from bone bind calcium atoms with a range of capacities. Calcium appears to induce conformational changes in bone acidic glycoprotein-75 which influences its self-association and binding to different substrata.

Thermal adaptation in biological membranes: interacting effects of temperature and pH

The interacting effects of pH and temperature on membrane fluidity were studied in plasma membranes isolated from liver of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20°C. Fluidity was determined as a function of temperature under conditions of both constant (in potassium phosphate buffer) and variable pH (in imidazole buffer, consistent with imidazole alphastat regulation) from the fluorescence anisotropy of two probes: 1,6-diphenyl-1,3,5-hexatriene, which intercalates into the bilayer interior, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene which is anchored at the membrane/water interface. The temperature dependence of the anisotropy parameter for 1,6-diphenyl-1,3,5-hexatriene in plasma membranes of 20°C-acclimated trout was greater when determined in phosphate (ΔAP per °C=-0.047) than in imidazole buffer (ΔAP per °C=-0.022); similar, but less significant, trends were noted with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene. In contrast, the temperature dependence of fluidity (ΔAP/°C in the range-0.0222 to-0.027) did not vary with buffer composition in membranes of 5°C-acclimated trout. In phosphate buffer, anisotropy parameter values for 1,6-diphenyl-1,3,5-hexatriene were significantly lower in 5°C-than 20°C-acclimated trout, indicating a less restricted probe environment following cold acclimation and nearly perfect compensation (∼91%) of fluidity. Temperature-dependent patterns of acid-base regulation were estimated to account for 11–40% of the fluidization evident in membranes of 5°C-trout, but a period of cold acclimation was required for complete fluidity compensation. In contrast, no homeoviscous adaptation was evident in imidazole buffer, indicating that membrane fluidity is sensitive to buffer composition. Accordingly, vesicles of bovine brain phosphatidylcholine, suspensions of triolein, and plasma membranes of 5°C-acclimated trout were consistently more fluid in imidazole than phosphate buffer. Membranes of 5°C-acclimated trout were enriched in molecular species of phosphatidylcholine containing 22:6n3 (at the expense of species containing 18:1n9 and 18:2n6) compared to membranes of 20°C-trout; consequently, the unsaturation index was significantly higher (3.29 versus 2.73) in trout maintained at 5 as opposed to 20°C. It is concluded that: 1) the chemical composition of the internal milieu can significantly influence the physical properties of membrane lipids; 2) temperature-dependent patterns of intracellular pH regulation may partially offset the ordering effect of low temperature on membrane fluidity in 20°C-acclimated trout transferred to 5°C, but not in 5°C-acclimated trout transferred to warmer temperatures; 3) the majority of the thermal compensation of plasma membrane fluidity resulting from a period of temperature acclimation most likely reflects differences in membrane composition between acclimation groups; 4) imidazole apparently interacts with trout hepatocyte plasma membranes in a unique way.