Buffer-catalyzed reactions

The absorbance change (AM) at 340 nm can be used to determine the amount of pymvate remaining. The lactate dehydrogenase [9001-60-9] catalyzed reaction can also be used in the reverse direction to measure lactate. The reaction takes place in a buffer of pH 9—10 that neutralizes Hberated H". ... 

The role that acid and base catalysts play can be quantitatively studied by kinetic techniques. It is possible to recognize several distinct types of catalysis by acids and bases. The term specie acid catalysis is used when the reaction rate is dependent on the equilibrium for protonation of the reactant. This type of catalysis is independent of the concentration and specific structure of the various proton donors present in solution. Specific acid catalysis is governed by the hydrogen-ion concentration (pH) of the solution. For example, for a series of reactions in an aqueous buffer system, flie rate of flie reaction would be a fimetion of the pH, but not of the concentration or identity of the acidic and basic components of the buffer. The kinetic expression for any such reaction will include a term for hydrogen-ion concentration, [H+]. The term general acid catalysis is used when the nature and concentration of proton donors present in solution affect the reaction rate. The kinetic expression for such a reaction will include a term for each of the potential proton donors that acts as a catalyst. The terms specific base catalysis and general base catalysis apply in the same way to base-catalyzed reactions. 

The experimental detection of general acid catafysis is done by rate measurements at constant pH but differing buffer concentration. Because under these circumstances [H+] is constant but the weak acid component(s) of the buffer (HA, HA, etc.) changes, the observation of a change in rate is evidence of general acid catalysis. If the rate remains constant, the reaction exhibits specific acid catalysis. Similarly, general base-catalyzed reactions show a dependence of the rate on the concentration and identity of the basic constituents of the buffer system. 

Johnson et al. (1962) measured the quantum yield of Cypridina luciferin in the luciferase-catalyzed reaction for the first time, using a photomultiplier calibrated with two kinds of standard lamps. The measurement gave a value of 0.28 0.04 at 4°C in 50 mM sodium phosphate buffer, pH 6.5, containing 0.3 M NaCl. The quantum yield... 

Finally, students can be critics of published work, and perhaps have already encountered papers in the literature with questionable features. I invite reference to the paper, On the Mechanism of Catalysis by Ribonuclease Cleavage and Isomerization of the Dinucleotide UpU Catalyzed by Imidazole Buffers [Anslyn, E. Breslow, R. J. Am. Chem. Soc. 1989, III, 4473 1482]. A useful exercise is to list any flaws. Any such criticisms can then be compared with those raised in the article, Imidazole Buffer-Catalyzed Cleavage and Isomerization Reactions of Dinucleotides The Proposed Mechanism Is Incompatible with the Kinetic Measurements [Haim, A. J. Am. Chem. Soc. 1992,114, 8383-8388]. 

An IL solvent system is applicable to not only lipase but also other enzymes, though examples are still limited for hpase-catalyzed reaction in a pure IL solvent. But several types of enzymatic reaction or microhe-mediated reaction have been reported in a mixed solvent of IL with water. Howarth reported Baker s yeast reduction of a ketone in a mixed solvent of [hmim] [PFg] with water (10 1) (Fig. 16). Enhanced enantioselectivity was obtained compared to the reaction in a buffer solution, while the chemical yield dropped. 

Enantiomeric or specific synthesis of cyanohydrin is influenced by the reaction medium, cyanide source, water content, buffer pH, enzyme, and temperature during the HNL-catalyzed reaction. To maximize the enantiomeric excess of the cyanohydrin product, care must be taken to minimize the parallel chemical (nonenzymatic) condensation and racemi-zation of products. 

For these reasons, in the experimental study of the kinetics of enzyme-catalyzed reactions, T, shear and PH are carefully controlled, the last by use of buffered solutions. In the development, examples, and problems to follow, we assume that both T and pH... 

Many rate constants in aqueous solutions are pH or pD sensitive. In particular, enzyme catalyzed reactions often show maxima in plots of pH(pD) vs. rate. The example in Fig. 11.5 is constructed for a reaction with a true isotope effect, kH/kD = 2, and with maxima in the pH(pD)/rate dependences as shown by the bell shaped curves. These behaviors are typical for enzyme catalyzed reactions. When the isotope effect is obtained (incorrectly) by comparing rates at equal pH and pD, the values plotted along the steep dashed curve result. If, however, the rate constants at corresponding pH and pD (pD = pH + 0.5) are employed, a constant and correct value is obtained, kH/kD = 2. Thus for accurate measurements of the isotope effects one must control pH and pD at appropriate values (pD = pH + 0.5 in our example) using a series of buffers. In proton inventory experiments (see below) buffers should be employed to insure equivalent acidities across the entire range of solvent isotope concentration (0 < xD < 1), xD is the atom fraction of deuterium [D]/([H] + [D]). 

The pH of a buffered mobile phase is significant not only because retention of ionizable compounds is dependent upon pH but because bonded phase columns are made via an acid catalyzed reaction. Manufacturers recommendations are to avoid any system with a pH lower than 2 or accept the risk of reversing the bonding reaction. However, the effective pH experienced by a column in a water organic mixture is difficult to predict. We have operated at a pH of 1.5 in this laboratory with no adverse effects to the column, but exercise the simple precaution of not allowing the acidic mobile phase to remain on the column overnight or static for long periods of time (1 hr). We have particularly noticed that the CN column is stable at low pH s. 

Rowlett and Silverman used a Brpnsted plot to examine the interaction of external buffers with human carbonic anhydrase II. The buffers act as proton acceptors in the removal of the proton generated by the enzyme-catalyzed reaction. The Brpnsted plot displays a plateau at a value of about 10 for the catalytic rate... 

The equilibrium constant of an enzyme-catalyzed reaction can depend greatly on reaction conditions. Because most substrates, products, and effectors are ionic species, the concentration and activity of each species is usually pH-dependent. This is particularly true for nucleotide-dependent enzymes which utilize substrates having pi a values near the pH value of the reaction. For example, both ATP" and HATP may be the nucleotide substrate for a phosphotransferase, albeit with different values. Thus, the equilibrium constant with ATP may be significantly different than that of HATP . In addition, most phosphotransferases do not utilize free nucleotides as the substrate but use the metal ion complexes. Both ATP" and HATP have different stability constants for Mg +. If the buffer (or any other constituent of the reaction mixture) also binds the metal ion, the buffer (or that other constituent) can also alter the observed equilibrium constant . ... 

The rate of a general acid-catalyzed reaction is equal to 2ha ha[HA] multiplied by some function of the substrate concentration(s) where [HA] is the general acid concentration and A ha is the corresponding catalytic rate constant. Experimentally, general acid catalysis can be distinguished from specihc acid catalysis by analysis of the effect of buffer concentration on the overall reaction rate. See also Specific Acid Catalysis Catalysis General Base Catalysis... 

Buffer solutions that are isosmotic with respect to some standard, typically chosen such that suspended cells will neither shrink nor expand. Sodium chloride solutions (0.90% weight/volume or 0.155 M) at 37°C is often used to represent physiological conditions. These buffer systems are also important in studies of intact cells and membranal organelles likewise, many pharmaceutical formulations must be prepared as isotonic solutions. Most enzyme-catalyzed reactions are affected by ionic... 

Any electromechanical device that utilizes an automated feedback servomotor to regulate the addition of titrant (a standardized solution of acid or base within a syringe) into a reaction vessel or sample to maintain pH. The rate at which the syringe expels its contents allows one to determine the rate of a chemical reaction producing or consuming protons. There are many such enzyme-catalyzed reactions whose kinetics can be examined with a pH Stat. For maximal sensitivity, one must use weakly buffered solutions. In his classical kinetic investigation of DNA bond scission by DNase, Thomas measured the rate of base addition in a pH Stat. The number of bonds cleaved was linear with time, and this was indicative of random scission. 

In 1996, Detty and coworkers reported on the oxidation of sodium halides to positive halogens with hydrogen peroxide in two-phase systems of dichloromethane and pH 6 phosphate buffer, catalyzed by organoteUurium catalysts 237 (Scheme 181). Mixtures of 1,2-dihalocyclohexane (238) and 2-halocyclohexanol (239) were formed upon reaction of the positive halogens formed with cyclohexene. From the three tellurium catalysts... 

Environmental Fate. Extensive information is available on the general reactions of isocyanates that may pertain to the environmental fate of HDI (Chadwiek and Cleveland 1981 Kennedy and Brown 1992). However, investigations of the environmental fate of isocyanates have focused primarily on TDI and MDI (Duff 1983, 1985 Gilbert 1988 Holdren et al. 1984). Only one laboratory study was located in the available literature specifically on the ehemieal reaetions of HDI (i.e., bicarbonate buffer-catalyzed hydrolysis) that may be relevant to the environmental fate of HDI in water (Berode et al. 1991). HDI is expected to react relatively rapidly with hydroxyl radieals in the atmosphere and to be rapidly hydrolyzed in water and moist soils and sediment. The signifieanee of atmospheric hydrolysis has not been evaluated. Additional field and laboratoiy studies are needed to adequately eharacterize the environmental fate of HDI in air, water, soil, and sediment. 

A liquid formulation is usually comprised of a buffering agent, a stabilizer (which may also serve as a tonicity agent), a surfactant, and an anti-oxidant when protein oxidation is significant. Chelating agents are employed when metal ion catalyzed reactions predominate. A preservative may be included when a multi-dose formulation is desired. 

The importance of buffers in all areas of science is immense. At the outset of this chapter, we saw that digestive enzymes in lysosomes operate best in acid, which allows a cell to protect itself from its own enzymes. If enzymes leak into the buffered, neutral cytoplasm, they have low reactivity and do less damage to the cell than they would at their optimum pH. Figure 9-3 shows the pH dependence of an enzyme-catalyzed reaction that is fastest near... 

Apparent Temperature Optimum. A rise in temperature has a dual effect upon an enzyme-catalyzed reaction it increases the rate of the reaction, but it also increases the rate of thermal inactivation of the enzyme itself. Like the pH optimum, the temperature optimum may in certain instances be altered by environmental conditions, e.g., pH, type and strength of buffer, etc. The term temperature optimum, therefore, is useless unless the incubation time and other conditions are specified. A more enlightening term is apparent temperature optimum, which indicates that the optimum has been obtained under a... 

It was once thought that the rate of equilibrium of the catalytic acid and basic groups on an enzyme with the solvent limited the rates of acid- and base-catalyzed reactions to turnover numbers of 103 s 1 or less. This is because the rate constants for the transfer of a proton from the imidazolium ion to water and from water to imidazole are about 2 X 103 s 1. However, protons are transferred between imidazole or imidazolium ion and buffer species in solution with rate constants that are many times higher than this. For example, the rate constants with ATP, which has a pKa similar to imidazole s, are about I0 J s 1 M-1, and the ATP concentration is about 2 mM in the cell. Similarly, several other metabolites that are present at millimolar concentrations have acidic and basic groups that allow catalytic groups on an enzyme to equilibrate with the solvent at 107 to 108 s-1 or faster. Enzyme turnover numbers are usually considerably lower than this, in the range of 10 to 103 s-1, although carbonic anhydrase and catalase have turnover numbers of 106 and 4 X 107 s 1, respectively. 

The purpose of each laboratory exercise in this book is to observe and measure characteristics of a biomolecule or a biological system. The characteristic is often quantitative, a single number or a group of numbers. These measured characteristics may be the molecular weight of a protein, the pH of a buffer solution, the absorbance of a colored solution, the rate of an enzyme-catalyzed reaction, or the radioactivity associated with a molecule. If you measure a quantitative characteristic many times under identical conditions, a slightly different result will most likely be obtained each time. For... 

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