Fluorescence stopped-flow study of the o-phthaldialdehyde reaction

The fluorogenic reaction involving three species, namely, a primary amine, o-phthaldialdehyde (OPA), and a thiol compound was studied with the fluorescence stopped-flow technique. The results are consistent with the reaction of the amine with a 1:1 adduct of OPA and the thiol compound. The equilibrium constant for the formation of the adduct, OPAME, from OPA and mercaptoethanol (ME) was determined to be 164 m^?1. A survey of the rates of reaction of OPAME with various amino acids demonstrated that with OPA: ME:amine equal to 1:2.4:1 (total OPA concentration 0.5 to 3.0 × 10^?3m), the reaction followed second-order kinetics, with k = 150 to 450 m^?1s^?1 at pH 9.O. The differences in rates are discussed in relation to structural differences between the amines. The reaction, when conducted under conditions of excess OPAME yielded pseudo-first-order kinetics, with rates consistent with the second-order rate constants. The rate of reaction of OPAME with alanine was maximal at pH 10.5–11, and a great excess of ME resulted in a slower rate. Slower rates were also observed if ME was replaced by dithiothreitol or 1-propanethiol.     

The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney

The steady-state kinetic parameters for the hydration of CO₂ catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number k_cat is 6.5 × 10⁵ s^?1 and the Michaelis constant is 7.5 mm for CO₂ hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 10⁵ s^?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 10⁸m^?1s^?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO₂ in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO₂ hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase.     

Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions

The kinetics of the Quin 2-Ca²⁺ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca²⁺ complex with a large excess of EGTA, EDTA or MgCl₂ resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s^?1 for the dissociation rate constant (k_?) at pH 7.2, 37°C, ± 3mM Mg²⁺. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k₊ was estimated to be ≥10⁹ M^?1s^?1, in agreement with the calculation from $\text{k}{}_{\mathrm{+}}\text{=}\text{k}{}_{\mathrm{?}}\text{K}$. These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca²⁺ transients.     

Amine-catalyzed elimination of beta-phosphoric esters from aldehydes derived from ribonucleic acid and model substrates.

The kinetics have been measured for several steps of the diamine-catalyzed elimination of the terminal nucleoside from periodate-oxidized RNA and from several model substrates. The general-acid-catalyzed, rate-determining step has a k_HA of 0.13 M^?1 min^?1 (HA = RNH₃⁺) for primary amines, and the specific-base-catalyzed reaction has a k_HH of 0.35 min^?1 (0.2 mm RNA) with ornithine catalysis and a k_HH of 0.077 min^? (0.2 mm RNA) with lysine catalysis. Lysine has a third catalysis component, with a k_AH of 12 min^?1 M^?2. The diamino acid α,γ-diaminobutyrate is not effective as a catalyst, due to cyclic gem diamine formation. Substituents on the 5′-phosphoric ester group do not affect the kinetics unless the substituent is a proton (e.g., as in AMP); thus, AMP is not an accurate model for this type of sequential degradation of RNA.There are two degradative pathways, the β-elimination path and a route that involves cleavage of the C-1′-0-C-4′ ether linkage before the phosphoric ester is eliminated. The direct β-elimination path predominates below pH 7.5, with a maximum near pH 6, and yields only one set of end products. Because of its rapid and predictable course, the latter reaction is preferred for sequential degradation of RNA. The structure of the catalytically active intermediate (general-acid-catalyzed reaction series) involves the primary amino group of ornithine (lysine) condensed with the dialdehyde terminus to form the carbinolamine, aldimine, and enamine intermediates leading to the elimination.The ether cleavage path is controlled by a specific-base (k_HB) intramolecular catalysis above pH 7, and a side reaction leads to lowered yields of phosphoric ester cleavage. A primary amine group is required, since 3-dimethylamino propylamine does not catalyze the ether cleavage.     

Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition

Decomposition of phenyl acridinium-9-carboxylate is monitored using electrogenerated chemiluminescence in a flow system. The formation of the pseudobase from the acridinium ester [AE] is described by rate = k′₁[AE] + k″₁[AE][OH^?]^0.5, where k′₁ = 0.020 ± 0.006 s^?1 and k″₁ = 2.1 ± 0.8 (L/mol)^?0.5 s^?1. Irreversible decomposition of the pseudobase is described by rate = k′₂[AE][OH^?], where k′₂ = 20.1 ± 3.8 (L/mol s). These kinetic equations, plus measurement of variation in emission intensity for constant acridinium ester concentration, are used to predict the resulting emission intensity v. pH behaviour given various contact times (in the 0.25 to 25 s range) for the acridinium ester to be in an alkaline solution prior to initiation of the chemiluminescence reaction.     

Kinetic control of co-operativity in the oxygen binding of Panulirus interruptus hemocyanin.

The kinetics of the oxygen reaction of Panulirus interruptus hemocyanin have been studied at pH 9.6 under conditions where the protein exists in the undissociated, co-operative state and in the dissociated, non-co-operative state.Temperature-jump relaxation measurements of the undissociated protein at high oxygen saturation levels show one relaxation process which has been assigned to the high oxygen affinity (R) state, the on and off kinetic constants being 3.1 × 10⁷m^?1s^?1 and 60 s^?1, respectively. Stopped-flow measurements of the oxygen dissociation reaction show (1) an autocatalytic time-course of the reaction at pH 9.6 and (2) an increase in the overall oxygen dissociation rate constant, as the pH is decreased from 9.6 to 7.0.Temperature-jump relaxation measurements of the dissociated protein show one relaxation process characterized by a very high oxygen dissociation rate constant (1500 s^?1) and a combination constant which is of the same order of magnitude as reported for undissociated protein (k_on = 4.6 × 10⁷m^?1s${}^{\mathrm{?1}}$). The behaviour of dissociated protein can be considered as characteristic of the low oxygen affinity (T) state.The results presented in this paper, together with data available for other hemocyanins as well as hemoglobins, lead to the conclusion that respiratory proteins show a common feature in the kinetic control of co-operative oxygen binding: the stability of the oxygen-protein complex is largely determined by the value of the dissociation rate constant, the oxygen combination process very often appearing to be diffusion controlled.     

Nicotinamide adenine dinucleotide (NAD+). Formal potential of the NAD+/NAD· couple and NAD· dimerization rate

The rate constant, k_d, for the dimerization of the free radical (NAD^·), produced on the initial one-electron reduction of NAD⁺, was measured by double potential-step chronoamperometry, fast-scan cyclic voltammetry (cathodic-anodic peak current ratio) and slow-scan cyclic voltammetry (peak potential shift) for a medium in which neither NAD⁺ nor its reduction products are adsorbed at the solution/electrode interface. All three methods give concordant values of k_d (approx. 3·10⁷ M^?1·s^?1), which are in reasonable accord with the values determined by pulse radiolysis but are considerably greater than values previously determined electrochemically. For the NAD⁺/NAD^· couple, the heterogeneous rate constant (k_s,h) exceeds 1 cm·s^?1 at 25°C and the formal potential (E⁰_c) vs. sce is ? 1.155 V at 25°C and ? 1.149 V at 1°C at pH 9.1, with an uncertainty of about ±0.005 V.     

Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.     

Inducibility of Rat Liver Drug-Metabolizing Enzymes as an Index of Food-Screeing for Safety Assessment

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat?K _m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s^?1, and 100 (mmol/L)^?1 s^?1 respectively.     

The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.     

Electrogenic events in the ubiquinone-cytochrome bc2 oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c₂ in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c₂ by ZH₂ is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c₂ reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH₂ and ferricytochrome c₂, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c₂ reduction at pH 7.0 and at 24°C was 2 · 109 M^?1 · s^?1, but this varied with pH, being 5.1 · 10⁸ M^?1 · s^?1 at pH 5.2 and 4.3 · 10⁹ M^?1 · s^?1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.     

The kinetics of intrinsic factor-vitamin B12 binding

The kinetic behaviour of intrinsic factor-vitamin B₁₂ binding has been examined under varying conditions using an albuminised charcoal separation technique. The overall reaction obeys second order rate laws. The intrinsic factor considered alone obeys first order laws; the velocity of reaction of vitamin B₁₂ is too fast for measurement by the technique described but by deduction obeys first order laws. Rate constants as three temperatures, (k₂ at 25°C=1.56·10⁸·mole^?1·s^?1) the activation energy (E=12.7 kJ·mole^?1) and Arrhenius constant (A=2.7·10¹⁰ 1·mole^?1·s^?1 have been calculated. There is the possibility of diffusion control of the reaction in which case the E and A values are invalid. The effect of pH on the reaction has been studied and the results discussed in relation to the pH studies of other workers whose results show disagreement. Albumin coated charcoal was shown to discriminate between intrinsic factor-vitamin B₁₂ and free vitamn B₁₂ over a wide pH range. The apparent under-estimation of intrinsic factor in dilute solution was shown to be due to adsorption of the intrinsic factor to plastic tubes.     

X-ray diffraction measurements of dipalmitoylphosphatidylcholine as a function of pressure

A kinetics of azide binding by horseradish peroxidase was studied by temperature-jump method. It was found that the reaction of the enzyme with azide is quite rapid, occuring in microsecond time range. This rate is unusually rapid in contrast to the usual hemoprotein ferric iron-ligand interactions so far reported. The resulting value for the apparent association and dissociation rate constants were k₁=6.8×10⁶ M^?1 s^?1 and k¹=3.5×10⁵ s^?1 at 23°C and pH 5.0 for the reaction. The pH dependence of the rate constants was also studied to show a strong linkage of the ligand binding with a proton uptake of a dissociable group on the enzyme.     

Cloning,expression and some properties of α-carbonic anhydrase from Helicobacter pylori

The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO₂ hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are k_cat=2.4×10⁵ s⁻¹, K_M=17 mM and k_cat/K_M=1.4×10⁷ M⁻¹ s⁻¹. The pH dependence of k_cat/K_M fits with a simple titration curve with pK_a=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO₂ hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k_enz, of 24 M⁻¹ s⁻¹ at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a k_enz value of 665 M⁻¹ s⁻¹ was obtained under similar conditions.     

Reaction of cythchrome c with one-electron redox reagents. I. Reduction of ferricytochrome c by the hydrated electron produced by pulse radiolysis

Pulse radiolysis-kinetic spectrometry has been used to investigate the reaction of hydrated electrons with ferricytochrome c in dilute aqueous solution at pH 6.5–7.0. Time resolutions from 2·10^?7 to 1 s were employed. Transient spectra from 320 to 580 nm were characterized with a wavelength resolution of ±0.5 nm. 1 In neutral salt-free solution, k(ferricytochrome c+e^?_aq)=(6.0±0.9)·10¹⁰ M^?1·s^?1 and k(ferricytochrome c+H)=(1.2±0.2)·10¹⁰ M^?1·s^?1. The reaction of ferricytochrome c with hydrated electrons is sensitive to ionic strength; in 0.1 M NaClO₄, k(ferricytochrome c+e^?_aq)=(2.4±0.4)·10¹⁰ M^?1·s^?1. In contrast, k(ferricytochrome c+H) is insensitive to ionic strength. Time resolution of three spectral stages has been accomplished. The primary spectrum is the first observable spectrum detectable after irradiation and is formed in a second-order process. Its rate of formation is indisting-uishable from the rate of disappearance of the electron spectrum. The secondary spectrum is generated in a true first order intramolecular process, k(p→s)=(1.2±0.1)·10⁵ s^?1. The tertiary spectrum is also generated in a true first-order process, k(s→t)=(1.3±0.2)·10² s^?1. The specific rates of both transformations are independent of the wavelength of measurement. The tertiary spectrum, observable 50 ms after initial reaction and remaining unchanged thereafter for at least 1 s, shows that relaxed ferrocytochrome c is the only detectable product. This product is not autoxidizable, as expected for native reduced enzyme. It is more probable that the intramolecular changes responsible for the p→s and s→t spectral transformations involve the influence of conformational relaxation of ferrocytochrome c upon electronic energy states then that they are intramolecular transmission of reducing equivalents from primary sites of electron attachment.     

Effect of pH on structure,function, and stability of mitochondrial carbonic anhydrase VA

Mitochondrial carbonic anhydrase VA (CAVA) catalyzes the hydration of carbon dioxide to produce proton and bicarbonate which is primarily expressed in the mitochondrial matrix of liver, and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis, and neuronal transmission. To understand the effect of pH on the structure, function, and stability of CAVA, we employed spectroscopic techniques such as circular dichroism, fluorescence, and absorbance measurements in wide range of pH (from pH 2.0 to pH 11.5). CAVA showed an aggregation at acidic pH range from pH 2.0 to pH 5.0. However, it remains stable and maintains its secondary structure in the pH range, pH 7.0–pH 11.5. Furthermore, this enzyme has an appreciable activity at more than pH 7.0 (7.0 < pH ≤ 11.5) with maximum activity at pH 9.0. The maximal values of k_cat and k_cat/K_m at pH 9.0 are 3.7?×?10⁶ s^?1 and 5.5?×?10⁷ M^?1 s^?1, respectively. However, this enzyme loses its activity in the acidic pH range. We further performed 20-ns molecular dynamics simulation of CAVA to see the dynamics at different pH values. An excellent agreement was observed between in silico and in vitro studies. This study provides an insight into the activity of CAVA in the pH range of subcellular environment.     

The Reaction of no With Superoxide

The rate constant for the reaction of NO with ·O₂^? was determined to be (6.7 ± 0.9) × 10⁹ 1 mol^?1 s^?1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 both by monitoring the loss of ·O₂^? and the formation of the product ^?OONO. The decay rate of ^?OONO, in the presence of 0.1 moll^?1 formate, ranges from 1.2s^?1 at pH 5 to about 0.2s^?1 in strong base, the latter value probably reflecting catalysis by formate.     

Kinetic studies on the binding of Streptomyces subtilisin inhibitor with subtilisin BPN′

The binding mechanism of Streptomyces subtilisin inhibitor and subtilisin BPN′ was studied kinetically with the stopped-flow method by monitoring the protein fluorescence increase due to complex formation. In the lower concentration range of proteins, the reaction followed the second-order kinetics. The pH dependence of the apparent second-order rate constant, k_on, suggested the involvement of the two ionizable groups of pK_a of 7.8 and 10 in the binding. The activation parameters were calculated from the temperature dependence of the apparent second-order rate constants. The value of the apparent activation energy (E_A = 39.7 kJ · mol^?1, 9.50 kcal · mol^?1) and insensitivity of k_on to the viscosity of the medium suggest that the binding is not a simple diffusion-controlled bimolecular association. Further studies with a much broader range of protein concentrations have revealed that the reaction tends to approach first-order kinetics as the inhibitor concentration increases. The binding reaction is, therefore, reconcilable with a two-step mechanism, in which a fast bimolecular association is followed by a slow unimolecular isomerization step; the dissociation constant of the first step, K_L, is estimated to be 1.2 × 10^?4m and the rate constant of the second step, k₊₂, to be 770 s^?1. It was also found that the increase of tryptophan fluorescence due to the complex formation occurs solely in the rate-determining unimolecular process.     

PHOTOSYNTHESIS-IRRADIANCE RELATIONSHIPS IN SEA ICE MICROALGAE FROM McMURDO SOUND,ANTARCTICA1

Sea ice microalgae in McMurdo Sound, Antarctica were examined for photosynthesis-irradiance relationships and for the extent and time course of their photoadaptation to a reduction in in situ irradiance. Algae were collected from the bottom centimeter of coarse-grained congelation ice in an area free of natural snow cover. Photosynthetic rate was determined in short term (1 h) incubations at ?2° C over a range of irradiance from 0 to 286 μE·m^?2·s^?1. Assimilation numbers were consistently below 0.1 mg C·mg chl a^?1·h^?1. The I_k's³ averaged only 7 μE·m^?2·s^?1, and photosynthesis was inhibited at irradiances above 25 μE·m^?2·s^?1. Photosynthetic parameters of the ice algal community were examined over a nine day period following the addition of 4 cm of surface snow while a control area remained snow-free. A reduction of 40% in P_m^B relative to the control occurred after two days of snow cover; α, β, I_k, and I_m were not significantly altered. Low assimilation numbers and constant standing crop size, however, suggested that the algal bloom may have already reached stationary growth phase, possibly minimizing their photoadaptive response.     

Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.