Stopped-flow kinetic analysis

Abu-Soud, H., Mullins, L. S., Baldwin, T. O., and Raushel, F. M. (1992). Stopped-flow kinetic analysis of the bacterial luciferase reaction. Biochemistry 31 3807-3813. 

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

For faster reactions the speed with which reactants can be mixed is a hmitation the mixing time must be less than the half-time. Stopped-flow techniques have been developed that permit remarkably short kinetic analysis times. Beckwith and Crouch described a stopped-flow kinetic analysis apparatus, with a mixing and dead time of less than 0.01 s, capable of analyzing 1000 phosphate samples per hour with a relative standard deviation of about 1%. Sample handUng, mixing, and gathering and evaluation of data were automated with the help of on-Une computer systems. 

Stopped Flow Kinetic Analysis A Direct Assay for Superoxide Dismutase Activity... 

We recognized the need for methodology to measure SOD activity directly that would be more accessible to the bench-top scientist than is the method of pulse radiolysis, another direct measure. Consequently, we developed methodology to measure the catalytic dismutation of superoxide by stopped-flow kinetic analysis.By this technique, we directly monitor the decay of superoxide spectrophotometrically in the presence or absence of a putative SOD mimic at a given pH. Kinetic analysis of this decay can determine whether the complex is a SOD mimic (decay of superoxide becomes first-order in superoxide and first-order in complex see equations 1 and 2), or is inactive (decay of superoxide remains second-order for its self-dismutation see equation 3). At least a tenfold excess of superoxide over the putative SOD mimic is used in the stopped-flow assay, to eliminate contributions due to a stoichiometric reaction of the complex with superoxide. A catalytic rate constant for the dismutation of superoxide by the complex can be determined from the observed rate constants of superoxide decay as a function of catalyst concentration. ... 

Figure 4 Catalytic rate constants (Katifof ihe dismutation of superoxide and the kinetic stabilities the dissociative rate constant) of the Mn(it)-based SOD mimics SC-52608, SC-54417, and SC-55858. The values are measured by stopped-flow kinetic analysis, and the values are measured by spectrophotometrically monitoring the exchange ofCu(ii)for the Mn(il) in the complex...

Monosubstituted Mn(ii)-based complexes, which catalyze the dismutation of superoxide as shown by stopped-flow kinetic analysis, were tested for antiinflammatory activity in the mouse acetic acid-induced colitis model. As can be seen from Table 3, all of the SOD mimics are anti-inflammatory. Histological analysis of the colonic tissue confirmed these results. Of particular importance is the observation that Mn(ii) complexes that have no SOD activity, specifically the Mn(ii) dichloro complexes of 1,4,7,10,13-pentaazacyclohexadecane and 1,4,7,11,14-pentaazacycloheptadecane, do not protect against the colonic inflammation induced by acetic acid when the compounds are administered in-tracolonically at a dose of 30mgkg These results are consistent with a role for superoxide as a mediator of neutrophil-dependent inflammation. Consistent with this hypothesis is the observation that SC-52608 inhibits neutrophil-dependent inflammation induced by the intradermal administration of leukotriene 64, a neutrophil chemoattractant. ... 

Zuberbiihler etal. reported stopped-flow kinetic investigations of the oxygenation reactions of a series of tefradentate copper(I) complexes in propionitrile, tetrahydro-furan, and acetone. The formation of [(L °)Cu (02 )]+ can only be followed below 203 K because at a higher temperature it occurs faster than the stopped-flow instrumental limit. On the basis of experimental results, the authors proposed a reaction mechanism, which involved four steps, at least three intermediates. On the basis of the kinetics analysis, spectra for species (intermediates and product) could be calculated. 

M. Pistonesi, M.E. Centurion, B.S.F. Band, P.C. Damiani, A.C. Olivieri, Simultaneous determination of levodopa and benserazide by stopped-flow injection analysis and three-way multivariate calibration of kinetic-spectrophotometric data, J. Pharm. Biomed. Anal 36 (2004) 541. 

M. Blanco, J. Coello, H. Iturriaga, S. Maspoch, J. Riba, E. Rovira, Kinetic spectrophotometric determination of Ga(III)-Al(III) mixtures by stopped-flow injection analysis using principal component regression, Talanta 40 (1993) 261. 

Saurina J, Hernandez-Cassou S, Tauler R, Multivariate curve resolution and trilinear decomposition methods in the analysis of stopped-flow kinetic data for binary amino acid mixtures, Analytical Chemistry, 1997, 69, 2329-2336. 

H. Kagenow and A. Jensen, Kinetic Determination of Magnesium and Calcium by Stopped-Flow Injection Analysis. Anal. Chim. Acta, 145 (1982) 125. 

Spin saturation transfer studies of the 31p NMR of the iso-propyl complex yield data regarding the rate of interconversion of the dihydride and molecular hydrogen complexes. Thus ki is calculated to be 63 s-1 (299 K) and k-i is calculated to be 12.4 s-l(300 K). These values are in reasonable agreement with the corresponding values determine by stopped-flow kinetics for the cyclohexyl complex as described above ki = 37 S l(298 K) and k-i = 18 S l (298 K). Activation parameters for conversion of the dihydrogen to dihydride reaction (kq step) were determined based on analysis of the spectral changes in the temperature range 288-310 K. The values Zij = 16.0 0.2 kcal/mole, AI = 10.1 1.8 kcal/mole and -19.9 6.0 cal/mole deg were determined. 

The reactivity of metal nitrosyl complexes (51) with thiols is of particular concern in the mobilization of NO to make it accessible for the vasodilation process. Very recently, it has been reported (52) that the S-atom of cysteine reacts to bind the N-atom of the nitrosyl complex of Ru-edta to form a 1 1 intermediate species. Stopped-flow kinetic studies revealed the formation of a transient species, whose rate of formation was found to be first order with respect both [Ru (pac)(NO)] and RSH. The values of rate constants ( 1) were formd to be in the range (0.2-5) x 10 M s at 25°C. Considering the spectral features and kinetic behavior of various [Ru (pac)(SR )] and [Ru (pac)NO] species as described in the preceding sections, and analysis for the products of the above reaction (N2O), the following mechanism (Scheme 15) for the redox reactions involving electron transfer fi om thiols to coordinated NO, that results in the formation of disrdfide (RSSR) and N2O, has been proposed for the reaction of [Ru (pac)(NO)] with thiols (RSH). 

Dr. M.T. Duarte (Institute Superior Tecnico) for the X-ray diffraction analysis of one of the isocyanide complexes, Dr.R. Henderson (Nitrogen Fixation Laboratory, Univ. Sussex) for stopped-flow kinetic studies. Dr. E.G. Bakalbassis and Prof. C.A. Tsipis (Aristotle Univ., Thessaloniki)for the extended Huckel MO calculations, as well as, from our laboratory, Dr. M.F.N.N. Carvalho (some isocyanide and aminocarbyne complexes, and stopped-flow studies). Lie. M.A.N.D.A. Lemos (electrochemical studies). Lie. S.P.R. Almeida and Lie. M.F.C. Guedes da Silva (some vinylidene and carbyne complexes, and electrochemical studies). 

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