Spectroscopy (stopped flow method)Spectroscopy:It is the analysis of the electromagnetic radiation emitted, absorbed, or scattered by molecules. It is used to analyze materials and to monitor changes in concentration in chemical kinetics. Since photons act as messengers from atoms, the atomic spectra can be used to obtain detailed information about electronic structure and molecules.
The energy of a molecule can change because of electronic transitions, and it can make transitions between its rotational and vibrational states. They are more complicated than atomic spectra and can give more information about electronic energy levels and about bonds length, strength, and angels (1).Stopped-flow technique:The solutions are mixed very rapidly and forced from syringes into a mixing chamber designed to ensure that the flow is turbulent and that complete mixing occurs very quickly.
Behind the chamber, there is an observation cell fitted with a plunger that moves back as the liquids flood in and comes up against a stop after a certain volume. Filling the chamber corresponds to a sudden creation of an initial sample of the reaction mixture. The reaction then continues in the thoroughly mixed solution and is monitored spectrophotometrically. After a very short time of the flow, the flow is stopped suddenly when the observation cell is filled by an opposing piston which is linked to a sensing switch that triggers the measuring device (2).
Small volumes of solutions are used, and the kineticequations for modeling the reactions are equivalent to those used inconventional methods in which concentration and time are measured.It is useful to study fast reactions that have half-lives as short as a few milliseconds. It is appropriate for studying biochemical reactions, such as studying enzymes. ?
Formation of Peroxynitrite
Peroxynitrite, an inorganic molecule of biological interest, is the product of the combination reaction between nitric oxide (?NO?^•) and superoxide (O_2^(•-))(4).?NO?^•+ O_2^(•-) ? ?ONOO?^-Increasing sensitivity can potentially allow the reaction to be measured, to be studied by slowing down the reaction rate through the use of lower concentration of reactants.the rate of the isomerization reaction depends little on the pressure ‘high pressure slows down the rate of reaction'(3).
With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa.The kinetics of the decay of hydrogen peroxynitrite are not very pressure-dependent: from stopped-flow experiments up to 152 MPa the rate of the isomerization reaction depends little on the pressure.
Materials:
Deionized water.
Buffers, which are saturated with argon immediately before use, from salts and acids.
Freshly prepared peroxynitrite.
Prepration: Peroxynitrite was prepared by a modification of biomimetic synthesis of nitrogen monoxide with solid potassium superoxide. In Brief, 30% mol nitrogen monoxide was added to 100% mol solid potassium superoxide that was stirred with sand under argon in a 100-mL Erlenmeyer flask. Laser Flash Photolysis: the following reactions could take place:(i) Isomerization: cis and trans- peroxynitrite: (ii) Photodissociation: (iii) Photoionization: Reactions 3-6 can be neglected and for spectroscopic reasons reactions 8 and 9 must be abandoned.Under the assumption that we observed the reverseof reaction 7.
The global analysis gives a rate constant of (1.5-2.0) × ?10?^10 M^(-1) S^(-1). Conventional second-order analysis at 25 different wavelengths and 4 different laser intensities resulted in a value of (1.6 ( 0.2) × 1010 M^(-1) S^(-1).The rate constant for reaction 7 of 1.6 × ?10?^10 M^(-1) S^(-1) is higher than those reported by other workers. These rate constants were measured under experimental designs that involve reaction cascades to arrive at the reactants.
The rapid one-step production of superoxide and nitrogen monoxide reported herein has the advantage of allowing measurement of the recombination rate not complicated by other processes.The high rate constant has important consequences for the generation of peroxynitrite in vivo. Under normal in vivo conditions, the concentrations of nitrogen monoxide and superoxide dismutase are on the order of 1 × ?10?^(-8) and 5 × ?10?^(-6) M, respectively, whereby superoxide dismutase scavenges 98-99% of all superoxide, and consequently very little peroxynitrite is formed.
Near an activated macrophage the nitrogen monoxide and superoxide concentrations may be 100 times higher, and the ratio of superoxide reacting with nitrogen monoxide to superoxide reacting with superoxide dismutase becomes 3:2. While considerations based on homogeneous solution kinetics do not necessarily apply to the inhomogeneous milieu inside or outside a cell, they do indicate that peroxyitrite formation is likely.According to the mechanism we can represent the rate law as:Rate = k [?NO?^•][O_2^(•-)]In addition to the determining step, a second-order curve was obtained which proves that the overall order of this reaction is 2.
