Stopped-flow spectrophotometer

The reaction of Ru(III) chelate with diimine is about 99 times more efficient than that of Ru(III) with hydrazine. Computer-simulated chemiluminescence time curves based on the kinetic data of the above reaction scheme exactly matched light-intensity time curves recorded in a stopped-flow spectrophotometer 166h At high hydrazine concentra-... 

HjO]/[AOT] ratios influenced the equilibrium sizes of CdS particles generated rates of CdS growth, determined in a stopped-flow spectrophotometer, were consistent with the rate-determining intermicellar exchange of solubilizates... 

The rate at which liquid crystalline bile salt monoolein aggregates were dispersed by bile salt solutions was determined by Quentin Gibson (Cornell University, Ithaca, N. Y.) using a rapid mixing stop-flow spectrophotometer designed in his laboratory (4). 

The thematic approach to isolating the deacylation step is to generate the acylen-zyme in situ in the stopped-flow spectrophotometer by mixing a substrate that acylates very rapidly with an excess or stoichiometric amount of the enzyme. The acylenzyme is formed in a rapid step that consumes all the substrate. This is then followed by relatively slow hydrolysis under single-turnover conditions. For example, acetyl-L-phenylalanine p-nitrophenyl ester may be mixed with chy-motrypsin in a stopped-flow spectrophotometer in which the enzyme is acylated in the dead time. The subsequent deacylation may be monitored by the binding of proflavin to the free enzyme as it is produced in the reaction.8... 

There are also nonthematic methods that allow the formation of acylenzymes under conditions where they are stable, so that they can be stored in a syringe in a stopped-flow spectrophotometer. For example, it is possible to synthesize certain nonspecific acylenzymes and store them at low pH.9 12 When they are restored to high pH, they are found to deacylate at the rate expected from the steady state kinetics. This approach has been extended to cover specific acylenzymes. When acyl-L-tryptophan derivatives are incubated with chymotrypsin at pH 3 to 4, the acylenzyme accumulates. The solution may then be pH-jumped by mixing it with a concentrated high-pH buffer in the stopped-flow spectrophotometer.1314 The deacylation rate has been measured by the proflavin displacement method and by using furylacrylolyl compounds. 

Two points should be noted (1) Because the rate constants are pseudo-unimolecular, there is a concentration dependence, so ka and koff may be resolved without the amplitude factor. (2) There is a lower limit to 1/r that is, 1/t cannot be less than koS. This sets a limit on the measurement of these rate constants. A good stopped-flow spectrophotometer can cope only with rate constants of 1000 s 1 or less, and many enzyme-substrate dissociation constants are faster than this. 

The hydrolysis of amides in the presence of acceptor nucleophiles gives the same product ratios as those found for the hydrolysis of the methyl ester (Ac-Phe-OCH3) under the same conditions (Table 7.4). Furthermore, these product ratios are the same as those expected from direct rate measurements of the attack of the nucleophiles on Ac-Phe-chy-motrypsin, generated in situ in the stopped-flow spectrophotometer (Table 7.5). 

A Multi-Microcomputer Controlled, Vidicon, Stopped-Flow Spectrophotometer... 

Me had a number of goals in mind when we set out to design a new vidicon rapid scanning stopped-flow spectrophotometer. 

The combination of rapid mixing and fast detection systems allows cationic polymerisations to be followed on an even shorter time scale than with adiabatic calorimetry. Recent commercial stop-flow spectrophotometers have a dead time of about 15 msec, an improvement of more than one order of magnitude over previous home-made models. This implies that reactions with half lives of less than 100 msec can be analysed kinetically with a good degree of accuracy. Hi -vacuum techniques are not compatible with these instruments and all operations are therefore carried out in an inert atmosphere. 

A stopped-flow spectrophotometer was used to obtain the kinetics data for the reaction of a nickel(II) complex NiL2 of a substituted bidentate diamine ligand (L = tetmeen = 2,3-dimethyl-2,3-diaminobutane) with cyanide ion. The reaction is biphasic one diamine ligand is replaced by two cyanide ligands, then the second diamine ligand is replaced ... 

At 25 °C, the initial reaction is much faster, indicating a considerable activation enthalpy, and the concentration range is limited to a value equal to or less than 1.35 mM for the time scale of our stopped-flow spectrophotometer. Again the rate is first order with respect to both enzyme and substrate. The amplitude of the initial phase at this higher temperature also increases with reductate concentration, but in a more pronounced manner. At the highest concentration used, this initial rapid phase represents 80% of the total reaction amplitude. 

Actinomycin D dissociation kinetics were measured on a Cary 219 spectrophotometer equipped with a magnetic stirrer and thermostated cell holders. Sodium dodecyl sulfate (SDS) was used to sequester dissociating actinomycin D, and the resulting Increase In absorbance was monitored at 452 nm as a function of time. Stop-flow studies (daunorubicin and daunorubicin/ actinomycin D) were conducted with a Durrum-Glbson Model 110 stopped-flow spectrophotometer equipped with a dual detector accessory and a Tektronix storage oscilloscope Interfaced with a PDF 11/34 computer. Experiments were done In a 0.01M Na phosphate buffer, 0.1M NaCl, 0.001M NaEDTA, pH=7. Dissociation time constants were computed with a multlexponentlal analysis computer program. 

Durrum-Gibson D-109 stopped- flow spectrophotometer interfaced to an IBM-PC XT (ti/2... 

The generation of O2 from potassium superoxide was also applied to stop-flow procedures. In this method O2 was dissolved in dimethyl sulfoxide and stabilized in 18-crown-6-polyether. This method is useful for mechanistic studies indeed, McClune and Fee (1976) were able to obtain catalytic rate constants for bovine copper/zinc superoxide dismutase as a function of pH in various buffers. More recently the mechanism of catalysis and of anion inhibition of iron superoxide dismutase from E. coli have been examined by this method using a specially constructed stop-flow spectrophotometer (Bull and Fee, 1985). A limitation of the method is that the pre-equilibrium state cannot be properly investigated because of the time resolution of the stop-flow equipment (== 5 msec). 

Fig. 3. Block diagram of data acquisition and analysis system. In our laboratories two systems based on the above have been built. One is a Durrum-Gibson stopped-flow spectrophotometer coupled through a Datalab DL901 transient recorder to a Commadore PET 32K minicomputer (used for data in Fig. 2). Another system consists of a Canterbury SF-3A stopped-flow instrument interfaced, via a Datalab DL901 transient recorder to an Exidy Sorcerer minicomputer. Software is available.

If the a-chymotrypsin-catalysed hydrolysis of 4-nitrophenyl acetate [10] is monitored at 400 nm (to detect 4-nitrophenolate ion product) using relatively high concentrations of enzyme, the absorbance time trace is characterised by an initial burst (Fig. 5a). Obviously the initial burst cannot be instantaneous and if one uses a rapid-mixing stopped-flow spectrophotometer to study this reaction, the absorbance time trace appears as in Fig. 5b. Such observations have been reported for a number of enzymes (e.g. a-chymotrypsin [11], elastase [12], carboxypeptidase Y [13]) and interpreted in terms of an acyl-enzyme mechanism (Eqn. 7) in which the physical Michaelis complex, ES, reacts to give a covalent complex, ES (the acyl-enzyme) and one of the products (monitored here at 400 nm). This acyl-enzyme then breaks down to regenerate free enzyme and produce the other products. The dissociation constant of ES is k2 is the rate coefficient of acylation of the enzyme and A 3 is the deacylation rate coefficient. Detailed kinetic analysis of this system [11] has shown... 

Figure 15-7 Stopjsed-flow traces of iron oxidation by EcFtn-(E129K + E130A). Final protein concentration 3 pM in 0.1 M Mes buffer pH 6.5 and 48 Fe(II) atoms per molecule. Spectra recorded over 50 seconds using photodiode array stopped-flow spectrophotometer. Maximum absorbance at 600 nm was obtained at 0.9 seconds.

Coagulation of Polystyrene Latex in a Stopped-Flow Spectrophotometer, J. Colloid Interface Sci. 1974, 49, 281-285. 

Paul, C., K Kirschner, et al. (1979). "Calibration of Stopped-Flow Spectrophotometers Using a Two-Step Disulfide Exchenge Reaction." Analytical Biochemistry 101 442-448. Roister, J. and H. Lachmann (1989). Spectrometric Titrations Analysis of Chemical Equilibria. Weinheim, VCH. 

Figure 3. View of the whole apparatus. Right, a Union Giken RA-1300 stopped-flow spectrophotometer, gas-pressure-driven type) in light-scattering mode left, a Union Giken RA-450 rapid data processor with a cathode ray monitor on the processer. The recorder is on the upper center shelf.

A newly designed, high-resolution rapid-scan-stopped-flow spectrophotometer has been used to study the sequential aquation reactions shown in equation (34), where ki = 86.6 s" k2 = 5.2 s and k = 0.145 s at 298.2 K in... 

Figure 3 shows a schematic diagram of a stopped-flow spectrophotometer (or spectrofluorimeter) coupled to an automatic analyzer controlled by a computer that also handles data acquisition and processing. ... 

Figure 3 Diagram of a stopped-flow spectrophotometer or spectrofluorimeter. (Reproduced with permission from Perez-Bendito D and Silva M (1988) Kinetic Methods in AnafyUca Chemistry. Chichester Ellis Norwood.)...

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