Stopped-flow method Temperature-Jump combination

To study rates of antibody-hapten reactions the principal methods employed have involved stopped flow or temperature jump techniques. The former was first used by Sturtevant et al. (64) and Day et al. (65). The temperature jump method has been employed by Froese, Sehon, and their collaborators (66-68) and more recently by Pecht et al. (69). Both methods are utilized in conjunction with very rapid optical measurements (in the millisecond range). For example, Sturtevant et al. took advantage of a spectral shift which occurs upon combination of anti-Dnp antibody with the dye, 2-(Dnp-azo)-I-naphthol-3,6-disulfonic acid (64). With the same hapten, and with e-Dnp-L-lysine and e-Dnp-6-aminocaproate. Day et al. (65) used the method of fluorescence quenching (Section VI,D) with a stopped flow apparatus. In the temperature jump technique the components are first equilibrated, a temperature increment is rapidly induced (up to 10°C in 0.1 isecond), and the rate of reequilibration at the new temperature is measured. Velocity constants can be estimated from the data the mathematical approaches required are described in the references cited. 

Clearly, this method cannot be applied to systems in which there are irreversible chemical processes. It is most suitable for situations involving simple ligand binding (such as NAD+ with a dehydrogenase), inhibitor binding, or conformational changes in the protein. There have been some attempts to combine the temperature-jump with the stopped-flow method. 

Temperature-jump can be combined with c-jump (for example, stopped-flow) methods. 

To circumvent the equilibrium requirement, which is the greatest limitation of temperature jump, attempts have been made to combine this technique with some others. The stopped-flow temperature jump, for example, has found use in studies of reactions involving the formation of intermediates on not-too-short timescales (>10 ms) [23]. In this method, the temperature jump is applied during the course of the stopped-flow reaction. The equilibrium between the reactants and intermediates is perturbed, which permits a direct study of the fast steps occurring prior to the rate-determining step [28]. 

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