Subzero temperature quenching and electrophoretic methods for the isolation of protein reaction intermediates

A quenching technique for the study of rapid protein reactions is described which consists of injecting a small volume of aqueous solution of reactants into a large volume (X10) of hydro-organic solvent cooled at subzero temperature and mechanically shaken. The protein reaction intermediates, stabilized at subzero temperature and brought into a hydro-organic solution, can then be separated by subzero temperature electrophoretic methods, such as isoelectric focusing, in the same solvent. The alkaline hydrolysis of 2,4-dinitrophenylacetate was studied by the use of this quenching technique in order to compare the quenching time and the rate constants of the reaction with those obtained by normal rapid quenching methods. It was found that first-order reactions having rate constants up to about 5 s-1 can be satisfactorily studied by this technique. The technique is not suitable for the study of faster reactions because of the high value of the quenching time (40-100 ms). The hybridization reaction of carboxyhemoglobins A and C in aqueous solution at 22 degrees C was studied as an example of the application of this quenching technique and of the isoelectric focusing method at subzero temperature to the isolation of unstable intermediates in a protein reaction.