On dielectric constant and enzymatic kinetic. IV. Dipolar ions. Ester hydrolysis by trysin and alpha chymotrypsin in glycine solutions.

A study was made on the effect of glycine on systems involving trypsin and BAEE(1) or TSAME on the one hand, or alpha-chymotrypsin with any of the substrates BAEE, TEE, or PEE, on the other. In all cases there was a linear relationship between the rate logarithm and the reciprocal of the dielectric constant of the glycine solution. The slopes were positive in the reactions of trypsin.

Effect of ionic strength on the kinetics of trypsin and alpha chymotrypsin.

The kinetic effects resulting from changes in the medium ionic strength on reactions involving trypsin or alpha-chymotrypsin are different. The reaction rate increases continuously as the ionic strength increases with alpha-chymotrypsin. With trypsin, the rate increases at low ionic strengths but as the ionic strength further increases a gradual inhibitory effect is observed.

On Dielectric Constant and Enzymatic Kinetics : III. Interrelationships of dielectric constant and pH.

The dielectric effects on trypsin and alpha-chymotrypsin activities have revealed that at pH 7.8 the active species of the former is the cation while that of the latter is the anion. The present study on the dielectric effects along the pH-activity curves shows that trypsin remains positive within the pH range of 5.

Effect of the medium dielectric strength on the activity of alpha chymotrypsin.

The rates of hydrolysis of TrEE, TEE, and ATEE(1) by alpha-chymotrypsin were determined in media of variable dielectric strength. Many substances which modify the dielectric constant of the medium, exert additional specific effects on the reaction rate, noticeable at more or less elevated concentrations. Notwithstanding, it is possible to differentiate the dielectric and specific effects by comparing the rates in solvents of distinct nature at relatively low concentrations.

The influence of the medium dielectric strength upon trypsin kinetics.

The use of aqueous alkali for the titration of esterolytic activity when the esters are dissolved in alcoholic solutions, results in an error due to changes in the ionization of the buffer. This is corrected by titrating with alkali in the same solvent as the substrate. Alcohols and other substances which change the dielectric strength of water modify the rate of hydrolysis of BAEE(1) and TSAME by trypsin to an extent proportionate to their effect on the dielectric strength.

The action of alpha chymotrypsin upon mixtures of esters and proteins at enzyme-saturating concentrations.

The behavior of alpha-chymotrypsin has been studied in the simultaneous presence of two different substrates, each present in the reaction mixture at its saturation level. Mixtures of two esters were hydrolyzed at rates intermediate between the rates of hydrolysis of each ester when present alone, suggesting, in this case, competitive hydrolysis. In contrast, the rates of hydrolysis in mixtures of casein with gelatin or of either protein with an ester were equal to the sum of the rates of hydrolysis of the separate substrates, indicating in these cases independent hydrolysis.

Simultaneous hydrolyses of esters and proteins at saturation levels.

A direct titration method for the determination of proteolytic activity is discussed. This involves the potentiometric measurement of the volume of 0.08 N NaOH required to maintain a constant pH (8.