Search

To evaluate contrast sensitivity and disability glare in patients with dry eye using the Contrast Glaretester 1000.

The rates of hydrolysis of TrEE, TEE, and ATEE1 by α-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. Thus, the effect of varying the dielectric strength could be studied within wider ranges (ΔD = 20 with TrEE and ca. 28 with ATEE) than in the previous study of trypsin (ΔD = 12). The dielectric effect on α-chymotrypsin is the opposite of that observed with trypsin. In both cases there is a linear relationship between the logarithm of the rate of hydrolysis and the reciprocal of the dielectric constant. The slope is negative with α-chymotrypsin and positive with trypsin. According to expressions relating the dielectric constant to the rate in non-enzymatic reactions, the behavior of α-chymotrypsin is like that of a negative ion, while trypsin behaves as a positive ion. The enzyme activity appears to depend upon the arrangement of charges in the enzyme and substrate molecules, rather than on the presence of certain atomic groupings in the substrate.

The dielectric effects on trypsin and α-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 5.5 to 8.5. Conversely, α-chymotrypsin is positive from pH 5.5 to 6.6, negative from 6.6 to about 8.1, and at pH 8.25 becomes positive again. The first point of inversion in charge sign shifts from 6.6 to 7.15 with the addition of 0.05 M phosphate buffer. The point of inversion does not seem to be modified significantly by changes in the substrate structure. At pH values near the point of inversion the plots of rate log vs. 100/D are broken lines formed by various straight portions, the slope of each varying progressively from a maximum positive to a maximum negative value. This suggests an effect of resonance possibly attributable to an imidazole group. As an attempt to explain the two observed points of sign inversion in α-chymotrypsin, the possibility is suggested that different enzyme configurations are disclosed by the combined action of pH and dielectric constant. On this theoretical basis, it i6s feasible that more than one isoionic point exists.

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 BAEE1 and TSAME by trypsin to an extent proportionate to their effect on the dielectric strength. The reaction rate increases with diminished dielectric strength and vice versa. At low concentrations of substance there seems to be no specific effect other than that derived of the variation in dielectric strength. At higher concentrations, the enzyme might be denatured. In addition, it is probable that specific effects of each substance might intervene. The Coulombic and thermic energies of activation were calculated for the two esters in various solvents. The plot of the logarithm of rate constant vs. reciprocal of dielectric constant yields a straight line with positive slope. This behavior is similar to that of a non-enzymatic positive ion-dipole reaction. Trypsin reacts like a positive ion. The possible influence of the dielectric strength on the regulation of the equilibria involved in the interconversion of the various forms of trypsin in solution (active, inactive, denatured) is discussed.

The behavior of α-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. The activity of the α-chymotrypsin preparation used could not be attributed to contamination with other enzymes. Studies of the effect of soy bean inhibitor on chymotrypsin indicate that the mechanism of inhibition with protein substrates differs from that when esters are used, providing further evidence that α-chymotrypsin reacts differently with esters and proteins. These results indicate that if chymotrypsin forms specific complexes with its substrates, it must possess at least three distinct active sites. However there is independent chemical evidence that the proteolytic and esterolytic activities of this enzyme reside in the same active center. If this is true, the experimental observations reported here cannot be explained unless it is supposed that this enzyme does not form specific Michaelis complexes with its substrates.

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 (8.0) during the time of the hydrolysis. It is a sensitive method which presents several advantages; viz., it measures simultaneously protease and esterase activity, it follows the hydrolysis very closely and from the first stages; the titration is continuous and on the same sample. This method determines a constant fraction of the groups titratable by formol titration. The ratio formol: direct titration is represented by a factor "f" which is presumed to be distinct for each protein-enzyme system. Kinetic studies, using this method, revealed that the rates of hydrolysis of mixtures casein-gelatin on one hand, casein-BAEE or gelatin-BAEE on the other, are always larger than those of the corresponding isolated substrates. In many cases the resulting rates are equal or nearly equal to the sum of the individual rates, even though the mentioned rates have been determined within the saturation zones for every substrate. The former observations are inconsistent with the theory of the formation of an intermediary enzyme-substrate compound, unless it is assumed that the enzyme has a specific active group for each substrate.

The kinetic effects resulting from changes in the medium ionic strength on reactions involving trypsin or α-chymotrypsin are different. The reaction rate increases continuously as the ionic strength increases with α-chymotrypsin. With trypsin, the rate increases at low ionic strengths but as the ionic strength further increases a gradual inhibitory effect is observed. The effects produced by different salts of various valence types (from uni-univalent to uni-trivalent or tri-univalent) are essentially the same, and they are a function of the square root of the ionic strength. The quantitative differences among the various salts may be accounted for on the basis of individual properties of the ions, such as the size of the hydrated ion, "association," etc. The effects of salts on the enzymic reactions described herein are amenable to the same electrostatic treatment applicable to non-enzymatic reactions. By applying Brönsted's basic kinetic concepts and the Debye-Hückel law of electrolyte activity, it appears that the salt effects are mainly due to changes in the dissociation of ionizable groups. This appears to be a general method for analyzing the effect of inorganic ions on enzymic reactions.

A study was made on the effect of glycine on systems involving trypsin and BAEE1 or TSAME on the one hand, or α-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. In those catalyzed by α-chymotrypsin, the slopes were positive at pH 6.5 or lower, and negative at pH 7.5. However, the effects of glycine differ quantitatively from those of urea or other solvents. The presence of salt modifies somewhat the glycine effects. A low ionic strength increases the effect of glycine on trypsin, but if the inhibition caused by the ionic strength is relatively strong, the addition of glycine partially neutralizes the salt effect. Addition of salt to systems containing α-chymotrypsin always resulted in a diminished effect of glycine. An attempt is made to interpret the anomalies of glycine effects on the basis of its dipolar ion structure.