F.F. Nord

On the mechanism of enzyme action. XLVI. The effect of certain ions on crystalline trypsin and reinvestigation of its isoelectric point

Abstract Calcium and manganese ions are effective protectors of crystalline trypsin in alkaline solutions. Other ions seem to have no significant influence. The effect of calcium is due to a decrease in the rate of autodigestion of trypsin but does not affect the rate of tryptic digestion of other proteins. The isoelectric point of trypsin is in the neighborhood of pH 11, as determined by electrophoresis analyses. This high isoelectric point is correlated with the amino acid composition of trypsin and its amide nitrogen content.

On the mechanism of enzyme action. LVIII. Acetyltrypsin, a stable trypsin derivative

Abstract Acetylated trypsin is homogeneous in the ultracentrifuge and presents a slight inhomogeneity in electrophoretic migration. Its isoelectric point is about pH 3.8. Owing to blocking of the ϵ-amino groups of lysine, acetylated trypsin is stable in alkaline media and is not subject to self-digestion. Its stability in acid media is, however, smaller than that of the unmodified enzyme. Its thermal stability is also slightly lower than that of trypsin. Ca++ ions stabilize both enzymes. The pH optimum of digestion for casein is shifted to more alkaline pH values when the enzyme is acetylated. The temperature optimum is slightly lower. The Michaelis-Menten constant for trypsin and acetyltrypsin with casein is Km = 4.4 × 10−6M and Km = 3 × 10−5M, respectively. Acetyltrypsin combines also with ovomucoid and is inhibited by it, though to a much lesser degree than the unmodified enzyme. The dissociation constant of the equimolecular acetyltrypsin-ovomucoid is 1.3 × 10−6M using hemoglobin as substrate, and 3.5 × 11−7M with casein. The inhibition seems therefore to be competitive.

On the mechanism of enzyme action. LX. Acyl derivatives of trypsin

Abstract Four new acyl derivatives of crystalline trypsin were prepared by reacting the anhydrides of propionic, butyric, citraconic, and itaconic acids with the native enzyme. The properties of these derivatives were compared with those of the original and the acetylated enzyme. The specific proteolytic activity of all samples is comparable to that of the native enzyme, the greatest difference existing when hemoglobin is used as a substrate. All derivatives are stable in the alkaline pH region, contrary to the native enzyme, which is rapidly self-digested. The pH optimum is shifted toward more alkaline pH values, the shift being greatest for the derivatives of the dicarboxylic acids. Michaelis-Menten constants, when casein served as a substrate, and the detailed kinetic study of hydrolytic action upon benzoyl arginine ethyl ester have shown that there is probably no steric hindrance caused by any of the substituents in either formation or the decomposition of the enzyme-substrate complex.

On the mechanism of enzyme action. LXI. The self digestion of trypsin, calcium-trypsin and acetyltrypsin.

Abstract The self-digestion of trypsin proceeds by second-order kinetics. Acetylation stabilizes the enzyme by reducing the self-digestion to a first-order reaction. The addition of calcium ions to trypsin also stabilizes the enzyme, the order of reaction being intermediate, i.e., 1.5. The protective effects of acetylation and calcium ions are additive, and acetyltrypsin in the presence of calcium ions resists inactivation at 25 °C. and pH 8.5. The number of free amino groups liberated in the process of self-digestion was also determined. In the initial stages of self-digestion, trypsin liberates the lowest number of groups per mole of enzyme inactivated. This number increases with calcium-trypsin and even more so with acetyltrypsin. Acetyltrypsin in the presence of calcium ions, while maintaining its enzymatic activity, still undergoes partial digestion, as evidenced by the liberation of a substantial number of free amino groups. This finding suggests the presence of an enzymatically active degradation product of the native crystalline trypsin.

On the mechanism of enzyme action. LIII. The amphoteric properties of trypsin

Abstract Several methods were employed for the preparation of salt-free trypsin samples which were used to determine the electrometric titration curves of the enzyme. These curves point to a maximum acid-binding capacity below pH 2. Stoichiometric analysis indicates the presence of 6 carboxyl groups per 10,000 g. of proteins, 1 imidazole group, and 13 hydroxyl-binding groups. Calcium has a specific effect on the titration curves by increasing the acidity of the carboxyl groups in the pH range 3.5 – 5. This effect is not shown by potassium, sodium, or even the bivalent magnesium ion. It is attributed to the formation of a specific complex between the enzyme and the calcium ions, involving the carboxyl groups of the protein. The equally specific protective effect of calcium on the self-digestion of trypsin can therefore be explained by assuming the formation of a complex which stabilizes the enzyme. There is good qualitative agreement between the pH dependence of the electrophoretic mobility of the enzyme and the titration curves. In addition to stabilizing the enzyme, calcium also slightly increases its activity. The investigation of the dependence of the proteolytic activity of trypsin on the pH in the presence or absence of calcium at various temperatures indicates that both effects of calcium ions, the protective effect and the increase in activity, are due to a shift of the equilibrium between native and denatured enzyme in favor of the active form.

On the mechanism of enzyme action. LVII. Interaction between trypsin and ovomucoid

Abstract Trypsin, ovomucoid, and their equimolecular complex have sedimentation constants of 2.5, 2.4, and 3.7 – 3.9 S , respectively. While trypsin can bind only one ovomucoid molecule, ultracentrifugal and electrophoretic data show that more than one molecule of trypsin can react with each molecule of ovomucoid. The dissociation constant of the equimolecular complex is 5.8 × 10 −9 M and of the complex involving two trypsin and one ovomucoid molecule is about 2.5 × 10 −6 M , as dedetermined by activity measurements. The isoelectric points of the two proteins and their equimolecular complex are at about pHs 10.8, 4.3, and 9, respectively. The complex behaves on electrophoretic examination as an independent protein, stable over an extended pH range, and dissociating only around pH 3.8. The trimolecular complex has a mobility intermediate between that of the equimolecular complex and free trypsin. It has a more limited pH range of stability, and dissociates readily around pH 6. The presence of either of the complexes does not cause a change in the electrophoretic mobilities of the other components present. The inhibition of trypsin by ovomucoid is considered to be another instance of competitive substrate inhibition.

Investigations on proteins and polymers. X. Composition and fractionation of ovomucoid.

Abstract Ovomucoid was shown to be a heterogeneous protein consisting of three major and two minor components. The relative proportion of the five components was determined as well as their isoelectric points, the latter ranging from pH 4.41 to pH 3.83. One of the three major components could be isolated in a state of electrophoretic purity whereas the other two were substantially enriched. Electrophoresis-convection yielded a total of seven different fractions, the carbohydrate content, sedimentation behavior, and antitryptic activity of which were determined. These data have shown that at least the three major components are a striking example of electrophoretically discrete proteins originating from the same source and possessing the same biological properties.

Investigations on proteins and polymers. VII. The denaturation of egg albumin.

Summary The denaturation of egg albumin, in the pH range of 0.9–3.4, over the temperature range 25.0–44.4°C., has been found to follow first-order kinetics, over a wide range of initial protein concentrations and for a wide range of the total denaturation process. Two distinct ways of denaturation have been detected. The first involves the suppression of the dissociation of one hydrogen ion in the protein molecule, while the secon involves two such suppressions. In a moderately acid solution, only the first such dissociation is of significance, while the second dissociation enters into prominence in more acid regions. From the respective rate constants, the apparent heats of activation, ΔH*, were found to be 36.7 and 50.0 kcal./mole.

On the mechanism of enzyme action. LXIII. Specificity of acetylation of proteins with C14 anhydride.

Abstract The acetylation of trypsin and serum albumin in acetate, bicarbonate, and carbonate buffers was studied using radioactive acetic anhydride. The reaction is most specific for amino groups in the acetate buffer, though even under this condition some aliphatic hydroxyl, though no phenolic groups are esterified. In more alkaline buffers a more complete acetylation of the amino groups is achieved, but some phenolic hydroxyl groups are also esterified. As a valuable test for O -acyl groupings in proteins the reaction with hydroxylamine was applied. O -Acyl groups were found to be subject to spontaneous hydrolysis which is not influenced by simultaneous proteolytic digestion.

On the mechanism of enzyme action. XLVII. Effects of high intensity electron bombardment on crystalline trypsin

Summary The effect of ionizing radiations on enzymes in solution is much more complex than on enzymes in the dry state. The radiation dose required to cause a certain amount of inactivation of the enzyme will be as much as 170 times higher in the dry state, than in some dilute solutions. Any intermediate values can be obtained by changing the conditions of the irradiation of the enzymes in solution. The factors influencing the socalled “solvent” effect were found to be: concentration and nature of the buffer solution, pH, and temperature. The inactivating effect of electron bombardment on trypsin is considerably enhanced by increasing the acidity of the enzyme solution. This is in contrast with the maximum stability of this enzyme at pH 2.3 when not exposed to radiation. Calcium ions have no specific effect on the inactivation of trypsin caused by electrons, although, under normal conditions, these ions stabilize the enzyme. It can be concluded, therefore, that factors which contribute toward an increased stability of an enzyme under normal conditions have no correlation with the effects of ionizing radiation on the enzyme.