Arginase Studies.—i. The Relation Between the Activity of the Enzyme and the Concentration of Hydrogen Ions

Abstract

June 1924 we reported that "the activity of arginase commences at pH 4X8, rises to a maximum at or near pH 9-8, and then falls so abruptly that at pH 11 X5 it is practically zero" (1). Incidental mention of the rather unusual position of the optimum was made in a paper published shortly afterwards by HUNTER and DAUPHINEE (2). Later, in 1927, we reported (3) to the American Society of Biological Chemists that the pH-activity curve of arginase, besides being as a whole strikingly unsymmetrical, took in its longer ascending limb the unusual form of two successive sigmoid curves of unequal size. The experimental results upon which these several statements were based have been repeatedly confirmed in each of two laboratories, but they have not hitherto been published. It is the purpose of this paper to make good the omission.The relation of arginase activity to pH has been studied, it should be mentioned, not only by ourselves, but also by EDLBACHER and BONEM (4), HINO (5), and EDLBACHER and SIMONS (6). The work of EDLBACHER and BONEM, published in 1925 and therefore almost contemporaneous with our own, placed the optimum where we had found it-at pH 9 5 to 9-8. The later experiments of EDLBACHER and SIMONS (1927) indicated the distinctly lower (and, we believe, less nearly correct) value of 9-0. HINO (1926) reported an optimum at 7-3 to 7-5. Oddly enough, a single experiment of our own (7), the first attempted, had given in 1922 a result not very different from that of HINO; but in no subsequent trial have we ever observed a maximal effect, for the early stages of the reaction, at any pH lower than 9-6.As a matter of fact, the true optimum is in all probability rather above 9*8 than below it. 1 Based upon a thesis presented by J. A. MORRELL in partial fulfilment of the requirements for the degree of Ph.D. at the University of Toronto.Hunter and Morrell MATERIALS AND GENERAL METHODS. (1) Arginase.-As a source of arginase we used a dry fat-free liver powder prepared from beef liver by the method of WIECRowSKI (8). As occasion arose, 10, or sometimes 15, grm. of such a powder were extracted for 24 hours with 100 c.c. of 50 per cent. glycerol. The extract was filtered through a bed of paper pulp, and the perfectly clear, light brown, slightly acid filtrate was titrated with N NaOH until its pH, electrometrically determined, was 7 0. Arginase solutions thus prepared and neutralised retained their activity unaltered for many weeks. In some experiments they were used directly, in others after dilution to an appropriate degree with water. They contained, of course, a considerable quantity of protein as well as other inactive material; but for our present purpose a high degree of purity in the enzyme was not considered to be essential. The several crude enzyme solutions from time to time employed varied considerably in activity; it is therefore seldom possible to compare one experiment with another upon the simple basis of the amount of liver extract in use.(2) Urease.-Active solutions of urease were obtained by extract...

Determination of the arginase activities of homogenates of liver and mammary gland: effects of pH and substrate concentration and especially of activation by divalent metal ions

“…Similar optima for liver arginase were reported by Felix & Schneider (1938) and for both liver and jack-bean enzyme by Damodaran & Narayanan (1940). Forliver arginase, more alkaline optima were given by Edlbacher & Bonem (1925) (pH 9-5-9-8), Hunter & Morrell (1933b) (pH 9-8) and Mohamed & Greenberg (1945) (pH 10-0); and more acid optima by Hino (1926) (pH 7-3--7-5), Edlbaeher & Simons (1927) (pH 9-0) and Hellerman (pH, 8-0). The latter value may be compared with the value pH 9-0 given by Stock?…”

“…Alternatively, the tissue can be homogenized with saline containing the correct concentration of Mn++, and any subsequent necessary dilution carried out with saline containing 2 mg. Mn++/ml. In the presence of Mn++ (or Co++) under these conditions, a fine flocculent precipitate is formed in the homogenate, removal of which would almost certainly result in loss of enzyme activity (see, e.g., Hunter & Downs, 1944). This precipitate can be uniformly dispersed by vigorous shaking just before pipetting.…”

The Temperature Coefficient and the Apparent Energy of Activation of the Enzymatic Hydrolysis of Arginine; With Additional Observations Upon the Stability of Arginase Under Various Conditions

“…At pH 9-6 velocity coefficients, if calculated by the formula which fits the data at 7-1, fall so rapidly, even at 20°, that the accurate estimation of initial values is quite impossible. This fall is in accord with previous experience, and expresses doubtless the destructive effect of excessive alkalinity (1). To make possible under these circumstances an estimate of the relative initial velocities at 20°and 30°it is necessary to use in the calculations such a value of m as will make k at 200 reasonably constant.…”

“…3. The velocity and temperature coefficients of the (1,2), that even at room temperatures the enzyme undergoes at pH 9-8 a spontaneous inactivation; but it may be taken to show that the inactivation caused by excess of hydroxyl ions is not, within the range of temperature in question, appreciably accelerated by heat. The values of Q for 00 to 200 may therefore be accepted as expressing the full accelerating influence of rise of temperature upon reaction velocity.…”

Arginase Studies.—ii. The Influence of Hydrogenion Concentration Upon the Stability of the Enzyme

“…FROM indirect considerations HUNTER and MORRELL (1) were led to conclude that in alkaline media arginase undergoes a more or less rapid irreversible destruction. In the present paper we report experiments designed to test directly the influence of both acid and alkaline environments upon the stability of the enzyme.…”