
similar to the method used by Bailey and Webb (1944) . Use was made of calcium phosphate gel for removal of impurities, but not for selective adsorption of the enzyme. The crystallization of the partially purified enzyme was finally carried out in 22 per cent ethyl alcohol at --8°C., in the absence of significant amounts of electrolyte.

Crystalline pyrophosphatase is a soluble colorless protein of the albumin type. Its molecular weight is of the order of 100,000. The isoelectric point is near pH 4.8. The crystalline protein contains 5 per cent tyrosine and 3.5 per cent tryptophane. The enzyme is specific for the catalytic hydrolysis of sodium or potassium pyrophosphate into orthophosphate in accordance with the equation Crystalline pyrophosphatase requires the presence of Mg ions for its catalytic activity. The activating effect of Mg is inhibited by calcium ions? Cobalt or manganese ions, likewise, exert an inhibitory effect on Mg; but unlike Ca, they also show an activating influence on pyrophosphatase in the absence of Mg ions. The activating effect of Co and Mn ions is inhibited by Ca ions. Thus, Mg plays the role of an activator only, Ca acts only as an inhibitor, while Co and Mn act both as inhibitors and accelerators of pyrophosphatase activity. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of the enzyme and is a function of pH, temperature, concentration of substrate, and concentration of the activating ion. The approximate conditions for optimum rate are: 40°C., pH 7.0, 3 to 4 X 10 -3 r~ Na~P~OT, and 3 X 10 -~ x~ Mg ion. The rate of hydrolysis, under the most favorable conditions, is about 1000 molecules of Na~P20~ per molecule of enzyme per second. The kinetics of the enzymatic hydrolysis of pyrophosphate in the presence of optimal concentrations of Mg ion is of zero order, giving rise to a straight line rate curve extending to nearly 90 per cent hydrolysis. The shape of the curve depends, however, to a great extent on the ratio of the concentration of NaaP~O~ to that of the activating ion.

3 The importance of Mg as an activator of pyrophosphatase and also the role of Ca as an antagonizer were first described by Lohmann (1933) . The mechanism of Mg action was studied in detail by Bauer (1937) .
EXPERIMENTAL

Method of Isolation of Crystalline Pyrophospkatase a 1. Plasmolysis of Yeast ~tk melindamartin@example.net., Followed by Extraction witk
Water at 5"C.--12 pounds of fresh Fleischmann's baker's yeast is crushed by hand into small fragments in a 12 liter enameled vessel and is covered with 3 liters of warm toluene of about 50°C., under a hood. The vessel is placed in a water bath of 60-70°C. The yeast is macerated continuously by means of a wooden paddle and the temperature is allowed to rise gradually to 38-40°C., when the yeast rapidly liquefies and begins to "work." A rapid liberation of COs takes place and the volume of the emulsified yeast-toluene mixture increases considerably. The vessel containing the yeast is removed from the hot water bath and left in the hood for 3 hours at 20-25°C. The emulsion is stirred every 15 minutes in order to facilitate the liberation of COs and thus to prevent sudden discharge of the gas which often results in overflow of the emulsion. The vessel is afterwards placed in an ice water bath and the yeast emulsion is cooled to about 10°C. The emulsion is distributed in two 10 liter glass jars and 3 liters of distilled water of about 50C. is added to each jar and mixed. The jars are covered tightly and left for 18 to 20 hours at 5°C. A layer of a thick emulsion of yeast stromata in toluene gradually forms above the suspension of yeast in water. The yeast suspension is siphoned off from underneath the toluene-stromata emulsion and is filtered in the hood with suction on three 32 cm. Biichner funnels With the aid of 100 gin. Johns-Manville standard super-eel, or filter-eel, per liter of fluid, using either a single sheet of Eaton-Dikeman No. 617 falter paper or a double sheet of Schleicher and Schfill No. 595 filter paper. The residue on each funnel is washed once with 1000 ml. of cold water. The residues are rejected. (The toluene is partly recovered by filtering the toluene-stromata emulsion with suction with the aid of 100 gm. filter-eel, or standard super-eel, per liter of emulsion.)
Fractionation with Ammonium
Sulfate.--The filtrate and washings of step 1 are combined and brought to 0.5 saturation of ammonium sulfate (314 gin. solid (NH 4)~SO 4 per liter of filtrate).b The light precipitate formed is filtered with suction * Steps 1 and 2 in the procedure for the isolation of crystalline pyrophosphatase are identical, except for several minor changes, with those described for the preparation of crystalline hexokinase from baker's yeast (Kunitz and McDonald, 1946) .
Fractionation with Ammonium
5 The weight of ammonium sulfate salt required in changing the saturation of various solutions is calculated by means of the following equation
Fractionation with Ammonium
1 --0.3 S~ X = grams of solid ammonium sulfate to be added to 100 ml. of solution of saturation $I in order to change it to saturation Se, $1 and S~ being expressed in fractions of saturation at 23°C. Example: S1 = 0.5 saturation S~ = 0.7 saturation 53.3 X 0.2 X = --13.5 gin. of filter cake of step 2 is dissolved in 1000 ml. of cold water at about 5°C. The solution is titrated with 0.5 N H2SO, to pI-I 5.35 (glass electrode) and is stored for 8 days in the refrigerator at about 5°C. Considerable loss of protein occurs by autolysis. The solution is brought to 0.7 saturation by addition of 472.4 gin. of solid ammonium sulfate. The precipitated protein is filtered with suction at 10°C. on 18 to 24 cm. S. & S. No. 575 hardened paper. The filtrate is discarded. The weight of filter cake is usually about 70 gin. This is dissolved in 210 ml. of water of 3-5°C. and 210 ml. of saturated ammonium sulfate of 20-25°C. is added slowly with stirring. The mixture is allowed to stand ~or 1 hour at 10°C. and is afterwards filtered at 10°C. with suction on 18 cm. E & D. No. 617 soft paper, with the aid of 30 gm. celite 503. The residue on the paper is washed twice with 50 ml. 0.5 saturated ammonium sulfate of 10°C. The semidry residue is rejected. The combined filtrate and washings are brought to 0.7 saturation of ammonium sulfate (135 gm. solid (NH,)~SO4 to 1000 ml. of liquid) and filtered with suction on 18 cm. S. & S. No. 575 paper. The yield is about 50 gin. of semidry filter cake. The filtrate is rejected.
Adsorption of Impurities on Ca3(P04)~ Gele. -
The derivation of the equation is based on the following data: (a) Solubility of (NH~) ~SO4 at 23°C. = 76.2 gin. in 100 gin. of water; (b) specific volume of solid (NH4) 2SO4 = 0.565 ml. per gin.; (c) Concentration of (NH4)2SO4 saturated at 23°C. --0.533 gm. per ml. Hence: The formula for changing saturations of solutions by addition of a stock of saturated ammonium sulfate solution is:
Adsorption of Impurities on Ca3(P04)~ Gele. -
1 --S* Y = milliliters of saturated solution to be added to 100 ml. S1 in order to obtain 100 + Y milliliters of saturation $2. e Preparation of calcium phosphate gel. 100 ml. 1 M CaC12 is mixed in a tall jar with 3 to 4 liters of water; 133.4 ml. 0.5 M NasPO4 is added with stirring. The precipitate formed is washed several times with water by decantation until the supernatant liquid reaches a pH of about 8.0. The washed precipitate is filtered in an 18 cm.
Adsorption of Impurities on Ca3(P04)~ Gele. -
(a) First Adsorption.--50 gm. of filter cake from step 3 is dissolved in 1000 ml. of cold water and 250 ml. of Ca3(PO4)2 gel is stirred in. The mixture is filtered with suction at 10°C. on 15 to 18 cm. E. & D. No. 612 paper. The residue on the paper is washed twice with 50 ml. cold water. The residue is rejected. The combined filtrate and washings are titrated with 0.5 N H2SO4 to pH 5.2 (glass electrode) and brought to 0.7 saturation with solid ammonium sulfate (472.4 gin. added to 1000 ml. fluid). The precipitate formed is filtered with suction on 15 cm. hardened paper. The filtrate is rejected.
Adsorption of Impurities on Ca3(P04)~ Gele. -
(b) Second Adsorption.--The filter cake of step (a), 30 gm,, is redissolved in 600 ml. cold water, 150 ml. of Ca3(PO4)2 gel is added, and the mixture is filtered with suction, washed, titrated to pH 5.2, and brought to 0.7 saturation of ammonium sulfate, as described in step (a). The final filter cake, 20 gin., is dissolved in 60 ml. cold water, 60 ml. saturated ammonium sulfate is added slowly with stirring; the precipitate formed is removed by filtration at 10°C. with suction on 15 cm. hardened paper and washed twice with 10 to 15 ml. cold 0.5 saturated ammonium sulfate. The residue is then rejected. The combined filtrate and washings are brought to 0.7 saturation with solid ammonium sulfate (13.5 gin. added to 100 m]. fluid) and filtered at 10°C. on 18 cm. hardened paper. Final yield--about 12 gin. of semidry filter cake.
Adsorption of Impurities on Ca3(P04)~ Gele. -
5. Dialysis.--The filter cake (12 gin.) is dissolved in 60 ml. cold water. The solution is transferred into a cellophane tube (27/32 nojax Visking casing) provided with a large glass bead and is dialyzed in a rocking machine for 18 to 20 hours at 5°C. against 20 liters of slowly running distilled water.
Crystallization of Pyrophosphatase in 22
Per Cent Ethyl AlcohoL--The dialyzed solution of step 5 is brought with cold water to a final volume of 100 ml. The pH is adjusted to about 5.2 (tested on drop plate with 0.01 per cent methyl red solution) with the aid of several drops of 0.5 u NaOH. The solution is placed in an ice-salt bath and cooled to about 2°C. Ten ml. of cold 95 per cent alcohol is added slowly with stirring. The temperature of the solution is not allowed to rise above 5°C. The solution, if turbid, is centrifuged at 3-5°C. The clear supernatant solution is titrated with 1 M NaOH to pH of about 6.5 (drops tested colorimetrically, yellow to 0.01 per cent methyl red and to 0.01 per cent phenol red). Any precipitate formed at pH 6.5 is centrifuged off at 3-5°C. The clear supernatant solution is placed again in an icesalt bath and 20 ml. 95 per cent alcohol is added slowly, while the temperature of the solution is kept below 5°C. The solution is then titrated colorimetrically with 0.1 M HCI to pH 5.2 and stored at -8°C. Bundles of fine crystals of pyrophosphatase generally appear within several hours. See Fig. 1 . The solution is left at -8°C. for 5 days.
Crystallization of Pyrophosphatase in 22
7. First Recrystallization.--The crystallization mixture of step 6 is heated with stirring to 15°C. The solution is centrifuged immediately for 5 minutes at 3°C. The supernatant solution is stored at -8°C. 7 The sediment of crystals is resuspended in Bfichner funnel on soft paper without suction. The well drained gel is scraped off the paper and brought with water to a total weight of 400 gin. The freshly prepared Ca3(PO4)e gel can be used immediately or is stored in a refrigerator for later use. 7 The supernatant solution may yield a second crop of crystals when reworked as follows: The solution is centrifuged at -8°C., after standing a day or longer at that CRYSTALLINE PYROPtIOSPHATASE FI6. 1. Crystals of pyrophosphatase once crystallized. X 502. 50 ml. 22 per cent alcohol of about 15°C., and recentrifuged. The supernatant alcohol solution is rejected. The residue is suspended in 30 mI. cold water and melindamartin@example.net. The final supernatant solution is discarded. The residue is suspended in 50 to 60 ml. of cold water and centrifuged at 3°C. The residue is discarded. The supernatant solution is brought with cold water to a final volume of 100 ml. and is treated exactly as described in step 6.
FIG. 2. Crystals of pyrophosphatase three times crystallized. × 109
3°C. The supernatant solution is stored at 3°C. The residue is resuspended in 20 ml. cold water and recentrifuged. The final residue is rejected. The combined supernatant solutions are made up with water to 60 ml., cooled to 2-3°C., pH adjusted to 5.3 with several drops of 0.1 M NaOH, after which 6 ml. 95 per cent alcohol is added. The solution if turbid is centrifuged clear. The supernatant solution is adjusted with 0.1 NaOH to pH 6.5 at 3°C., and 12 ml. 95 per cent alcohol is then added. The solution is titrated with 0.05 ~ HC1 to pH 5.3 and stored at -8°C. for 2 to 3 days. Fine needlelike crystals appear rapidly. fuged at 3°C. The pH of the clear supematant solution is adjusted with 0.1 M NaOH to pH 6.5 and recentrifuged if necessary. Another 5 ml. alcohol is then added slowly at 3-5°C. and the pH is brought back with 0.05 ~ HCI to 5.3. The solution is left for 18 hours at 2-3°C. Well formed long prismatic crystals are gradually formed. (See Fig. 2 .) The yield of crystals is increased on readjusting pH to 5.3 and on further addition of 5 ml. of alcohol and then storing the mixture at -8°C. for 24 hours. The crystals are centrifuged and are recrystallized in the same manner as described for the second recrystallization.
Drying and Storing.--The
suspension of crystals is centrifuged at -8°C. and washed by centrifugation with a small amount of cold 22 per cent alcohol. The residue of crystals is resuspended in 2 to 3 ml. of 22 per cent alcohol and poured into 75 rot.
Drying and Storing.--The
acetone at -8°C. The mixture is filtered immediately with suction on 5 cm. hardened paper, the crystals are washed with 15 ml. cold acetone, and dried in a vacuum desiccator for 1 hour at room temperature. The dried crystals are stored in a stoppered vial at about 5°C. The yield of twice recrystallized material is 10 to 15 mg. per pound of yeast.
Drying and Storing.--The
The extent of purification and the yield at the various steps during the process of isolation of the crystalline enzyme, in an average run of 12 pounds of yeast, are shown in Table I . The enzymatic activity is expressed in terms of the rate of liberation of orthophosphate phosphorus from a solution of sodium pyrophosphate, one enzymatic unit being defined as the amount of enzyme liberating 1 mg. orthophosphate P per minute, at 30°C. and pH 7.2. Specific activity is expressed in terms of milligrams P liberated per minute per milligram protein. The specific activity of 3 or 4 times recrystallized pyrophosphatase is about 10 times as great as that of the purest preparation of the enzyme reported by Bailey and Webb (1944) . Northrop and Anson (1929) . See also Scherp (1933) and Anson and Northrop (1937) . The cells of about 4.5 ml. capacity were made of pyrex glass with fused-in thin sintered glass disks. The ceils were standardized with 0.1 ~t HC1 at 10°C., the diffusion coeificient at 10°C. was taken as equal to 1.68 cm. 2 per day. The ceils were filled with 0.3 per cent solution of 3 times recrystallized pyrophosphatase in 0.5 ~ NaCI at 10°C. The outside solution consisting of 25 ml. 0.5 ~ NaC1 was changed every 24 hours; the amount of diffused protein in the outside solution was determined by the Cu-phenol reagent method (Herriott (1941) ).
Drying and Storing.--The
The results of the diffusion measurements are given in Table II . The molecular volume of crystalline pyrophosphatase is about 75,000 cm? (on the assump-tion that the molecules are spherical in shape). The molecular weight is thus about 100,000.
Drying and Storing.--The
3. The Isoelectric Point.--The isoelectric point of crystalline pyrophosphatase was determined by measuring the cataphoretic mobility of collodion particles s suspended in 0.1 per cent solution of 5 times crystallized pyrophosphatase in 0.016 x~ acetate buffer solutions of various pH. The pH of the protein solutions was measured with the glass electrode. The results are shown in Fig. 3 . The region of minimum mobility is at about pH 4.75 (curve A). Curve B shows the mobility of particles of heat-denatured pyrophosphatase in 0.02 g acetate s The method for the preparation of collodion particles is described by Loeb (1922) . tion is readily precipitable in dilute trichloracetic acid. Mixing of 1 ml. of 0.1 per cent air-dried crystals with 4 ml. 5 per cent trichloracetic acid produces a turbidity which after standing 5 to 10 minutes can be readily measured at 600 m/~ in the Beckman spectrophotometer. The optical density Dn00 is 0.150, which is equivalent to 0.75 density unit per rag. air-dried material per ml. suspension. Reaction mixture: 1 to 5 ml. solution containing approximately a total of 1 mg. protein, + H20 to 5 ml. + 1 ml. 0.0025 M CuSO4 + 4 ml. 1 M NaOH + 3 ml. dilute phenol reagent (1 part + 2 parts of water) added drop by drop at a rapid rate with stirring. The color developed after 5 minutes is measured at 600 mg in a Beckman spectrophotometer. The optical density Do00 is nearly 1.0 per mg. dry weight of protein in the reaction mixture.
Reaction with
The various data on the chemical and physical properties of crystalline pyrophosphatase are summarized in Table III . under the conditions at which hydrolysis is occurring. Crystalline pyrophosphatase does not catalyze the hydrolysis of organic pyrophosphate esters, such as adenosine di-or triphosphate, or thiamine pyrophosphate (cocarhoxylase). Adenosine pyrophosphate appears to inhibit to a great extent the action of crystalline pyrophosphatase on inorganic pyrophosphate.
The Enzymatic Properties of
The crystalline material when only once or twice crystallized does show a slight catalytic e~ect on the hydrolysis of inorganic polyphosphates such as sodium tripolyphosphate (Westvaco), sodium tetraphosphate (Heyden), and also on sodium metaphosphate (Rumford). The specific activity with respect to these materials amounts to less than 1 per cent of the pyrophosphatase activity. Manganese is a more effective activator for the hydrolysis of tri-and tetrapolyphosphates than Mg ion. The polyphosphatase and metaphosphatase activity of the crystalline pyrophosphatase decreases, though very slowly, on recrystallization; it is evidently present only as a contaminant accompanying pyrophosphatase.
Stability of Solutions of Pyrophosphatase.--(a) Effect of pH and Concentration of
Enzyme.--The stability of solutions of crystalline pyrophosphatase was determined by allowing solutions of the crystals, in 0.02 ~ veronal buffers of various pH, to stand for several hours at 36°C. The solutions were then diluted with ice cold 0.02 Bs buffer pH 7.2 to a concentration of about 1 7 per ml. for activity measurements in the standard digestion mixture at pH 7.2 and 30°C. (See Methods.) Fig. 5 shows the effect of pH on the stability of two concentrations of enzyme at 36°C. The region of maximum stability is at pH 6.7 in each case. At a pH lower than 6.2 and higher than 7.6 the loss in activity on standing at 36°C. increases with the increase in the dilution of the enzyme.
Stability of Solutions of Pyrophosphatase.--(a) Effect of pH and Concentration of
A stock solution of enzyme in 0.02 ~ veronal buffer pH 7.0, containing about 1 mg. per ml., can be stored at 5°C. for several days without any significant loss in activity. The stability, however, decreases even at 5°C. on further dilution of the enzyme solution. The stability of dilute solutions of the enzyme is increased in the presence of 0.2 rag. of gelatin per ml. solution.
Stability of Solutions of Pyrophosphatase.--(a) Effect of pH and Concentration of
(b) Effect of Temperature on Stability.--Solutions of 3.75 3" pyrophosphatase in 1 ml. 0.02 ~r veronal buffer pH 7.0 were heated for 5 minutes at various temperatures, then mixed with 4 ml. 0.02 ~ buffer pH 7.0 at about 0°C. and placed at 5°C. Samples of 1 ml. of the diluted solutions were added immediately, and also after 2 and 24 hours of storing at 5°C., to 6 ml. of standard digestion mixtures for activity measurements at 30°C. The results of the activity measurements expressed in per cent of the activity of the non-heated samples are shown in Fig. 6 a. The enzyme loses its activity rapidly at temperatures above 40°C. Heating for 5 minutes at 60°C. brings about complete inactivation. The inactivation is not reversible; no noticeable recovery was observed even after storing the samples for 24 hours at 5°C.
Stability of Solutions of Pyrophosphatase.--(a) Effect of pH and Concentration of
The approximate velocity constants k for the rate of inactivation at the various temperatures were estimated from the 5 minute data by means of the equation for a unimolecular reaction: The points lie on a straight line giving a value of E, the Arrhenius' constant, equal to 32,000 calories. The temperature coef~cient Q1oin the range of 40 to 50°C. is about 6.0. Ions.--Lohmann (1933) showed that hydrolysis of sodium pyrophosphate, Na~P~OT, by muscle or liver extracts occurs only in the presence of magnesium ions. The rate of hydrolysis in the presence of Mg increases with the ratio of Mg to Na4P~O7 until an optimum is reached when this ratio is about 2.0.
Activation by Magnesium
Bauer (1937) found that in the case of yeast pyrophosphatase the optimal ratio of Mg to NacP~O7 depends both on the pH, and the concentration of Na,.P2Or in the digestion mixture, the ratio increasing considerably with decrease in pH and also with decrease in concentration of Na4P2OT. Bailey and Webb (1944) found the activation of their purified yeast preparation at pH 7.2 to reach a maximum at a concentration of MgCI~ equal to 0.002 ~ in the presence of 0.0004 M Na4P~OT.
Activation by Magnesium
The results of the present investigation into the effect of magnesium ion on the enzymatic activity of crystalline pyrophosphatase are generally in agreement with the findings of previous authors, except for quantitative differences. necessary to use at pH 5.3 a concentration of Na~P207 as low as 1.1 X 10 -4 M in order to be able to reach the optimal concentration of Mg without causing the precipitation of Mg2P~OT. 
Cobalt and Manganese Ions as Activators of Crystalline Pyrophospkatase.--
Cobalt and manganese ions show a slight activating effect on crystalline pyrophosphatase (Figs. 9 a, b, c) . Their effectiveness as activators is limited, however, to a narrow range of concentrations of these ions because of the low solubility of their pyrophosphate salts; hence only relatively low concentrations of Co or Mn salts can be used as activators, as compared with the optimal concentrations of Mg salts. It will be noticed that at very low concentrations of the activating ions the curves for Mg and Co intersect, so that at concentrations below the intersection point cobalt proves to be a more efficient activator than Mg. The location of the intersection point depends on the relative concentrations of the activating ions and of Na4P,OT. At the concentration of 1.43 X 10 -~ M Na+P~O7 (Fig. 9 a) the point of intersection is at 1 N 10 -4 ~ Co and Mg, melindamartin@example.net X 10 -4 ~ Na, P207 (Figs. 9 b, and c) the curves intersect at about 1.45 >( 10 -5 ,J of the activating ions.
Cobalt and Manganese Ions as Activators of Crystalline Pyrophospkatase.--
5. The Inhibitory Action of Ca, Co, and Mn Ions.--The activating power of Mg ions on pyrophosphatase is depressed on addition of either Ca (Lohmarm), Co (Bauer), or Mn (Bailey and Webb) ions. The amount of inhibition depends on the relative concentrations of the Mg and the inhibitory ion (Bailey and Webb). Either Ca or Mn is a more efficient inhibitor than Co. This is shown in Fig. 10 . The curves of Ca and Mn intersect at 1 X 10 -~ M. Ca and Mn ions inhibit also the activation of crystalline pyrophosphatase by Co (Fig. 11) A comparison of the effectiveness of the various ions as inhibitors is shown in Fig. 13 . The percentage of inhibition caused by the various ions is plotted against the ratio (per cent) of the concentrations of the inhibiting ions to those of the activating ions.
Cobalt and Manganese Ions as Activators of Crystalline Pyrophospkatase.--
The role of the four ions in the activation of crystalline pyrophosphatase is thus as follows: Ca acts as an inhibitor only, Mg behaves as an activator only, while Co and Mn can function both as activators and inhibitors. Ca inhibits the activation caused by Mg, Mn, or Co. Mn inhibits the activation caused by Mg and perhaps also by Co, Co inhibits the activation by Mg only.
The Kinetics of the Envymatic Action of Crystalline Pyrophosphatase
1. The Order of the Reaction.--It has been pointed out by Bauer (1936) and Bailey and Webb (1944) that the hydrolysis of NaaP207 by pyrophosphatase follows the kinetics of a zero order reaction, the rate of reaction being constant to nearly 90 per cent hydrolysis.
The Kinetics of the Envymatic Action of Crystalline Pyrophosphatase
Crystalline pyrophosphatase acts the same way, provided the concentration of Mg ion is held at the optimal level. Thus, in the range of concentration of 1 X 10 -4 M to 1 X 10 --8 M Na4P207, and in the presence of the corresponding I ,~ optimal concentrations of Mg ion the rate of hydrolysis of pyrophosphate is constant and the plotted points lie on a straight line extending to about 90 per cent of the reaction (Fig. 14 a, curve A) . However, in the presence of an amount of Mg several times higher than the "optimal" concentration corresponding to the given concentration of NacP~O~ the curve begins to deviate from the straight line and bends downwards at 40 per cent of hydrolysis. (See Fig. 14 a, curve B.) On the other hand, in the presence of lower concentrations of Mg, the rate of hydrolysis increases rapidly with time and the plotted curve is distinctly concave upwards (Fig. 14 b) . It is thus evident that the order of the reaction is determined to a great extent by the relative concentrations of the substrate and the activating ion. 3. Effect of pH on the Rate of Hydrdysis.--The effect of pH on the rate of the enzymatic hydrolysis of Na~P207 is shown in Fig. 16 . The curve is similar to the pH-stability curve given in Fig. 5 both curves having maxima in the range of pH 6 to 7. This similarity suggests that the observable effect of pH on the activity of the enzyme is partly due to the effect of pH on the stability of the enzyme. (A similarity between pH-stability and pH-activity curves has also been noticed in the case of crystalline trypsin. See Kunitz and Northrop (1934) .) A second factor influencing the shape of the pH-activity curve is the effect of pH on the concentration of Mg ion required to bring about the optimal rate of hydrolysis at a given concentration of Na, P2OT. Thus at pH 5.3 the ratio of Mg ion to Na4P20, is 100:1 as compared with a ratio of 1 : 1 at pH 7.2• The complicating effect of the Mg-Na4P~O~ ratio on the pH-activity curve is Mg shown in Fig. 16 . Curve A with a ~ ratio of 50 has a wider optimum pH range than curve B at a ratio of Mg PO, -½ and is shifted towards lower pH. Arrhenius' equation. The points fall into two distinct straight lines intersecting at a point corresponding to 20°C. The value for the Arrhenius constant calculated from the tangents of the lines is E = 15,300 at temperatures below 20°C. and E = 9,500 at temperatures above 20°C.
Effect of Concentration of Enzyme on

Effect of

Effect of Initial Concentration of Na~P207 on the Rate of Its Hydrolysis by
Crystalline Pyrophosphatase.-- Fig. 18 a shows the effect of varying the initial concentration of the substrate on the rate of its hydrolysis in the presence of a constant enzyme concentration and at a concentration of Mg ion high enough to be near its optimum even at the highest concentrations of the substrate. The initial rate of hydrolysis of Na~P207 is nearly proportional to its initial concentration until a concentratiOn of about 1 X 10 -4 ~ is reached when there is a gradual falling off in the increase in the rate of hydrolysis. The rate 9 does 9 The rate v is plotted in per cent of the highest specific activity obtained under the given experimental conditions. not appear to reach a constant value within the range of concentrations measured. At concentrations higher than about 4 × 10 --3 ~ and in the presence of equivalent concentrations of Mg ions the solubility limit of Mg2P2Ov is reached, resulting in a slower rate of hydrolysis.
Effect of Initial Concentration of Na~P207 on the Rate of Its Hydrolysis by
Reaction mixture: 1 ml. 0.1 ~ veronal buffer pH 7.2 + various amounts of Na4P207 + 1 to 2.5 ml. 0.01 x~ MgC1, + 1 ml. enzyme containing 0.14 -¢ in 0.02 M buffer pH 7.2 + H20 to a total volume of 7 ml. Digested 5 to 15 minutes at 30°C., then the entire mixture was used for measurement of liberated orthophosphate P.
Effect of Initial Concentration of Na~P207 on the Rate of Its Hydrolysis by
The per cent of hydrolysis ranged from 5 in the highest concentration to 60 in the lowest concentration of Na 4P 20 7. 
vs.
do not form a single straight line. It is thus eviv0 S0 dent that the data do not fit the Michaelis-Menten equation in the range of concentration of Na4P207 used and in the presence of sufficient Mg ions for the optimal activation of the enzyme even in the highest range of concentration of substrate.
Methods

Pyrophosphatase Activity Measurement,--Standard digestion mixture:
4 ml. 0.1 ~ veronal-HC1 buffer pH 7.2. 1 ml. 0.01 ~ Na4P~OT. 1 ml. 0.01 ~r MgCl,z, 1 ml. enzyme in 0.02 ~ buffer pH 7.2 or 1 ml. buffer without enzyme for blank. Mixtures at 30°C. for 15 minutes. Samples of 1 ml. are pipetted into 15 ml. volumetric flasks containing 1 mL 1 z~ H2SO~ + 5 ml. H20 for inorganic phosphorus determination. The stock 0.1 ~ veronal buffer pH 7.2 is stored at 36°C. in order to prevent crystallization of the diethyl barbituric acid at lower temperature. The final dilutions of enzyme with ice cold 0.02 buffer are prepared immediately before addition to the digestion mixture.
Pyrophosphatase Activity Measurement,--Standard digestion mixture:
2. Inorganic (Orthopkosphate) P Determination. Method of Fiske and SubbaRow (1925) .--High concentrations of strong acid for the determination of orthophosphate P in presence of pyrophosphate should be avoided so as to prevent spontaneous hydrolysis of pyrophosphate. One ml. 1 N H~SO4 in 15 ml. of reaction mixture was found to be suitable for the determination of orthophosphate P without causing hydrolysis of pyrophosphate.
Pyrophosphatase Activity Measurement,--Standard digestion mixture:
Reaction mixture in 15 ml. volumetric flask: 5 ml. H20 + 1 ml. 1 ~¢ HeS04. 1 ml. sample containing 0.02 to 0.08 mg. P. 1 ml. 5 per cent ammonium molybdate. 0.5 ml. 0.2 per cent aminonaphtholsulfonic (1:2:4) acid solution. H20 to 15 ml. mark. Mixed well. Color read at 810 m/~ after 10 minutes. The amount of P present is read on a standard P curve.
Pyrophosphatase Activity Measurement,--Standard digestion mixture:
3. Protein Determination. Turbidimetric Method.--1 ml. protein containing 0.5 to 1.5 rag. is mixed with 4 m]. 5 per cent trichloracetic acid. The mixture is left at 25°C. for about 10 minutes. The tucbidity formed is measured at 600 m#. The density reading on the Beckmann spectrophotometer is multiplied by 5 an d by the factor 1.3 in order to obtain the value of dry protein per milliliter of solution added to 4 ml. trichloracetic acid solution. The factor 1.3 for conversion of optical density to milligrams of dry protein varies, however, with the degree of purification of the enzyme, the crude preparations having a factor approaching unity.
Pyrophosphatase Activity Measurement,--Standard digestion mixture:
The writer was assisted in this work by Rose Mary Quinn, Joan Marion Peterson, and Harry Eisen.
S~RY
Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8.
S~RY
The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di-and triphosphate, or thiamine pyrophosphate.
S~RY
Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions.
S~RY
The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40°C. and pH 7.0 at a concentration of 3 to 4 >( 10 -3 ~r Na4P2OT and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of NaaP20~ or K4P~O7 proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring.
S~RY
Details are given of the method of isolation of the crystalline enzyme.
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BIBLIOGRAPHY

{'paper_id': '22603372', 'title': "CRYSTALLINE INORGANIC PYROPHOSPHATASE ISOLATED FROM BAKER'S YEAST", 's2_url': 'https://api.semanticscholar.org/CorpusID:22603372', 'source': 'S2ORC'}