Reagents

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rupted by the addition of 2 ml. of dinitrosalicylic acid reagent. The tube ..... 1 G. L. Baker, G. H. Joseph, Z. I. Kertesz, H. H. Mottern, and A. G. Olsen, Chem.

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149

Amylases, a and

By

P E T E R BERNFELD

Assay Method A large number of valuable methods have been described for the assay of amylase. They are based on one or another of the following phenomena observed in the enzyme digests: (1) increase in reducing power of a solution of amylopectin or soluble starch; (2) change of the iodine-staining properties of the substrate; (3) decrease of the viscosity of a starch paste. All three phenomena are characteristic for the action of a-amylases; only the first one, however, can be used for the assay of E-amylase. The assay method described below 1is based on the increase in reducing power and is applicable for both a- and B-amylases. Although any method for the determination of reducing sugars may be used, the one described here has proved to be simple, reliable, and rapid. It was first employed by Sumner ~ for the assay of saccharase.

Reagents Dissolve 1 g. of potato amylopectin, or 1 g. of Schoch's B fraction from corn, 3 or, if neither is available, 1 g. of soluble starch, Merck, in 100 ml. of 0.02 M phosphate buffer, pH 6.9, containing 0.0067 M NaC1 (for assay of human salivary a-amylase). For assay of sweet potato/~-amylase, dissolve 1 g. of the substrate in 0.016 M acetate buffer, pH 4.8. Dissolve at room temperature 1 g. of 3,5-dinitrosalicylic acid in 20 ml. of 2 N NaOH and 50 ml. H~O, add 30 g. of Rochelle salt, and make up to 100 ml. with distilled H~O. Protect this solution from COs. Procedure. I ml. of properly diluted enzyme is incubated for 3 minutes at 20 ° with 1 ml. of the substrate solution. The enzyme reaction is interrupted by the addition of 2 ml. of dinitrosalicylic acid reagent. The tube containing this mixture is heated for 5 minutes in boiling water and then cooled in running tap water. After addition of 20 ml. of H20, the optical density of the solution containing the brown reduction product is determined photometrically, by means of a green filter,4 and a blank is prepared i G. Noelting and P. Bernfeld, Helv. Chim. Acta $I, 286 (1948). 2 j. B. Sumner and S. F. Howell, J. Biol. Chem. 108, 51 (1935); J. B. Sumner, J'. Biol. Chem. 62, 287 (1924-25). a T. J. Schoch, Advances in Carbohydrate Chem. 1, 247 (1945); see also Vol. I I I [2]. 4 Filter No. 540 in an Evelyn photoelectric colorimeter, or filter No. 54 in a KlettSummerson photoelectric eolorimeter.

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in the same manner without enzyme. A calibration curve established with maltose (0.2 to 2 mg. in 2 ml. of H20) is used to convert the colorimeter readings into milligrams of maltose. Amylase Activity. Amylase activity is expressed in terms of milligrams of maltose (Cl~H22011-H20) liberated in 3 minutes at 20 ° by 1 ml. of the enzyme solution, even though, in the case of a-amylatic action, the actual reaction products are dextrins rather than maltose. Specific activity is expressed as amylase activity per milligram of protein. Protein is determined by either micro-Kjeldahl or according to Lowry) When the Kjeldahl method is used with solutions containing ammonium sulfate, NH3 is previously eliminated by distillation in the presence of MgO.

Purification Procedure of Human Salivary ~-Amylase Human salivary amylase has been chosen as an example to describe the purification and the properties of an a-amylase, because the purification procedure of this enzyme is readily reproducible and the starting material is easily obtained. 6 General Remarks. All operations are carried out between 0 and + 4 °. Precipitants are added to the enzyme solution under slow but efficient mechanical stirring of the latter. The time required for the addition is indicated in the text in parentheses. When the addition of precipitant is completed, the stirring is always continued for 15 minutes, before the centrifugation is begun. The centrifugations are done at about 3000 r.p.m. for 15 minutes, unless otherwise stated. All acetone concentrations indicated below are expressed as per cent by volume; all ammonium sulfate concentrations as per cent saturation. Starting Material. Human saliva is collected in the presence of a few drops of 2-octanol to prevent foaming. On the average, one individual can produce 50 ml. of saliva in I hour by chewing large pieces of paraffin. The saliva is cleared by centrifugation for 1 hour. It can be stored for several days in the cold in the presence of a few drops of toluene. Step 1. First Acetone Fractionation. Acetone is slowly added (45 minutes) to 1500 ml. of centrifuged saliva to give a final concentration of 48 %. The precipitate is discarded after centrifugation, the solution is brought to 70 % acetone (45 minutes), and the precipitate, after centrifugation and discarding of the supernatant solution, is dissolved in 350 ml. of 0.07 M sodium acetate. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 6 K. H. Meyer, E. H. Fischer, A. Staub, and P. Bernfeld, Helv. Chim Acta 31, 2158 (1948).

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Step ~. Second Acetone Fractionation. The precipitate obtained by adding acetone to the above solution to a final concentration of 50 % (in 20 minutes) is discarded after centrifugation, and a second precipitate obtained at 70 % acetone (20 minutes) is collected and dissolved in 250 ml. of 0.07 M sodium acetate. Step 3. First Ammonium Sulfate Precipitation. 10 ml. of a solution of ammonium sulfate, saturated at 0 °, is added rapidly to the enzyme solution from the previous step. The clear solution is then brought to pH 8.0 by the addition of a few drops of 0.1 N NH~OH. Then a solution of ammonium sulfate, saturated at 0 ° and previously adjusted to pH 8.0 with NH4OH, is added rapidly to raise the concentration of ammonium sulfate to 45.5 % saturation. After 30 minutes of slow stirring, the precipitate is removed by centrifugation for 30 minutes and is dissolved in 80 ml. of 0.07 M sodium acetate. Step 4. Second Ammonium Sulfate Precipitation. The operation outlined in the last paragraph is repeated with the enzyme solution of step 3, starting with the addition of 3 ml. of saturated ammonium sulfate to the enzyme solution, followed by adjustment of the clear solution to pH 8.0 and precipitation of the enzyme at 40 % saturation of ammonium sulfate. The precipitate is dissolved in 40 ml. of 0.2 M sodium acetate. Step 5. Third Acetone Precipitation. The solution of step 4 is adjusted to pH 7.0 with a few drops of 0.1 N NH4OH. Then acetone is added (15 minutes) to a final concentration of 70%, and the precipitate is collected by centrifugation and dissolved in 15 ml. of H20. The purpose of this step is to remove from the enzyme solution the bulk of ammonium sulfate by discarding it with the supernatant fluid. Step 6. Ion Exchange. The remaining sulfate ions are replaced with acetate ions by pouring the enzyme solution through a column (6 inches long, 6/~ inch inside diameter) containing 20 ml. of anion exchange resin Amberlite IRA-400 which has been previously charged with acetate and washed with H20. The resin is maintained in place between two cotton plugs. The flow rate is adjusted to 3 ml./min. When the enzyme solution has disappeared below the upper cotton plug, 1 ml. of H~O is added. In order to recover the enzyme from the column, this operation is repeated with four 1-ml. portions of H~O and then with larger amounts, each time the liquid level reaches the upper cotton plug. Fractions of 5 ml. each are collected, and all those fractions having an optical density of 0.5 or more at 280 m~ are combined (usually fractions 2 through 7). Step 7. Fourth Acetone Precipitation. Ammonium acetate is now the only electrolyte remaining in the enzyme solution; it does not interfere with the protein precipitation of step 7. In order to concentrate the enzyme solution and simultaneously to remove the salt, acetone is added

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(within 10 minutes) to make a final concentration of 70 %. After centrifugation, the supernatant fluid is decanted and discarded, and the centrifuge tube containing the precipitate is inverted for 5 minutes, in order to drain the adhering solution. After the inside walls of the tube have been wiped off with a filter paper, the precipitate is triturated with 2 ml. of 0.1 N NaOH, added gradually, until a clear solution is obtained (final pH 8.0 to 8.4. Crystallization. The above solution is kept in the icebox. About 60 % of the enzyme deposits in crystalline form overnight, 80% in 2 days; 1500 ml. of saliva yields approximately 150 rag. of crystalline amylase. Reerystallization. The centrifuged crystals are washed three times by rapidly triturating them with cold 30 % acetone, immediately followed by centrifugation. The crystals are suspended in H20 (1 ml. for 70 rag. of protein), and 0.04 N NaOH is added to raise the pH to 11.0. The crystals dissolve slowly; gentle continuous shaking hastens the dissolution. The pH decreases as the enzyme dissolves, and it should be maintained at 11.0 by occasional addition of small amounts of 0.04 N Na0H. After all the protein is dissolved, the solution is neutralized with 0.1 N acetic acid and immediately centrifuged to remove small amounts of insoluble material. The enzyme crystallizes upon standing in the ice chest; it may be necessary to add seeding crystals. Properties of Human Salivary a-Amylase

Action. The enzyme hydrolyzes ~-l,4-glucosidic bonds in polyglucosans (amylose, amylopeetin, glycogen, and dextrins). The location in the molecule of the bond to be hydrolyzed is selected at random, but the terminal bonds are split much more slowly. The initial stage of action of this enzyme is characterized by a rapid decrease of the molecular weight of the substrate, resulting in a rapid change of its iodine-staining properties (dextrinizing activity) and, when starch paste is the substrate, in an extremely rapid loss of the viscosity; these phenomena are produced by the cleavage of a very small number of glucosidic bonds; i.e., they are accompanied by only a slight increase in the reducing power (saccharifying activity). The final products of action are maltose, limit dextrins of low degree of polymerization (3 to 7), and small amounts of glucose. The a-l,6-glucosidic bonds (branching points) are not hydrolyzed by this enzyme. These all appear in the limit dextrins. Purity of Crystalline Salivary Amylase. After several recrystallizations of the enzyme, the specific amylase activity remaining in the mother liquor becomes equal to the specific amylase activity of the crystals. The crystalline enzyme migrates as a single protein on electrophoresis. It does not contain any maltase, nor could the presence of any other starch-

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TABLE I SUMMARY OF PURIFICATION PROCEDURE OF SALIVARY a-AMYId~E

Step of purification

1. 2. 3. 4. 5. 6. 7.

Centrifuged saliva First acetone Second acetone First (NH0sS04 Second (NH~)sS04 Third acetone Ion exchange Fourth acetone Crystallization Recrystallization

Amylase activity Protein,* per ml. mg./ml, 410 1,620 1,920 3,540 5,720 12,800 6,980 63,000

3.3 4.2 2.9 4.1 6.1 13.4 7.3 66.0

Over-all Specific enzyme amylase activity recovery, % 125 385 663 865 940 955 955 955

90 77 45 36 30 30 30 24

--

--

980

--

--

1000

19

o From Kjeldahl N determination, assuming in all the fractions the same nitrogen content as found in pure amylase (15.8%). digesting enzymes, such as B-amylase or a-l,6-glucosidase, be detected. Crystalline h u m a n salivary ~-amylase, therefore, a p p e a r s to be a pure protein. Chemical Composition. T h e crystalline e n z y m e contains 15.8 % N, less t h a n 0.01% P, and only traces of S. There is no indication for the presence of a prosthetic group in this enzyme. The solubility at 2* is 0.2 to 0.5 g. of crystalline protein in 100 ml. of H ~ 0 buffered to p H 8.5. The light absorption spectrum exhibits a m a x i m u m of the optical density a t 280 mu, and a slight irregularity at 292 m~. T h e r e is practically no light absorption above 310 mu. The electrophoretic mobility of the enzyme in glycine buffer, p H 10.14, of ionic strength u = 0.1, is u = 3.75 × 10 -s em. ~ volt -1 sec. -1. T h e isoelectric point is near 5.3. Effect of pH on the Stability. An aqueous solution of the crystalline e n z y m e does not lose a n y activity during storage for 20 hours at 20 ° when the p H is between 4.5 and 11.0. Effect of pH on the Enzyme Activity. T h e enzyme is active between p H 3.8 and 9.4 with a distinct o p t i m u m at p H 6.9. Effect of Temperature on the Enzyme Activity. T h e a c t i v i t y of the crystallized enzyme increases with the t e m p e r a t u r e to a b o u t 40 ° and decreases at higher temperatures. T h e t e m p e r a t u r e coefficient is 2.3 between 10 and 20 ° , and 2.0 between 20 and 30 ° . The energy of activation, calculated f r o m these data, is 13,350 cal./mole.

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Activators. The activity of pure human salivary a-amylase decreases to about 15% of its original value when the enzyme has previously been dialyzed for 2 days against dilute NH40H (pH 9 to 10) and when the substrate has been dialyzed against 0.01 M phosphate buffer. The full enzyme activity is restored upon addition of C1- to a final concentration of 0.01 M, and about 80% of the original activity is recovered in the presence of 0.001 M C1-. Other anions, having a similar but weaker effect, are, in the order of decreasing activation power: Br-, NO3-, and I-. No activation has been detected with S04--, C03H-, PO4H--, CNS-, or CH3C02-. This activation is entirely independent of the type of cation present. Comparison with Other Amylases. ~ Crystalline human salivary a-amylase is identical with crystalline human pancreatic a-amylase. 8 Human salivary amylase hydrolyzes the substrate in the same way as does crystalline pig pancreatic a-amylase. 9 These two latter enzymes also have an identical optimum pH of action, and both require C1- for their full activity. They differ markedly from each other, however, in their specific amylase activity, in their electrophoretic mobility, in their solubility, and in their pH tolerance. Crystalline human salivary a-amylase exhibits the same substrate specificity as the crystalline a-amylases from malt, 1° from B. subtilis, 11 and from A. oryzae.12 The end products obtained by extensive action of these three latter enzymes on the substrate, however, are of somewhat higher molecular weight than those obtained with salivary amylase. Furthermore, their optimum pH of action is more acid, and most of their physical and chemical properties are different from those of salivary amylase. The ratio of saccharifying to dextrinizing activity is the same for all these a-amylases. This ratio is about one-sixth of that observed with B-amylases.

Purification Procedure of Sweet Potato ~-Amylase This enzyme was the first amylase obtained in crystalline form. The original purification procedure of Balls et al. ~a is given here with some minor modifications. General Remarks. When not otherwise stated, all operations are carried out at room temperature. Overnight storage of enzyme material, however, is done at 4 ° . 7 p. Bernfeld, Advances in Enzymol. 12, 379 (1951). 8 p. Bernfeld, F. I)uekert, and E. H. Fischer, Helv. Chim. Acta 33, 1064 (1950). 9 E. H. Fischer and P. Bernfeld,Helv. Chim. Acta 31, 1831 (1948); K. H. Meyer, E. H. Fischer, and P. Bernfeld, Arch Biochem. 14, 149 (1947); Experientia 3, 106 (1947). 10S. Schwimmerand A. K. Balls, J. Biol. Chem. 179, 1063 (1949). 11K. H. Meyer, M. Fuld, and P. Bernfeld, Experientia 3, 411 (1947). 13E. H. Fischer and R. de Montmollin, Helv. Chim. Acta 34, 1987 (1951). 13A. K. Balls, M. K. Walden, and R. R. Thompson, J. Biol. Chem. 173, 9 (1948).

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Step 1. Extraction of Sweet Potatoes. 10 lb. of sweet potatoes is washed, ground to a coarse mixture in a meat grinder, and then homogenized in small portions for 5 minutes in a Waring blendor with the addition of 2500 ml. of H~O in the process. The solid is separated by centrifugation and discarded. Step ~. Heating at 60 °. About 1900 ml. of the turbid extract and a few drops of 2-octanol are placed in a 3000-ml. Erlenmeyer flask which is immersed in water of 85° . The flask is continuously shaken by hand until the temperature of the enzyme solution reaches 60° (8 to 10 minutes). The flask is immersed in water of 60°, kept there for 7 minutes without shaking, and then placed in cold running water, so that the temperature of the enzyme solution drops to 40 ° in about 5 minutes, and to 28° in 5 more minutes. The heated juice is stored under toluene for 4 days at 4 ° and then centrifuged. The precipitate is discarded. Step 3. Precipitation with Basic Lead Acetate. The turbid enzyme solution is thoroughly mixed with a quantity of a lead subacetate suspension (200 g. Pb(CH3CO~)~.Pb(OH)~, Merck, per liter) that causes a precipitation of 25 to 33 % of the amylase present. This quantity, which has to be determined in preliminary experiments, amounts to about 30 ml. of lead subacetate suspension per 1000 ml. of amylase solution. The precipitate formed is separated by centrifugation at 3000 r.p.m, and discarded. Step ~. First Ammonium Sulfate Precipitation. Crystalline ammonium sulfate is added to the turbid enzyme solution to 70% saturation, the total volume of the mixture is measured, and the precipitate is collected by spinning the mixture for 60 minutes at 20,000 r.p.m, in a Spinco Model L ultracentrifuge (45,000 X g.). The clear supernatant liquid is decanted and discarded after its volume is measured. The precipitate is diluted with 380 ml. of water to make it 25 % saturated with ammonium sulfate. The amount of water to be added is calculated from the volume of the precipitate (210 ml.) estimated by difference, and from the assumption that the precipitate is 70 % saturated with ammonium sulfate as if it were a solution. The mixture, which has a pH of 5 to 5.2, is centrifuged at 3000 r.p.m., the precipitate is further diluted with 500 ml. of water, which dissolves it partially, then made 25 % saturated with ammonium sulfate, and centrifuged again. The precipitate is discarded, and the turbid supernatant fluids are combined and clarified by high-speed centrifugation at 20,000 r.p.m. Step 5. Second Ammonium Sulfate Precipitation. Crystalline ammonium sulfate is added to 70% saturation. After centrifugation at 3000 r.p.m., the supernatant fluid is discarded and the precipitate diluted with water to 38 ml. Step 6. Dialysis. The turbid amylase solution is dialyzed for 2 days against four 2-1. portions of distilled water, During this time, the enzyme

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dissolves entirely, and the volume of the e n z y m e solution increases to a b o u t 100 ml. T h e a m y l a s e solution is then clarified b y 10 minutes' centrifugation at 18,000 r.p.m. Crystallization. T h e e n z y m e solution is diluted to 150 ml. and m a d e 2 0 % s a t u r a t e d with a m m o n i u m sulfate, the p H being 5 to 5.2: After cooling to 0 °, the p H is lowered to 3.2 b y the addition of N hydrochloric acid, and the precipitate is r e m o v e d b y centrifugation at 18,000 r.p.m, for 10 minutes at 0 °. S a t u r a t e d a m m o n i u m sulfate solution is then added to the s u p e r n a t a n t fluid to 25 % saturation, which raises the p H to 3.7, and seed crystals are added. T h e e n z y m e solution is placed in a dialysis b a g (Visking seamless regenerated cellulose tubing) and dialyzed at 0 ° against t w e n t y times its volume of 3 0 % s a t u r a t e d a m m o n i u m sulfate, p H 3.8. After several hours, the concentration of a m m o n i u m sulfate is gradually raised to 4 5 % s a t u r a t i o n over a period of 12 hours, b y adding s a t u r a t e d a m m o n i u m sulfate solution of p H 3.8 to the solution outside the dialysis tubing. Microscopic examination of the mixture reveals the presence of crystals along with the a m o r p h o u s m a t t e r . TABLE II SUMMARY OF PURIFICATION PROCEDURE OF SWEET POTATO f3-AMYLASE

Step of purification 1. 2. 3. 4. 5. 6.

Crude extract Heating at 60° Lead subacetate First (NH4)2SO~ Second (NH4)2SO4 Dialysis Crystallization Recrystallization

Specific Over-all Amylase activity Protein? Volume, amylase enzyme per ml. ~ mg./ml, ml. activity recovery, % 146 165 114 222 5750 685 ---

10.2 8.6 2.3 2.15 40.8 3.4 ---

3850 3850 3080 1160 38 130 ---

14.3 19.2 49.5 104 140 201 480 560

-113 63 46 39 16 13 11

One amylase activity unit corresponds to 0.25 of Balls '13 units. b From measurements according to Lowry, ~ using crystallized salt-free chymotrypsin (Worthington Biochemical Lab.) as the reference protein.

Recrystalli.zation. This procedure is a d e q u a t e l y described b y E n g l a r d and Singer. 1~ Six to eight recrystallizations are necessary to obtain a p r o d u c t which is free from a m o r p h o u s material. Propeffdes of S w e e t Potato ~-Amylase

Action. f~-Amylase hydrolyzes ~-l,4-glucosidic bonds in polyglucosans (amylose, amylopectin, glycogen, and dextrins). I n contrast to the action 14S. Englard and T. P. Singer, J. Biol. Chem. 187, 213 (1950).

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of a-amylase, only the penultimate bond from the nonreducing end group of the substrate molecule is selected for cleavage by E-amylase. Thus, one molecule of maltose after the other (as E-maltose after Walden inversion) is detached from the substrate until the enzyme encounters an obstacle, i.e., a branching point. The enzyme action is stopped at this point. Accordingly, amylose is completely converted into maltose when the reaction is carried out properly./6 Amylopectin yields about 60% of maltose and 40 % of a high molecular weight limit dextrin, giving a purple iodine color. All following properties, unless otherwise stated, have been reported by Balls et al. 13 Purity of Crystalline Sweet Potato E-Amylase. Enzyme recrystallized eight times behaves like a homogeneous protein in a phase-solubility test and on electrophoresis; by ultracentrifugation, an impurity was detected, constituting about 3 % of the total protein. 14 After several recrystallizations, the enzyme is free from a-amylase and maltose and contains no more traces of an acid phosphatase. Chemical Composition. The crystallized sweet potato E-amylase contains 15.1% N (Kjeldahl), 0.83% amino N, 1.16% amide N, 6.0% argio nine, 7.0 % tyrosine, 0.79 % cystine ~- cysteine, 4.32 % methionine, and 0.66 % ash. There is no indication that the enzyme contains a prosthetic group or is a heavy metal complex. The diffusion constant has been measured to be D20 -- 5.77 X 10-7 cm2 s e c . - t . 14

The molecular weight calculated from sedimentation and diffusion data is 152,000 _ 15,000.14 The isolectric point, as determined by electrophoresis, is pH 4.74 to 4.79.14 The turnover number has been calculated to be 250,000 glucosidic linkages hydrolyzed by 1 molecule of enzyme per minute at 30 ° and pH 4.80.14,16

Effect of pH on the Enzyme Activity. In acetate buffer, the enzyme is most active between pH 4 and 5; in citrate buffer the optimum is between pH 5 and 6. Activators and Inhibitors. Unlike the animal a-amylases, sweet potato /~-amylase does not require C1- or any other anions for its activity. In contrast to malt a-amylase, the E-amylases are not activated by Ca ++. Reagents recognized as selective for - - S H groups are powerful inhibitors of sweet potato/~-amylase, le i.e., 5 X 10-7 M p-chloromercuribenzoate, 10-b M AgNO3, 10-6 M HgC12, 10-3 M o-iodosobenzoate, and 10-8 M CuS04. The inhibition by 1.18 X 10-6 M p-chloromcrcuribenzoate can 15 p. Bernfeld and P. Gfirtler, Helv. Chim. Acta 31, 106 (1948). le S. Englard, S. Sorof, and T. P. Singer, J. Biol. Chem. 189, 217 (1951).

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be partially reversed b y 2 × 10 -5 to 2 X 104 M - - S H glutathione or b y 1.18 X 10 -4 M 1,2-dithiopropanol. 18 Comparison with Malt B-Amylase. T h e crystalline B-amylases f r o m sweet p o t a t o and f r o m m a l t 17 exhibit the same ratio of saccharifying to dextrinizing activity, indicating an i d e n t i t y in the e n z y m e specificity between these two amylases. T h e y differ f r o m each other, however, in m a n y of their physical and chemical properties. ITK. H. Meyer, E. H. Fischer, and A. Piguet, Helv. Chim. Acta 34, 316 (1951).

[18] Pectic Enzymes By

Z. I. KERTESZ

Introduction and N o m e n c l a t u r e I n this discussion the n o m e n c l a t u r e a d o p t e d b y the American Chemical Society 1 is used. T h u s " p e c t i n s " or "pectinic a c i d s " designate those water-soluble polygalacturonic acids of v a r y i n g m e t h y l ester contents and degree of neutralization which show colloidal properties and are capable of forming, under certain conditions, gels with sugar and acid. 2 " P e c t i c a c i d s " are polygalacturonic acids of colloidal nature b u t essentially free of m e t h y l ester groups. " P r o t o p e c t i n " is the water-insoluble p a r e n t pectic substance which occurs in plants and which, upon limited acid hydrolysis, yields pectins of various sorts. T h e e n z y m e " p r o t o p e c t i n a s e " supposedly hydrolyzes protopectin into pectin and perhaps cellulose. There is v e r y little exact knowledge concerning protopectin, and even the existence of a separate chemical e n t i t y of this sort is doubtful. F o r this reason protopectinase will not be discussed here except to state t h a t it supposedly occurs in some higher plants and in m a n y microorganisms. P l a n t tissue slices rich in water-insoluble pectic constituents are used as a s u b s t r a t e of protopectinase. 3 1 G. L. Baker, G. H. Joseph, Z. I. Kertesz, H. H. Mottern, and A. G. Olsen, Chem. Eng. News 22t 105 (1944). Z. I. Kertesz, "The Pectic Substances," Interscience Publishers, New York, 1951. For other recent reviews of the field of pectic enzymes, see H. J. Phaff and M. A. Joslyn, WaUers~einLabs. Cammuns. 10t 133 (1947); Z. I. Kertesz and R. J. McColloch, Advances in Carbohydrate Chem. 5, 79 (1949); H. Lineweaver and E. F. Jansen, Advances in Enzymol. 11, 267 (1951); Z. I. Kertesz in "The Enzymes" (Sumner, and Myrbiick, eds.), Vol. I, Part 2, p. 745, Academic Press, New York, 1951; H. Deuel, J. Solms, and H. Altermatt, Vierteljahrsschr. nat~rfarsch. Ges. Zi~rich 98, 49 (1953). 3 F. R. Davison and J. J. Willaman, Bolan. Gaz. 83, 329 (1927).