Determination of Peroxidase Activity DEANA. PACK,^ The Birdseye Laboratories, Gloucester, Mass.
T
H E preservation of fruits and vegetables by freezing and subfreezing temperature storage often requires the inhibition or control of enzyme action. The degree of success with which these measures are applied determines to some extent how well the color, flavor, odor, and other characteristics of the fresh product are retained during storage. A study of these problems has required the adaptation or development of exact methods of determining enzymes applicable to the particular products. Because of the rather general distribution of peroxidase and the need for control of its activity, a quantitative method of investigation was required. As the Guthrie (1) method seemed simple and required no expensive apparatus, it was studied. A preliminary study showed that this method did not give the maximum peroxidase activity of many productsfor example, strawberries gave little or no peroxidase activity, This difficulty was corrected in part hy thoroughly extracting the enzyme from all the tissue sample and making the determination a t the optimum hydrogen-ion concentration for strawberry peroxidase. The method as now worked out has the advantage of estimating, on a dry-weight basis, the total peroxidase activity of the entire sample in the presence or absence of catalase and a t the optimum hydrogen-ion concentration for the peroxidase of the particular product examined.
EXPERIMENTAL WORK The Guthrie method was changed in the following respects: (1) An extract of the entire tissue sample replaced the pressed juice sample; (2) McIlvaine's dibasic sodium phosphate and citric acid buffer was used instead of sodium hydroxide and citric acid buffer; (3) the reaction of the medium was adjusted to the peroxidase optimum for the particular tissue examined; (4) the amount of all reagents and extracts was reduced 50 per cent, except for toluene which was added as required for complete solution of the indophenol; (5) an enzyme sample in which the peroxidase had been inactivated was added with each reagent blank; and (6) the results were expressed as milligrams of indophenol per decigram of dry substance in the enzyme sample. In order t o obtain maximum peroxidase activity, the samples were ground thoroughly with water or buffer solution and fine sand in a mortar, made up to volume with water or buffer solution, and aliquot samples used for representative determinations. This method gave a more complete extraction of the enzyme than is possible by using only the pressed juice. The point was illustrated by the analyses of two samples of similar tissue and equal weight taken from an individual pear. The peroxidase in samples A was extracted by grinding, while that in samples B was extracted by freezing, thawing, and 5500 pounds pressure per square inch (387 kg. per sq. cm.). The amount of peroxidase in each sample was represented by the milligrams of indophenol produced per decigram of dry weight of samples A and B.
The results are given in Table I for determinations made a t both 6.0 and 6.4 pH. ACTIVITYOF PEARTISSUE TABLEI. PEROXIDASE SAMPLE
REACTION OF MIXTURIO, PH
INDOPHENOL PRODIJCED~ Mg.ldg.
A B
6.0 2.76 6.0 0.465 6.4 3.52 6.4 0.604 About 83 per cent of the peroxidase was not in the juice
i:
a
While it was noted that the a-naphthol-p-phenylenediamine mixture was more sensitive near the neutral point, this sensitivity varied with the kind of buffer used and finally with the
* Present address, General Foods Corp., Battle Creek, Mich.
compounds used to make up each buffer. Seven different kinds of buffer mixtures were examined. McIlvaine (2) and Sorensen (3) buffers were found to be less sensitive to the substrate than the other buffers a t p H values near the optimum for peroxidase. The magnitude of the complete blanks varied from 20 to 50 per cent of the total indophenol formed, depending upon the kind of enzyme extraction as well as the proximity of the solution to neutrality. The use of the McIlvaine or the Sorensen citrate-sodium hydroxide buffer mixture made it possible to determine the peroxidase activity a t a higher p H value. This modification adds to the usefulness of the method, since it is no longer limited to a reaction of p H 4.5. Table I1 gives the amount of indophenol produced a t the higher and lower p H values by aliquot samples from extracts of various products. TABLE11. EFFECTOF HYDROGEN-ION CONCENTRATION UPON PEROXIDASE ACTIVITY REACTION AT OPTIMUM PH IndoPRODUCT SAMPLE pH phenoln
REACTION AT OB NEAR PH4.6 IndopH phenol
Mo./&. 1 6.8 1.98 4.5 Strawberry 2 6.8 2.33 4.5 Strawberry 3 6.2 4.95 4.5 Pear Pear 4 6.2 5.01 4.5 5 6.4 113.0 4.4 Asparagus 6 6.4 112.2 4.4 Asparagus 7 7.0 80.7 Potato 4.4 a McIlvaine buffer used. b Citric acid and sodium hydroxide buffer used.
Mu./&. 0.03b 0.02b 0.42b 0.45b
20.04
21.0" 7.24
CATALABE ACTIVITY
OF PEROXIDABB SAMPLE
INOXYQEN Cc. 0.02 0.02 0.00 0.00 1.98 1.98 0.20
These results show that one may substitute the McIlvaine buffer and increase the pH value with an increase of the peroxidase activity. The blanks with the McIlvaine buffers were lower than those with the citric acid and sodium hydroxide buffers. As the peroxidase of many fruits and vegetables is wholly or partly inactive if determined a t pH 4.5, determinations should be made a t the optimum pH for the peroxidase being examined. This optimum for strawberries, pears, cauliflower, asparagus, and potatoes was found to be between 6 and 7 pH. One may compare the peroxidase activity of these tissues a t the optimum p H values with the activity of the same extracts buffered to a lower pH value (Tables I1 and 111). These results show a notable increase of peroxidase activity a t or near the optimum pH value. Peroxidase determinations made by the Willstatter and Stoll method also showed that pear peroxidase was more active a t 6.2 than a t 4.5 pH. The catalase in the peroxidase sample may be inactivated a t a suitable low temperature with only a slight loss of peroxidase activity. This temperature varies with the product; thus, 60' C. was used for asparagus, while 50' C. was sufficient for pear extracts. The following experiment was performed to show that pear peroxidase was more active a t 6.2 than a t 4.5 pH in the absence of catalase: A 1 per cent chloroform-water extract of pear tissue, having a pH value of 4.56, was heated for 12 minutes at 50' C. to inactivate the catalase. To determine the absence of catalase, samples five times as large as the peroxidase samples were buffered to 6.2 pH and the chloroform was removed from each sample by shaking just previous t o the catalase determination. Aliquot samples of this extract, which gave no catalase activity, were used for determination of its peroxidase activity at 6.2 and at 4.5 pH. The McIlvaine buffer was used for the 6.2 pH and the citric acid with sodium hydroxide buffer for the 4.5 pH determinations. 170
May 15, 1934
INDUSTRIAL AND ENGINEERING CHEMISTRY
The results given in Table I11 show that pear peroxidase was also more active a t the optimum pH value of 6.2 after the catalase had been destroyed. TABLE111. PEROXIDASE ACTIVITY OF PEARTISSUE AFTER CATALASE HADBEENINACTIVATED BY HEAT SAMPL~
PEROXIDASE PH VALUE OF ACTIVITY REACTION (INDOPHENOL PRODUCED) 6.2 6.2 6.2 6.2 4.5 4.5 4.5 4.5
Mg. 3.62 3.62 3.69 3.75 0.46 0.44 0.37 0.47
The optimum pH of 6.2 for pear peroxidase was determined experimentally by making peroxidase determinations under controlled conditions and pH values ranging from 4.4to 8.4. Aside from this, no constant relation has been noted between peroxidase and catalase activity, and it is doubtful if the presence of catalase interferes directly with the peroxidase determination. If this is true, the catalase in peroxidase samples need not be inactivated. The reagents were used in the same relative proportion as outlined by Guthrie but halved in amount. To insure complete and rapid solution of the indophenol, it seemed best to vary the amount of toluene with amount of indophenol to be dissolved. The indophenol production is linear with the enzyme concentration in dilute solutions. It is best to adjust the enzyme sample so as to not exceed 3 mg. of indophenol per sample. In order to make the determination of peroxidase comparable, a blank consisting of all reagents and peroxidase-inactivated extract was carried along with each test. The peroxidage in these blanks was inactivated by boiling for 20 minutes just previous to the determination. The only change in the order of procedure was the addition of the substrate to the enzyme extract and finally the addition of the buffer with the hydrogen peroxide. To avoid part of the sensitivity effect of the reagents, the solutions should be held a t a temperature of 25' C. before mixing.
171
The results were expressed as milligrams of indophenol produced by the peroxidase per decigram of dry substance in the enzyme sample. The dry-weight base was used because the water content of fresh, frozen, and stored frozen products varies considerably. PROCEDURE RECOMMENDED The suggested procedure for the determination of peroxidase activity is as follows: Care should be exercised to obtain comparable samples of the product for both dry-weight and peroxidase determinations. The weighed peroxidase sample should be ground thoroughly with fine sand and afterwards made up t o a definite volume with water or buffer solution. A measured volume of this enzyme solution is boiled for 20 minutes to destroy the peroxidase and then made up t o the measured volume for peroxidase-free samples and indicated as the blank solution. A required volumetric sample of the enzyme solution is placed in a 100-cc. flask and to this are added 6.25 cc. of the substrate solution containing 0.02975 gram of p-phenylenediamine hydrochloride in water with 0.595 cc. of 4 per cent a-naphthol in 50 per cent alcohol. (Solutions are made up in these proportions for several samples at one time, brought together, and filtered just before using.) The reaction is started by the addition of 8.75 cc. of a solution containing 6.25 cc. of the optimum pH buffer and 2.5 cc. of 0.05 N hydrogen peroxide. The reaction progresses at 25" C. for 10 minutes and is stopped by the addition of 2.5 cc. of a 0.1 per cent aqueous solution of potassium cyanide. A blank determination, made up from all the reagents and the required volume of the blank solution, is carried along with each peroxidase sample. The indophenol produced is dissolved in toluene and separated from the aqueous solution by centrifugalizing for 1 minute. The amount of indophenol in the sample is determined by colorimetric comparison with a standard containing 50 mg. of indophenol per liter of toluene. The amount of indophenol produced by the sample, less that produced by its blank, gives the initial peroxidase activity. From the dry-weight determination, the peroxidase activity is reported as milligrams of indophenol per decigram of dry substance in the peroxidase sample. LITERATURE CITED (1) Guthrie, J. D., J . Am. Chem. SOC.,63, 242-4 (1931). (2) McIlvaine, T. C., J . Bid. Chem., 49, 183-6 (1921). (3) Sorensen, S. P. L., Ergebnisse Phusiol., 12, 393 (1912). RECEIVED December 11, 1933. Presented before the Division of Biological Chemistry at the 86th Meeting of the American Chemical Society, Chicage, Ill., September 10 t o 15, 1933.
A Stirrer for Solvent Extraction JOHN A. PATTERSON, JR., University of Pennsylvania, Philadelphia, Pa.
I
3' THE extraction by means of immiscible solvents, such
the upper layer. This type of stirrer was designed and sucas ether-water or carbon tetrachloride-water, the need cessfully used for the treatment of organic liquids with sulfor intimate contact between the liquid layers is apparent. furic acid, where the ratio of acid to organic liquid was Figure 1 shows two modifications of a stirrer designed very low and uniform treatment was essential. in this laboratory. When the stirrer is rotated as indicated by On the right is shown the modification for downward flow. the arrows, liquid is drawn from one layer and sprayed in fine The principle of operation is the same. I n this case the jets, droplets through the second phase. I n this way intimate B-B, rotate in the heavier liquid, and the lighter solvent is contact, with a large interfacial surface, is obtained. This drawn through four small openings, A-A, in the upper part stirrer is particularly advantageousfor continuous extractions, of the hollow shaft. This stirrer has been used with entire since the body of the liquid which is being satisfaction for continuous ether extractions sprayed is practically undisturbed and may of aqueous solutions. The ether is added conbe drawn off continuously during the extractinuously below the water surface, the excess ether being drawn off through an overflow. tion. On the left are shown the details of the The stirrers used in t h i s l a b o r a t o r y are made from 5-mm. inside diameter glass tubing. stirrer used to lift a heavier liquid and spray it through the lighter liquid layer. When the The jets are 1 cm. from the center and have stirrer is rapidly rotated counterclockwise, the about 0.5-mm. openings, turned a t right angles n to the cross arms (parallel to the direction of m o v e m e n t of t h e j e t s , B-B, through the liquid cause a decrease i n p r e s s u r e in the rotation). I n the down-flow type the openQ-B hollow shaft of the stirrer. The heavier liquid, ings A-A a r e a p p r o x i m a t e l y 1 mm. in L d into which the open end of the shaft dips, is diameter. drawn up into the jets and sprayed through FIGURE1 RECEIVED February 3, 1934.
.d..-i;
