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.
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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, P a .
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.
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