Quantitative Field Test for Estimation of Peroxidase Agricultural Research Administration,
W. B. DAVIS U. S. Department of Agriculture, LOB Angeles, Calif.
B
L A N C H I S G is now regarded as an essential operation in the dehydration of many vegetables. One of its objects is to inactivate enzymes that might otherwise cause deterioration of the product. The degree of blanching to which a given product is subjected is ordinarily specified b y stating the time and temperature of the heat treatment. It is difficult to describe the blanching effect accurately in these terms, however, because of the difference in types of blanchers used and variation in the heat required by vegetables from different sources. Attempts have therefore been made to define the heating which a product has undergone b y observing the extent to which certain easily measured enzymes have been destroyed thereby. Tests for oxidizing enzymes, such as the well-known tests with guaiacol and benzidine, have been tried as indicators of the proper degree of blanching, but the results have been rather unsatisfactory, probably because such tests were not sufficiently quantitative to permit a graded measurement of the heat effect. T o be satisfactory, such a test must be not only quantitative, but applicable to vegetables possessing color, and so simple and rapid as to be useful in the field. The method presented here is thought to meet these requirements. TABLE
present in the tissue and that which is added, and by the quantity of peroxidase. Ascorbic acid is the most important of the reducing substances present in the tissues of vegetables. A decrease in the quantity of ascorbic acid in vegetables caused b y blanching or dehydration would decrease the reaction time. A relatively large quantity of thiosulfate must therefore be used, so that changes in the amount of ascorbic acid do not introduce large errors. In the few cases where this is impracticable, the decrease in naturally occurring reducing substances may be found b y iodine titration, or the reaction time may be found without the addition of thiosulfate and a correction thus made for the natural reducing substances. A representative sample of the tissues weighing 25 grams is reduced to a fine state of subdivision in the Waring Blendor, using 140 ml. of buffer mixture as the extraction medium. The liquid is filtered through four thicknesses of cheesecloth. The cloth is squeezed practically dry by hand after the main bulk of the liquid flows through rapidly. Sufficient buffer mixture is added to the filtrate to make 200 ml. To a 50-ml. aliquot of the well-mixed extract, measured in a graduated cylinder, is added 1 ml. of 0.9 per cent hydrogen peroxide solution (prepared by diluting a pure 30 er cent solution containing no stabilizer) from a quick-flowing byow-out type of graduated pipet. The stop watch is started when the pipet is half empty. An ordinary watch or clock with second hand may be used instead of the stop watch. The time required for the appearance of the blue-black color, or the first appearance of change in color, is recorded as the reaction time. The reciprocal of this time is a measure of the enzyme activity. The buffer solution used in these investigations consisted of a mixture of 20 ml. of 2 per cent soluble starch, 10 ml. of 0.1 N sodium thiosulfate, 4.5 grams of potassium iodide,. and sufficient 0.2 N acetate buffer (pH 4.7) t o make 1 liter. This mixture will keep for 24 hours. I n Table I1 are given some representative results by this method. Two-gram samples of dried, blanched cabbage and mustard greens and 3-gram samples of dried, blanched beets and carrots, and 25-gram samples of the same blanched vegetables undried were used. All the extracts were colored, the beet extract more than the others, but in none was the end point indefinite; in fact, the quick “flash” of the end ppint is very striking. However, the method is capable of modification to give clear extracts if ever found necessary.
BY HEAT I. PEROXIDASE DESTRUCTIOX
(Water extract of cabbage) To Reach End Point Immersed in Boiling Water Sec. Sec. 12 0 11 15 13 30 90 60 46 60 5 118 120 57 120” 115 180 a
20 hours after cooling.
I n selecting a n enzyme suitable for the test it is necessary to avoid certain enzymes, such as catalase, which are so unstable as to be completely inactivated b y a little heat, as well as to avoid those seriously affected thereby. Peroxidase (8), which has a certain apparent thermostability (Table I), was selected for the test.
TABLE11. APPLICATION OF FIELDTEST FOR PEROXIDASE DESTRUCTION IN VEQETABLES Vegetable
Bach and Chodat (1) found that peroxidase in. a Tveak acetic acid medium catalyzed the oxidation of potassium iodide by hydrogen peroxide, releasing iodine. The iodine released in a given time was titrated with sodium thiosulfate solution. More recently Jayle (5) modified the method by adding a known amount of ascorbic acid to the system which also contained starch. The time necessary to free sufficient iodine in the buffered mixture to oxidize all the ascorbic acid mas indicated by the sudden appearance of a blue-black starch-iodine color. This time is a measure of the peroxidase activity. Sodium thiosulfate, which is more stable and more easily obtained than ascorbic acid, may be used as the reducing substance. I t is also more convenient to prepare a solution containing the potassium iodide, starch, sodium thiosulfate, and buffer mixture for use as the extraction medium. Only the peroxide needs to be added to such an extract, after which a record is made of the time required to form the blue-black color. In most cases, the strength of the iodide and the thiosulfate solutions may be adjusted, so that the end point can be observed in a conveniently short time.
Blanched
Min. Purple cabbage
Mustard greens
Beets
Carrots
0 1 2 5 0 0.5 1
4 0 10 20 0
1 6
To Reach End Point Before dryinga After drying% Sec. Sec. Sec. Sec. 4.0 4.5 6.5 7.5 62 97 29 25 72 145 173 68 655 290 300 480 7.0 7.5 1s 20 285 306 209 255 352 382 335 330 453 655 452 560 62 4s 67 33 250 215 255 215 315 295 322 265 225 230 223 217 230 180 180 222 216 251 235
Duplicate samples run.
The data in Table I1 show that the destruction of peroxidase is progressive and changes over a wide range with the blanching. The extent of blanching may therefore be easily defined, even when the blanching has been greatly overdone, as was the case with cabbage heated 5 minutes.
When other constituents are kept constant, the speed of the reaction is determined b y the reducing substances, both those 952
December 15, 1942
ANALYTICAL EDITION
953
It is sometimes observed that the reaction time for dehydrated material is less than that for the freshly blanched material. This may be due to loss of ascorbic acid or t o regeneration of the enzyme. Table I gives evidence of such regeneration in cabbage extract. This test is not supposed to fix the proper amount of blanching, but only to show when that amount has been obtained. The proper amount of heating naturally varies with each kind of vegetable, but when it has once been determined there should be no difficulty in specifying this amount in terms of the peroxidase value, and in using the peroxidase value as a means of factory control thereafter.
plicable to control of the blanching time of cabbage, which has been a problem in the dehydration field. No doubt the test may be used in other fields, such as blanching before quickfreezing. As a result of this work a new method for the determination of ascorbic acid is suggested, the details of which will be presented in another paper.
Summary
(1) Bach, A., and Chodat, R., Ber., 37, 1342-8 (1904). (2) Falk, K. G., McGuire, G., and Blount, E., J . B i d . Chem., 38, 229-
The method described appears to meet the requirements for a simple, sTVifttest for the blanching of \,egetables, even if extracts of them are highly colored. It is particularly ap-
Acknowledgment The aid of A. K. Balls, of the Enzyme Research Laboratory of this bureau, is gratefully acknowledged.
Literature Cited 44 (1919).
(3) J a y k &fax, ComPt. rend. SOC. bioi., 128, 1074-6 (1938). .kGRICULTURAL
Chemical Research Division Contribution NO. 87.
Preparation of Diphenylthiocarbazide and Diphenylthiocarbazone (Dithizone) OLIVER GRURIMITT AND RALPH STICKLE Western Reserve University, Cleveland, Ohio
A
LTHOUGH in recent years diphenylthiocarbazide and especially diphenylthiocarbazone (2, 5 ) have become important organic reagents for the analysis of certain metals, the only directions for the preparation of these compounds in the literature are essentially those of Fischer ( 3 , 4 , 6 ) ,who prepared them for the first time. Fischer’s method is limited to the preparation of very small quantities in low yields. The following method is based on Fischer’s procedure, but with modifications that permit a substantia1,quantity of product t o be made at one time. The reactions involved are: 2 CeHL’JHKHz
+ CS2 +CsH6NHh’HCSNHsPI”CsHa ll
I
(CeH6NHNH)zCS
+ HzS I1
2 (CeH5SHKH)&S
‘xH C&X=NCNHKHC~HS + +
Q
I11
CeHbNHNHCNHz
/I
+ CeHsNHz
S The condensation of phenylhydrazine and carbon disulfide yields I, the phenylhydrazine salt of P-phenyldithiocarbazic acid, which on heating forms diphenylthiocarbazide (11). This substance in the presence of alcoholic sodium hydroxide undergoes mutual oxidation-reduction, yielding diphenylthiocarbazone (111).
Experimental Fifty-four grams (50 cc., 0.5 mole) of freshly distilled phenylhydrazine and 200 cc. of benzene are mixed in a 500-cc. threenecked round-bottomed flask fitted with an efficient Nichrome wire stirrer ( 7 ) ,reflux condenser, and dropping funnel. From the funnel 20 grams (16 cc., 0.27 mole) of carbon disulfide are added with vigorous stirring in the course of 15 minutes, The mixture
of the salt (I) and benzene is then heated on the steam bath with stirring a t gentle reflux either under the hood or with the condenser connected to a gas trap (8) until the evolution of hydrogen sulfide practically ceases (about 3.5 hours). Near the end of this heating period the evolved gas should be tested frequently with moist lead acetate paper. The reaction may be considered complete when the test paper does not turn dark until exposed for several seconds. (Overheating is indicated by the formation of a very dark color and the rapid evolution of ammonia must be avoided. Ammonia is slowly evolved durin heating and this side reaction reduces the yield somewhat At this point the benzene layer is usually green in color and there is a heavy crystalline precipitate. The mixture is thoroughly cooled at 0” to 10” C. and suction-filtered, and the solid is washed with two 15-cc. portions of benzene. The yield of crude diphenylthiocarbazide is 48 to 50 grams, 80 to 83 per cent of the theoretical. If diphenylthiocarbazone is the desired product, the crude diphenylthiocarbazide may be used without further purification. However, if the diphenylthiocarbazide is to be used as a reagent, it must be crystallized from 900 cc. of 95 per cent ethanol. As the alcoholic solution decomposes slowly at its boiling point, it should be boiled no longer than necessary to dissolve practically all of the solid. The solution is filtered hot, thoroughly cooled at 0” to l o ” , and suction-filtered, and the fine needlelike crystals of diphenylthiocarbazide are washed with three 20-cc. portions of benzene. The yield of almost colorless product is 28 to 30 grams, 47 to 50 per cent of the theoretical; melting point about 145-150” C. with decomposition. No additional product of suitable purity can be obtained from the filtrate from this crystallization. To prepare diphenylthiocarbazone the crude diphenylthiocarbazide is dissolved in a solution of 200 cc. of methanol and 80 cc. of 25 per cent sodium hydroxide. [In the mutual oxidationreduction of diphenylcarbazide to diphenylcarbazone Slotta and Jacobi, ( 9 ) , obtained improved yields by adding a small amount of dilute hydrogen peroxide to the alcoholic alkaline solution. In the case of diphenylthiocarbazone added hydrogen peroxide has no effect.] This solution is refluxed on the steam bath for 5 minutes (longer heating will materially reduce the yield), poured into 1 liter of water, and filtered. The filtrate is acidified with dilute sulfuric acid (I volume of concentrated acid to 4 volumes of water); approximately 120 cc. of acid are required. The black precipitate of crude diphenylthiocarbazone is removed by suction filtration and then dissolved in an ammonia solution made from 100 cc. of concentrated ammonium hydroxide and 250 cc. of water. This dark red solution of the ammonium
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