Table I.
Time for Appearance of Color Due to Oxidation of Guaiacol“ and Gum Guaiacb by Peroxidase in the Presence of Hydrogen Peroxidec
Peroxidase Concn.,
%
0 .O l d 0 .O O l d
0.000,3d 0.000; Id O.Ol*
0.003, 3a 0.001e 0.000,56 0.000,ld 0.000,01d 0,000, 003d 0.000,1~ 0. 000,056 0.000,01~
b
6
Seconds, Mean 1.0 4.0 7.6 15 1.4 2.7 5.5 7.8 1.2
5.5
Replicates Falling within fc,
U
Guaiaco1 fO.0 f0.6 f0.4 fl.0 50.3 50.3 f0.6 50.6 Gum Guaiac f0.2 f0.4
13 51 1.7 f0.3 2.7 f0.2 8.5 f0.7 0,000,005a 14.0 f0.8 0.000,001~ 43 f1.3 0.57, guaiacol in 507, ethyl alcohol. 0.337, gum guaiac. 0.087, hydrogen peroxide. Peroxidase in distilled water. Peroxidase in pH 6 . 5 phosphate buffer.
and their statistical significance are presented in Table I for unbuffered peroxidase solutions and for peroxidase dilutions prepared in 0.1M phosphate buffer at p H 6.5; corresponding values for gum guaiac covering the range from 1.0 X to 1.0 X 10-6 weight % peroxidase are also given (Table I). Regression lines were calculated using the least mean square method ( 3 ) . Typical standard curves plotted gave: for unbuffered peroxidase with guaiacol, a negative slope of 0.588 and an S,
%
2u
100 100 70 100 75
f 0 f1.2
94
50.6
74 69
f1.2
90 60
50.8
100
91 55 77 45 60
Replicates Fa11ing within f2u, %
50.8
f2.0 50.6
5i.i
50.4
f 2 f0.6 f0.5
51.4
51.6 f2.6
100
100 100 100
100 94 ._
95 95 100
100 100 100 100 96
100
100
value of +0.016; for buffered (pH 6.5) peroxidase with guaiacol, a negative slope of 0.582 and an S , value of *0.010; for unbuffered peroxidase with gum guaiac, a negative slope of 0.679 and an S, of 4=0.017; and for buffered (pH 6.5) peroxidase with gum guaiac, a negative slope of 0.703 and an S,of 0.010 (where S , is the standard error of estimate in linear regression). DISCUSSION
This method, designed to simplify the
usual peroxidase techniques, markedly reduced the time required to carry out a single determination. The technique is very simple but requires some practice before the analyst becomes familiar with the method and detection of the first tinge of color. A distilled water blank should be carried out using the reagents. Intensely colored materials may interfere. The solutions, paper disks, extracts, and other materials used should be maintained at approximately room temperature or a t the temperature used when carrying out the determinations for the calibration curve. LITERATURE CITED
(1) Colowick, S. P., Kaphp, K. O.,
“Methods in Enzymology, Academic Press, New York, 1955. ( 2 ) Farkas, D. F., “High Temperature Enzyme Inactivation,” 11. Sc. thesis, Department of Food Technology, Massachusetts Institute of Technology, Cambridge. Mass.. 1955. (3) Mills, p.‘ C., “Statistical Methods,” 2nd ed., Henry Holt, Sen- York, 1938. (4) Polis, B. D., Shmukler, H. W.,J . Bzol. Chena. 201, 475-500 (1953). ( 5 ) Sumner, J. B., Somers, G. F., “Chemistry and Methods of Enzymes,” 3rd ed., Academic Press, S e x York, 1953. RECEIVED for review December 10, 1957. Accepted September 8, 1958. Contribution 1130, Massachusetts Agricultural Experiment Station. Research undertaken in cooperation with the Quartermaster Food and Container Institute for the A4rmedForces; this is paper 814 in the series approved for publication. Views or conclusions contained in this report are those of the authors and are not to be construed as necessarily reflecting the views or endorsements of the Department of Defense.
N e w Method for Catalase Determination MARCEL GAGNON,’ WARD M. HUNTING, and WILLIAM B. ESSELEN Department o f Food Technology, University o f Massachusetts, Amherst, Mass.
b A new, very rapid, and satisfactory method for determining catalase by flotation of disks of absorbent paper is described. A linear calibration curve was obtained when the logarithm of time was plotted against the logarithm of concentration of the enzyme. Other practical applications of the new method are discussed.
A
rapid. and simple method for determining the enzyme catalase quantitatively and qualitatively has NEW,
1 Present address, Alphonse Raymond LtBe, Montrkal, P.Q., Can.
144
ANALYTICAL CHEMISTRY
been developed. The literature shoB-ed that the assay for catalase was based on the measurement of the amount of oxygen liberated by the action of the enzyme. Sumner and Somers ( 5 ) reviewed the methods used for the measurement of catalase activity. Methods for the determination of catalase based on the previous reaction were developed and later modified by Norgarrd (S), Knott ( 2 ) , .Pack ( 4 ) , Balls and Hale ( I ) , Tressler and Evers ( 7 ) .and Thompson (6). The first four authors reported accurate methods, which were time-consuming and required specialized apparatus. Thompson’s method ( 6 ) , which is a modification of the procedure
of Tressler and Evers ( 7 ) ,m s rapid and necessitated less specialized equipment, but it required rigid temperature control and a complex manipulation. This new method for catalase determination is based on the liberation of oxygen due to the action of catalase on hydrogen peroxide. However, the volume of oxygen released is not measured as such. Rather, the buoyant effect of the liberated oxygen on paper disks saturated with the enzyme solution is measured, indirectly, by recording the time required for the disk to rise to the surface of a solution of hydrogen peroxide.
EXPERIMENTAL METHODS
Reagents and Equipment. Catalase (lyophilized) (Nutritional Biochemicals Corp., Cleveland, Ohio) prepared from beef liver and containing 40 t o 50 meq. of sodium perborate per 0.1 gram was used. A 0.10 weight % ’ solution of the enzyme was prepared and dilutions of 0.010, 0.0025, 0.0010, 0.00025, and 0.00010 weight 7@ were made. The original activity of the lyophilized enzyme was 100 units per mg. (1 unit = the amount of the enzyme which decomposes 1 mg. of hydrogen peroxide in 1 minute). Hydrogen peroxide, 3% solution, reagent grade, was prepared from 30% Superoxyl by dilution with distilled water. Paper disks, No. 57-GH specially prepared bv Schleicher and Schuell Co., Keene, N. H., from purest, highly absorbent paper for the assay of enzymes, catalysts, and other substances. Test tubes, inside diameter of a t least 16 mm. to allow for free motion of the paper disks. Stop watch, graduated to 0.2 second. Preparation of Calibration Curve. Paper disks held by sharp-pointed tweezers were saturated with the various dilutions of enzyme solution by dipping into the liquid. They were then immediately dropped into test tubes containing approximately 5 ml. of a 37@hvdrogen peroxide solution. The time which elapsed between the moment the disk touched the surface of the hydrogen peroxide as it fell toward the bottom of the tube and the time when it again reached the surface was recorded to the closest 0.2 second. The logarithm of the time elapsing before the disk is floated is plotted against the logarithm of the concentration (or activity) of the enzyme solutions. Enzyme Determination. T o find the activity of catalase in an unknown material, dilutions must be prepared and the procedure outlined above must be carried out. When the material is a solid or contains suspended particles, it should be comminuted in a Waring Blendor after addition of a known amount of distilled water or of an appropriate buffer solution. The solution may be filtered or centrifuged to obtain a clear liquid. To preDare the extracts, recommendations readily found in the literature, for the specific product to be examined, may be followed. The per cent catalase (based on the activity of lyophilized beef liver catalase or other purified enzyme preparation) can be obtained by interpolation on the calibration curve using the time, in seconds, required by the specific unknown sample to float the disk. Qualitative Procedure. The quantitative procedure is readily simplified for adaptation t o the detection of catalase activity. It is necessary only to dip a disk in a solution or slurry, or, in some cases, simply to moisten the disk by pressing it against the bruised tissue of a food product or other material and then drop it into the hydrogen
Table 1.
Average Time for Disk Flotation in 3% Hydrogen Peroxide at Various Catalase Concentrations and Their Statistical Significance
Catalase,
%
0 01000 0.00250 0.00100 0 00025 0.00010
Mean Value, Seconds 2.7 9.3 21.6 46.0
98
Replicatee Falling within U
0.5 0.8 2.5 1.6 12
peroxide solution. The flotation of the disk within seconds or minutes constitutes a positive test. A blank should be run on reagents and paper disks to establish the time limit beyond which the pad would rise in the absence of catalase. RESULTS
Mean values of times obtained for lyophilized catalase solutions from 0.010 t o 0.00010 weight yo,together with the statistical significance of the values, are reported in Table I. The calibration curve was constructed using the loglo values of the enzyme concentration expressed as per cent X lo4 and loglo values of the mean time in seconds found for each concentration. The regression line was calculated using the least mean square method. The straight line plotted using this equation gave a y intercept of 2.008 corresponding to nn enzyme concentration for the present data of 0.00010% catalase. The slope calculated was -0.762, and the S , value was =k0.076. (8, is the standard error of estimate in linear regression.) DISCUSSION
The flotation rate of a paper disk is affected by a number of factors which should be standardized by the analyst in quantitative applications. Highly udiform disks must be used. Disks which have been compressed or imperfectly cut, or are obviously defective should not be used. Complete and uniform saturation of the pads in the enzyme solution is very important. Care should be taken that the disk does not come in contact with nor adhere to drops of hydrogen peroxide on the sides of the test tube when dropped into the tube, especially when dealing with the higher enzyme concentrations. Uniform-diameter test tubes should be selected. Their diameter should be somewhat greater than that of the disk. The same solution of hydrogen peroxide used in preparing the calibration curve should be used throughout the set of samples tested. The temperature of the hydrogen per-
*u, c-
ia
75 67 67 50
7c
Replicates Falling within 2u
i.2U,%
1.0 2.5 5.0 3.2 24
100 100
95 100
100
oxide should be uniform, as large variations would affect the rate of reaction. A control should be carried out using disks soaked with distilled water or buffer (free of catalase). The time required for the control disk to rise will be taken as the higher time limit for detection or determination of catalase. Beyond this$ time the disk flotation is not related to the reaction times nor to the presence of the enzyme. At lower concentrations, the catalase activity is inhibited to a degree by the hydrogen peroxide (or the oxygen produced by the reaction) resulting in a deviation from the straight-line relationships found for higher concentrations. The standard solutions of catalase should be prepared a t the highest concentration possible, and all subsequent dilutions should be made directly from this solution, because of the rapid loss of activity associated with solutions of lorn concentrations. I n dealing with these low concentration solutions, measurements should be taken immediately to minimize loss of activity due to standing. Other Applications. This method is not limited t o the reaction of catalase with hydrogen peroxide, b u t may be adapted t o many other analyses where a gas is involved in the over-all reaction. The evolution of carbon dioxide, sulfur dioxide, oxides of nitrogen, methane, and other gases may respond to this simplified method in both analysis and control procedures. CONCLUSIONS
RIethods for the determination of catalase, in general, were somewhat cumbersome and time-consuming. The method described is believed to be an improvement over the previous ones, because no specialized equipment or special technical training is required. The high speed and ease of making the determinations render it possible to accumulate data readily in quantity large enough to obtain statistically significant results. The principle involved in the new disk flotation method for catalase determination or detection should find a wide application in apVOL. 31, NO. 1, JANUARY 1959
145
plied fields and in analytical chemistry wherever the evolution of gases is concerned. LITERATURE CITED
(1) Balls, .4.K., Hale, W. S., J . Assoc. Oflc. Agr. Chemists 1 5 , 483-90 (1932). (2) Knott, J. E., N . Y . Stale Agr. Espl. Sta. Illem. (Ithaca, N . Y.)106 (1927). (3) Norgarrd, .4.17. S., J . Bid. Chem. 38,
501 (1919).
(4) Pack, D. A., IND.EKG. CHEU., ANAL.ED. 4, 393 (1932). (5) Sumner, J. B., Somers, G. F., “Chemistry and Methods of Enzymes,” 3rd ed., p . 218, Academic Press, New York, 1953. (6) Thompson, R. R., IND.ENG.CHEM., ANAL.ED. 14,585 (1842). ( 7 ) Tressler, D. K., Evers, C. F., (‘Freesing Preservation of Fruits, Fruit Juices and Vegetables,” p. 228, Avi Publishing Co., New York, 1936.
RECEIVED for review December 7 , 1957. Accepted September 8, 1958. Contribution 1126, bfassachusetts Agricultural Experiment Station. Research undertaken in cooperation with the Quartermaster Food and Container Institute for the Armed Forces, assigned number 809 in the series of papers approved for publication. Views or conclusions contained in this report are those of the authors, and are not to be construed as necessarily reflecting the views or endorsements of the Department of Defense.
Rapid Spectrophotometric Determination of Manganese Triethanolamine and Peroxide Complexes of Manganese(ll1) E. R.
NIGHTINGALE, Jr.
Ethyl Corp., Detroit 20, Mich., and University o f Nebraska, Lincoln 8, Neb.
b In alkaline triethanolamine solutions, manganese(l1) is air oxidized to form a green manganese(ll1)-triethanolamine complex. At 438 mp the molar absorbance index of the complex is 1 99. The spectrophotometric measurement of the manganese(lll)-triethanolamine complex in alkaline solution offers a rapid and highly selective method for determining manganese. Evidence is presented for the existence of a manganese(ll1)-peroxide complex in alkaline solution.
Absorbances w r e measured using a Beckman Model DU quartz spectrophotometer. All measurements were made in 1-em. silica or Corex cells a t 1’ C. 25”
*
PROCEDURE
Although the manganese(II1)-TEA complex has been used for polarographic determinations of manganese (9,4, 6, 7, 8), the complex has not been inveetigated previously for use in a spectrophotometric or colorimetric determination.
Pipet an acid sample containing 0.02 to 0.2 mmole (0.001 to 0.01 gram) of manganese(I1) into a 150-ml. beaker. Evaporate, if necessary, to 25 ml., and add 5 nil. of triethanolamine. Add 10 ml. of 9M sodium hydroxide and stir well to dissolve any manganese(I1) hydroxide precipitated by a local excess of sodium hydroxide. If the sample contains more than 75 meq. of acid, use additional sodium hydroxide. Add 1 ml. of 0.1M potassium bromate, and heat the solution to boiling. Remove the sample from the hot plate, bubble air into the solution for 2 minutes, and cool to room temperature. Add 2 ml. of 2M sodium sulfite. Transfer the solution t o a 50-ml. volumetric flask and dilute to volume. The final solution is 0.6M in TEA (80% solution) and approximately 0.6M in sodium hydroxide. Measure the absorbance at 438 mp and determine the manganese concentration by comparison with standard samples.
REAGENTS A N D APPARATUS
RESULTS A N D DISCUSSION
Chemicals of C.P. grade and distilled water were used to prepare all reagent solutions. Although 9801, triethanolamine (Matheson Coleman & Bell, No. 2885) was used t o develop the analytical procedure, regular (80% minimum) triethanolamine is satisfactory for routine analyses. The triethanolamine can be dispensed conveniently from a 5-ml. hypodermic syringe fitted with a 13-gage needle. Spectra were recorded on a Cary Model 105 recording spectrophotometer.
Manganese(I1) reacts with triethanolamine to form a tan complex which is stable above p H 5.5. Triethanolamine is a weak base with a pK, of 7.77 (1). Below p H 5.5, the fraction of the triethanolamine in the basic form is too small to permit complex formation. Curve C in Figure 1 shows the absorption spectrum of a 1mM solution of the manganese(I1)-triethanolamine complex in O.6M triethanolamine a t a p H of 7.5. The spectrum of 2.0mM manganese-
W
MANGANESE(II) is oxidized by air in alkaline triethanolamine (TEA) solutions, a green manganese(111)-TEA complex (6) is formed according to the reaction: O2 Mn(TEA)2+*= HOzMn(TEA)2+a HEX
+
146
+
e
ANALYTICAL CHEMISTRY
(111)-triethanolamine complex in 0.6-11 triethanolamine and 0.6M sodium hydroxide is shown by curve A in Figure 1. A broad absorption maximum occurs a t 438 nip, nith a n absorption minimum a t about 377 nip. The positions of the maximum and minimum are not dependent upon the concentrations ol triethanolamine or sodium hydroxide. The previous polarographic proredures for determining manganese using triethanolamine (2, 4, 6, 7) have involved preparation of the manganese(111)-triethanolamine complex by air oxidation of the manganese(I1)-triethanolamine complex. All attempts in these laboratories to achieve rapid quantitative air oxidation at room tempernture have been unsuccessful for solutions containing more than 0.5 mmole of manganese. I n addition, the absorbance of the solution changes appreciably with time in a manner characteristic of the presence of a second colored species. The absorption maximum at 438 mu for curve A in Figure 1 increases about 3y0 in 60 minutes. The absorption minimum increases by about 39Yo,, and the position of the minimum shifts to longer wave lengths. The increase in absorbance may continue for several hours, after which it begins to decrease. After prolonged standing-e.g., 24 hours-the absorbance becomes constant, indicating quantitative formation of the manganese(II1)-triethanolamine complex. I n a n attempt to achieve quantitative oxidation of manganese(I1) more rapidly, hydrogen peroxide was added to an alkaline triethanolamine solution containing manganese, and a red complex was formed. Curve B, Figure 1, shows the absorption spectrum of the red com-
