down earlier for reference data. They are apparently incomplete, in that too few lines are recorded, and inadequate indication of relative intensities has been given. Frequently, too, these data are not consistent with those in the x-ray powder data file, or with those obtained in the present investigation. The pattern given by Penprase and Biles for Barbital is evidently that of Barbital 11. However, the three lines which these authors describe as strong (lines at 3.54, 3.34, and 3.07 A. in Table 11) are moderate, weak, and moderate in Huang’s estimation, and are given relative intensities of 15% or less in Table 111. The line a t 5.30 A,, the strongest line in Huang’s data, and in Table 111 is completely omitted by Penprase and Biles. This investigation has confirmed Huang’s assertion that Diel is monomorphic ( 2 ) , and the spacing of the strongest line is given as 6.54 A. in Table 111. Huang also reports 6.54 A. for this spacing, but Penprase and Biles report it as 6.40, which represents a difference of 0.3” in 28, although these authors quote a tolerance of 0.1” in 28. I n some cases, differences between Penprase and Biles’
data, and those in Table I1 are undoubtedly due to different methods of crystallization, because these workers crystallized their samples from aqueous alcohol which, it has been found in this work, does not always give reproducible results.
barbiturates, or a mixture of a barbiturate and some other drug. Despite these difficulties, the advantages of the method are its ease and simplicity, the use of a very small sample which is not destroyed, and the availability in most larger laboratories of the equipment needed.
CONCLUSION
The diffraction patterns of a number of barbituric acids crystallized by the standard method, used by toxicologists, have been established. That x-ray methods can be used for identification of barbituric acids has been shown (6), and Marvin and Garbarino have discussed the advantages and disadvantages of the method ( 7 ) . One of the chief difficulties is polymorphism, but this can be avoided by standardizing crystallization methods. A further difficulty, which has been demonstrated by Huang (4, 5 ) , is that mixed crystal formation can occur between different barbituric acids, and between barbituric acids and other compounds. This difficulty is unlikely to occur often, although some drug houses market preparations containing two different
LITERATURE CITED
(1) Am. Soc. Testing Materials, “Index to the X-Ray Powder Data File,”
Brindle)., G. W., ed., Philadelphia, Pa., ,nc7 lYJl.
(2) Huang, T-Y., Acta Pharm. Intern. 2 , 4 3 (1931). (3) Ibzd., p. 93. ( 4 ) Ibid., p. 173. ( 5 ) Ibzd., p. 259. (6) Huang, T-Y., Jerslev, B., Acta Pharmacol. et Toxicol. 7, 227 (1951). ( 7 ) Marvin, B., Garbarino, J. J., J . Criminal Law Criminal. 44, 525 (1944). (8) Penprase, W. G Biles, J. A., J . Am. Pharm. ASSOC., Sc‘i).Ed. 45, 585 (1956). (9) Simmons, J. S., Gentskow, C. J.,
“Medical and Public Health Laboratory Methods,” p. 549, Henry Kimpton, London, 1955.
RECEIVED for review April 28, 1955. Accepted August 11, 1958.
New Method for Peroxidase Determination WARD M. HUNTING, MARCEL GAGNON,’ and WILLIAM B. ESSELEN Department of Food Technology, University of Massachusetts, Amherst, Mass. ,A new, very rapid, and satisfactory method for determining peroxidase is based on the time for development of a colored spot in a paper disk. Either guaiacol or gum guaiac is used with hydrogen peroxide. A linear calibration curve is obtained when the log of time is plotted against the log of concentration of the enzyme.
A
rapid, and simple colorimetric method for determining peroxidase activity has been developed. Methods reported in the literature have been extensively and thoroughly revieived by Sumner and Somers ( 5 ) , Colowick and Kaplan ( f ) , and Farkas (2). Spectrophotometric, colorimetric, and manometric methods were described. The most practical and simplest quantitative and qualitative methods appeared to be the colorimetric ones, using mainly a guaiacol substrate in the presence of hydrogen peroxide. An attempt was made t O simplify the NEW,
Present address, Alphonse Raymond LtBe, MontrBal, P.Q., Can.
quantitative peroxidase assay using the well known and recommended guaiacol and hydrogen peroxide substrate. However, in an effort to increase sensitivity, gum guaiac mas used in place of guaiacol a t low enzyme activities. The new quantitative determination for peroxidase activity is rapid and accuratc. REAGENTS AND EQUIPMENT
Peroxidase prepared from horseradish was obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. A 0.01 weight yo solution of the enzyme was prepared and suitable dilutions were made from this. The original activity of the peroxidase used was approximately 30 units per mg. (4). Hydrogen peroxide. Solutions (0.08 and 0.5%) were DreDared from J. T. Baker’s ’ 3% reagent’ grade hydrogen Deroxide. Phosphate buffer, O.lM,,,pH 6.5. Guaiacol, 0.5% solution in 5070 ethyl alcohol. Gum guaiac, 0.33% solution in Soy0 ethyl alcohol. Paper disks, KO. 57-GH1 specially prepared by Schleicher and Schuell Co., Keene, N. H., from purest, highly
absorbent paper for the assay of enzymes, catalysts, and other substances. Stop watch, graduated to 0.2 second. PROCEDURE
Preparation of Calibration Curves.
Paper disks were placed on a clean glass plate and 0.05 ml. of a standard diluted solution of peroxidase mas added t o t h e disks by means of a graduated syringe. A drop of the guaiacol and a drop of 0.08% hydrogen peroxide were added to the disk. At low peroxidase concentrations, 0.33% gum guaiac and o.5y0 hydrogen peroxide solutions were used. The time was measured with a stop watch from the moment the drop of reagent touched the paper disk until the first tinge of color could be detected. This measured time in seconds gave a linear relationship when its log value was plotted against the log value of the given peroxidase concentration expressed as per cent by weight of the purified enzyme. Mean values of such measured times obtained for solutions covering the range from 1.0 X lo-* to 1.0 X weight yo peroxidase using guaiacol VOL. 31, NO. 1, JANUARY 1959
143
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.
