ON THE ABSORPTION SPECTRUM OF CATALASE*

BY KURT G. STERN

(From the Laboratory of Physiological Chemistry, Yale University School of Medicine, New Haven)

(Received for publication, June 22, 1937)

The qualitative study of the light absorption of catalase solu- tions under varying experimental conditions (1) led to the con- clusion that the iron contained in the hematin grouping of the enzyme exists in a stabilized trivalent state. Furthermore, it was possible to find criteria which permit a differentiation be- tween the somewhat similar spectra of catalase and of methemo- globin. In the same communication, the effect of a number of enzymatic inhibitors and of two substrates on the spectrum of catalase has been described.

The present paper reports the quantitative determination of the catalase spectrum in the visible range. Highly purified enzyme preparations from horse liver were analyzed by a recording photo- electric spectrophotometer. This procedure yields the extinction coefficient data as obtained at the concentration studied. The hemin content of the same enzyme solutions was assayed by means of a simple spectral calorimeter after quantitative transformation into pyridine hemochromogen. The correlation of the relative extinction values with the enzyme concentration (in terms of mM of iron per liter) gives the spectrum calculated on the basis of a concentration of hemin = 1 m&r per liter.

EXPERIMENTAL

Enzyme Preparations-Catalase from horse liver was prepared according to Zeile and Hellstrom (2), with slight modifications. Aluminum hydroxide gel was used as the adsorbent. Besides the enzyme these preparations contain traces of biliverdin and hepa-

* This work was aided by a grant from the Elizabeth Thompson Science Fund, the assistance of which is gratefully acknowledged.

561

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562 Absorption Spectrum of Catalase

toflavin and appreciable amounts of inert proteins. The concen- tration of the contaminating pigments is too small to interfere with the study of the light absorption in the visible region. The protein concentration, however, is too high to permit the study of the absorption spectrum of the enzyme in the ultraviolet region. The present work has therefore been limited to the visible range (400 to 700 mp). It should be mentioned that all preparations used for this work were clear.

Activity Determinations-The activity of the suitably diluted enzyme solutions was determined under standard conditions; i.e., with 0.015 N HzOz at pH 6.9 (final phosphate buffer concen- tration ~/75) and at 0’. Samples were withdrawn before and 5, 10, and 15 minutes after addition of the enzyme. The experi- mental mixtures were stirred electrically. The remaining hy- drogen peroxide was determined iodometrically (cf (3)). The velocity constants for a monomolecular reaction were calculated. Since these constants were not always identical for the entire course of the reaction, the value obtained for the sample with- drawn after 5 minutes was always used. Two or more deter- minations were carried out for each enzyme preparation. The values were then multiplied by the dilution factor of the solution in order to obtain the activity of the stock preparation. In Table III, second column, the mean values for the solutions em- ployed are tabulated.

The stock solutions had to be diluted up to 10,000 times for these activity determinations. The enzyme appears to suffer spontaneous inactivation on standing at room temperature at such high dilutions with distilled water. The dilutions were therefore prepared freshly for each experiment.

Absorption Data

Samples of a number of enzyme preparations were sealed in Pyrex ampules and sent to the Massachusetts Institute of Tech- nology, where they were analyzed in the recording photoelectric spectrophotometer of Hardy (4) soon after receipt. The undiluted solutions could be used for the green and red region of the spec- trum, but they had to be diluted with distilled water for the ex- amination in the blue-violet region. Nine complete curves were thus obtained, each covering the range of absorption between 400

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K. G. Stern 563

and 700 rnp for a different sample. Some of the results are re- produced in Fig. 1. For most of the measurements the “density” cam of the photometer was used, whereby a plot of wave-length against optical density as the ordinate is obtained. In one case

the “transmission” cam of the apparatus was used, which fur- nishes plots of wave-length against per cent transmission as the ordinate. The width of the beam of monochromatic light was 10 rng throughout. This rather broad wave band is made neces-

TABLE I

Ratio of Densities of Various Enzyme Solutions at Maxima of y and of OL Absorption Bands

Preparation No.

xxxv 6.56 0.51 12.8 XXXVII 4.65 0.38 12.2

XXXVIII 2.52 0.21 12.0 XL 3.12 0.24 13.0 XL1 7.65 0.62 12.3 XL11 3.48 0.24 14.5

XL111 4.72 0.39 12.1 XLIV 2.80 0.195 14.3

XLV 5.74 0.385 14.9

Density at 409 mp (Y band)

F&Co of densities

ho9 m,,

0022 m,,

Cataphoresis experiment

Fraction I :: & ; ;;i; / &iii / ipI%

The densities D40s ,,+ for Preparations XXXV to XLV were obtained

from the relative absorption curves after correction for dilution.

sary by the illumination intensity requirements of the photometer. The use of such a broad spectral interval renders the determinations less accurate than can be attained by apparatus in which absorp- tion measurements can be made with spectral intervals of 2 to 3 mp. Inasmuch as the layer of thickness in these measurements was always 10 mm., the recorded densities of the enzyme solutions vary with their concentration. Such curves permit the deter- mination of the position of the absorption maxima of the enzyme.

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564 Absorption Spectrum of Catalase

The maxima are situated at 622, 540, 505, and 409 mp. The approximate position of the first three maxima has previously been determined by direct spectroscopic observation (2). In blood and liver catalase solutions bands at 406 and 266 rnp have been described (5).

Correlation of Absorption Maxima of Enzyme Solutions--If the absorption maximum at 409 mp is due to the same compound which is responsible for the maximum at 622 rnp, it is to be expected that the ratio of the densities of the maxima of these bands is similar for different enzyme preparations and also after fractiona- tion operations on a given enzyme solution. Fractionation was attempted by treatment with acetone and by subjecting an ace- tone concentrate to cataphoresis in an apparatus of Michaelis, as modified by Bennhold (6). The cataphoresis was carried out at pH 6.78, where the enzyme molecule has a negative charge (7). The specific conductivity was 0.0091; the potential gradient was 0.95 volt per cm. The pigment was permitted to migrate in this field for 26 hours at 24” =t lo. Three fractions were obtained by pipetting off three layers of the enzyme solution contained in the anodic side chamber of the apparatus. The relative ab- sorption spectrum of each fraction was obtained by the recording spectrophotometer. Table I contains the values for the densities of these three fractions, and also for a number of different cata- lase preparations at 409 rnp and 622 rnp, and the ratio of these values. The satisfactory agreement of these ratios suggests that the same compound is responsible for both absorption maxima.

Calculated Spectrum at Standard Concentrations of Hemin

For the calculation of the absorption spectrum a knowledge of the concentration of the enzyme in the optically analyzed solu- tions is a requisite. The determination of the enzymatic activity of the preparations, of their protein content, or of their total iron content as a measure of the enzyme concentration is subject to criticisms. The assay of the hemin content of the catalase prep- arations should yield correct values for the enzyme concentration in view of the evidence presented for the constitution of the pros- thetic group of catalase as protohematin (8). The preparations are free from other hemin-containing pigments besides the en- zyme, especially from hemoglobin or methemoglobin.

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E(. G. Stern 565

Hemin Determinations-The hemin group is detached from the protein carrier of the enzyme by treatment with alkali. It is converted into pyridine hemochromogen and measured against standards prepared from crystalline blood hemin. The same prin- ciple has been used by Zeile and Hellstrom (2) for the correlation of the activity with the hemin content of catalase preparations. The latter authors used a spectrographic method for the estima- tion of the pyridine hemochromogen. Keilin and Hartree (9) determined the pyridine hemochromogen derived from catalase hematin by means of a Zeiss microspectroscope, equipped with a comparator mechanism. In the present work, a Bock-Benedict calorimeter in conjunction with a Zeiss pocket spectroscope was employed. The calorimeter has one variable and one fixed ab- sorption cell. Only the light penetrating the variable cell was permitted to enter the spectroscope which was brought in contact with the eye-piece of the calorimeter. The mirror of the instru- ment was illuminated with a 100 watt lamp. A 1 cm. absorption cell was placed before the comparison prism of the spectroscope. Here a miniature flash-light bulb, the distance of which from the cell could be varied, served as the light source. The slit of the spectroscope and the distance of the auxiliary light source were adjusted to yield two equally intense spectra projected above each other. The sample to be analyzed was placed in the variable cell; the hemochromogen standard was contained in the fixed cell. The stratum of fluid in the variable cell was varied by means of the movable plunger until the absorption bands of the hemo- chromogen appeared of equal intensity in both spectra. The depth of fluid in the variable cell was then read off the scale. The bands are so well defined and their width and intensity are so strictly dependent upon the concentration of the hemochromogen that the measurements may be made with a high degree of accu- racy, as far as the actual process of comparison is concerned. Unspecific background absorption does not interfere with the measurements, nor does a slight difference in brightness of the two spectra.

A typical example of an analysis is the following. Hemochromogen Standard-11.0 mg. of crystalline horse hemin

were dissolved in 0.1 N NaOH to make a volume of 10 cc. This served as the stock solution. A mixture was prepared containing

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566 Absorption Spectrum of Catalase

0.5 cc. of the hem&n stock solution, 7.1 cc. of 0.1 N NaOH, 2.5 cc. of redistilled pyridine, and water to make a volume of 25 cc. The 1 cm. absorption cell was filled with this mixture, and immediately before the measurement a few mg. of sodium hydro- sulfite were added. After mixing, the cell was covered with a glass plate.

Hemochromogen from Catalase Hematin-2.0 cc. of catalase Preparation 45 (activity, L = 5734) were placed in the variable cell of the spectral calorimeter. 1 cc. of 0.1 N NaOH, 0.5 cc. of pyridine, and a few mg. of solid sodium hydrosulfite were added. Equal intensity of the absorption bands of the unknown and of the standard was observed at a stratum of 12 mm. of fluid in the variable cell (mean of several readings). A duplicate analysis had the same result.

Calculation of Hematin and Iron Content of Catalase-If c is the concentration of hematin in the enzyme preparation, cl the hema- tin content of the standard, h the stratum of the hemochromogen solution prepared from the enzyme preparation, and hl the layer of the standard (= 10 mm.), and if it is assumed that Beer’s law holds in the present case, the equation for the calculation of the hematin concentration in the enzyme solution is

cl X b c=-x175

h .

where the factor 1.75 is the dilution factor of the enzyme solution in the mixture analyzed. In the present example where cl = 22 (mg. of hemin per liter), hl = 10 mm., h = 15 mm., c becomes 25.7 (mg. of hemin per liter). If the iron content of hemin is taken as 8.6 per cent, this corresponds to a content of porphyrin-bound iron (Fe,) of the catalase preparation of 2.21 mg. or 4.10M2 mM per liter.

In order to obtain correct results, it is necessary to reduce the pyridine ferrihemochromogen to the ferrohemochromogen with hydrosulfite immediately before the measurement and to see that an excess of the reductant is present, since otherwise fading will occur. It might be mentioned as an outcome of actual experience that ordinary calorimetry or photoelectric calorimetry when applied to the present case will lead to erroneous results, owing to varying degrees of unspecific background absorption in the

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K. G. Stern 567

hemochromogen preparations. The use of color filters transmit- ting light of 500 to 600 rnp could not prevent these errors.

Extinction Coeficients of Catalase Solutions-The optical den- sity of the catalase preparations, as directly measured by the recording spectrophotometer (see the section on absorption data), is correlated to the pigment concentration, c, expressed in mM of porphyrin-bound iron per liter, by means of the equation

D E(C = 1 m,v per liter) = __

axe

where D represents the observed optical density, d the depth of the cell, and c the concentration in mM per liter, as proposed by Drab- kin and Austin (10). The observed optical density, D, equals the log IO/I, where I0 represents the intensity of the incident light and I the intensity of the transmitted light. Inasmuch as the concentration of porphyrin-bound iron is found by analysis (see the preceding section), this equation does not imply any assumptions as to the molecular weight of the pigment or to the number of hemin groupings per molecule.

In this manner, the extinction coefficients for various catalase preparations have been calculated for the wave-lengths 622 mp and 409 mp, which represent the position of the two defined max- ima of the solut,ions.

An inspection of Fig. 1 shows that, in addition to the above maxima, there are present, two less defined elevations in the ab- sorption curves, corresponding to the two /3 bands of Warburg’s respiratory ferment (11). Owing to their lack of definition, their extinction coefficients have not been calculated here. Table II shows that the extinction at the peak of the Soret band (at 409 mp) is roughly 13 to 14 times higher than that at the peak of the long wave absorption band (at 622 mp). Such a ratio is not un- common for pigments containing hemin nuclei combined with proteins.

Relationship of Enzymatic Activity, Hematin Content, and Op- tical Density of Catalase Preparations-If the pigment present in the catalase preparations is identical with the enzyme, the hema- tin content and the optical density of these solutions should be proportional to their enzymatic activity. The evidence offered by Zeile and Hellstrom (2) for the hemin nature of cat,alase is

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568 Absorption Spectrum of Catalase

based on the demonstration that the content in porphyrin-bound iron of a given individual enzyme solution runs parallel to its enzymatic activity in the course of various fractionation proce- dures. On the other hand, Zeile himself has reported varying

1.6

FIG. 1. Absorption curves of three cat&se solutions as traced by the

recording photoelectric spectraphotometer of Hardy. Curve 1, enzyme Preparation XXXV, undiluted; Curve 1, a, same diluted 4 times; Curve 2, enzyme Preparation XXXVII, undiluted; Curve 2, a, same diluted 3 times;

Curve 3, enzyme Preparation XXXVIII, undiluted; Curve 3, a, same diluted 2 times. Definition for optical density as employed in the ordinate,

D = log lo/Z, where Zois the intensity of the incident light and I the intensity of the transmitted light.

values for the activity to hematin (k/FeP) ratio for different prep- arations from the same source and from different sources. For two different horse liver preparations the k/Fee ratio was 2500 and 3400; for a preparation from cucumber seedlings the ratio

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K. G. Stern 569

was 8000 (12). Recently, this question has been reinvestigated by Keilin and Hartree (9). With respect to the result of frac- tionation procedures, they could fully confirm the conclusions drawn by Zeile and Hellstrom. The comparison of different enzyme preparations led to differences in the k/Fep ratio compa- rable to those found by Zeile.

TABLE II

Extinction Coeficients of Catalase Preparations at iWaxima of Absorption Bands at 62.2 rnl.r and 409 mp

cata1sse Porphyrin-bound Preparation No. iron

‘622 m/i “409 mp (c = 1 nm per liter) (c = 1 mM per liter)

-

x 103 mnr per 1.

XXXVII 3.5 10.9 133 XL1 5.3 11.5 144 XL11 2.3 10.5 151 XL111 3.9 10.0 121 XLIV 1.4 13.9 200 XLV 4.8 8.0 120

Mean............................... 10.8 I 145

The content in porphyrin-bound iron of enzyme Preparation XXXVII

was determined 9 months after the absorption spectrum had been measured.

The good agreement of the extinction coefficients of this preparation, as referred to the iron content (this table) and to the catalytic activity (see Table III), with the average obtained from all measurements is evidence

of the stability of the preparations. In the case of the other solutions, mentioned in this table and in Table III, the analytical and the optical measurements were carried out within an interval of 3 months. Determina-

tions of the catalytic activity showed no significant change in the enzyme content within even greater periods.

The data obtained in the present work permit a further scru- tiny of this important question. They are presented, together with the k/Fep ratios, in Table III. They yield an average value of 2289, which is close to that obtained by Zeile and Hellstrom for their first preparation (2). In Table III there are included the ratios of the activity of the solutions to their optical density, D, at the two absorption maxima, at 622 rnp and 409 mp. The agreement among these ratios is not so satisfactory as in the case of the k/Fep ratios, but this may partly be due to changes in pig-

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570 Absorption Spectrum of Catalase

ment content of the solutions during the transport to the Mas- sachusetts Institute of Technology where they were optically examined.

If there was any doubt left as to the identity of pigment and enzyme in catalase preparations, this doubt has been removed by the changes observed in the pigment speckurn in the course of the enzymatic decomposition of ethyl hydrogen peroxide (13). It has been shown that the pigment participates quantitatively, at least as far as the absorption band at 622 rnp is concerned, in the formation of a labile enzyme-substrate complex.

TABLE III

Ratio of Enzymatic Activity to Content in Porphyrin-Bound Iron and to

Optical Density --.

I at 6% mp and 409 rnb

Cat&se Enzymatic Preparation No. activity, k

xxxv

XXXVII

XXXVIII

XL

XL1

XL11 XL111

XLIV XLV

_____

5300 4948 3675 3093

6466 2845

5390 1739

5734

Porphyrin- bound iron

nq. per 1.

1.97 2512

3.0 2155 1.27 2240 2.21 2439 0.77 2245

2.67 2148

-

_ _

‘or Various Catalase Preparations

k

Fe,

Mean. . .I 2289

-I-

k

O622 mp

10,392 808 13,021 1064 17,500 1460 12,888 991 10,429 845

11,854 817 13,820 1142

8,917 621

14,893 999

k -~ D409 m,s

.- 12,635 972

DISCUSSION

Two enzymes have been definitely shown to contain hematins as their prosthetic groups : the oxygen-transferring (“respiratory”) enzyme (11) and catalase (2). Warburg has determined the ab- sorption spectrum of the respiratory enzyme by an ingenious in- direct method (11). The present paper reports the absorption spectrum of catalase in the visible range as obtained by a direct method. A quantitative comparison between the spectra of the two biocatalysts is as yet not possible, because there is available only the spectrum of the carbon monoxide complex of the ferrous form of the respiratory enzyme and that of the ferric form of

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K. G. Stern 571

catalase. Recently, Warburg and his colleagues (14) have de- scribed the position of the long wave absorption bands of the respiratory enzyme in certain microorganisms by direct spectro- scopic 0bservation.l The cy bands of the ferric form are given as 639 mp for Acetobxter and as 647 mp for Azotobacter. If this is accepted, the LY bands of the ferric forms of both catalase and the respiratory enzyme are situated in the red region of the spec- trum, though their exact position differs. No extinction values are available for the absorption spectrum of the ferric form of the respiratory enzyme. The extinction of the two enzymes may be somewhat different, inasmuch as the prosthetic group of the respiratory enzyme is a pheohemin (green-red hemin), whereas that of catalase is protohematin IX (8).

Catalase and methemoglobin have an identical prosthetic group but different protein carriers. Catalase has been trans- formed into methemoglobin by the exchange of carriers (8). The a! bands of catalase and of methemoglobin are similar in position (622 rnp and 630 rnp respectively). Their extinction at the peaks of these bands is E (c = 1 mM per liter) = 10.8 for catalase and 4.7 for methemoglobin (16).

SUMMARY

1. The light absorption of purified liver catalase solutions has been determined by a recording photoelectric spectrophotometer in the range from 400 to 700 rnp. In addition to the known ab- sorption bands in the visible region, the records show a maximum of high extinction in the far violet at 409 rnp. The presence of this band, which corresponds to the Soret band of hemoglobin- like pigments, is additional proof for the hemin nature of catalase.

2. The absorption spectrum of catalase is obtained by correlat- ing the optical density values, as directly measured, to the con- tent of the enzyme preparations in porphyrin-bound iron, as found by a spectral calorimetric procedure involving the formation of pyridine hemochromogen. The extinction of catalase was found to be 10.8 at 622 mp and 145 at 409 rnp, if referred to 1 mM of porphyrin-bound iron per liter (e (c = 1 mM per liter)). This represents the average of the data obtained for six different horse liver catalase solutions.

1 See, however, the criticisms of Keilin (15).

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572 Absorption Spectrum of Catalase

3. A satisfactory agreement is found for the k/FeP ratios (cata- lytic activity to content in porphyrin-bound iron in mg. per liter) existing in these six catalase preparations. The average value for the ratio was found to be 2289 f 200. The agreement between the ratios of catalytic activity and the optical density at the maxima of the two well defined absorption bands was less satis- factory.

BIBLIOGRAPHY

1. Stern, K. G., J. Gen. Physiol., 20, 631 (1937). 2. Zeile, K., and Hellstrijm, H., 2. physiol. Chem., 192, 171 (1930).

3. Stern, K. G., 2. physiol. Chem., 204,259 (1932). 4. Hardy, A. C., J. Optical Sot. Am., 26,305 (1935). 5. Itoh, R., J. Biochem., Japan, 22, 139 (1935).

6. Bennhold, H., Ergebn. inn. Med. u. Kinderh., 42, 273 (1932).

7. Stern, K. G., 2. physiol. Chem., 208,86 (1932). 8. Stern, K. G., J. BioZ. Chem., 112,661 (193536). 9. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 121,

173 (1936). 10. Drabkin, D. L., and Austin, J. H., J. Biol. Chem., 112,51 (193536). 11. Warburg, O., Angew. Chem., 46, 1 (1932). 12. Zeile, K., 2. physiol. Chem., 196,39 (1931).

13: Stern, K. G., J. BioZ. Chem., 114,473 (1936). 14. Warburg, O., and h’egelein, E., Biochem. Z., 262,237 (1933). Warburg.

O., Negelein, E., and Haas, E., Biochem. Z., 266, 1 (1933). Warburg,

O., and Haas, E., Naturwissenschuften, 22,207 (1934). Kegelein, E., and Gerischer, W., Biochem. Z., 268, 1 (1934).

15. Keilin, D., Nature, 132,783 (1933); 133,290 (1934). 16. Austin, J. H., and Drabkin, D. L., J. BioZ. Chem., 112, 67 (1935-36).

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Kurt G. SternCATALASE

ON THE ABSORPTION SPECTRUM OF

1937, 121:561-572.J. Biol. Chem. 

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