AN ULTRACENTRIFUGAL STUDY OF CATALASE

BY KURT G. STERN AND RALPH W. G. WYCKOFF

(From the Laboratory of Physiological Chemistry, Yale University School of Medicine, New Haven, and the Laboratories of The Rockefeller

Institute for Medical Research, Princeton)

(Received for publication, May 5, 1938)

The standard method for the purification of catalase as de- veloped mainly in the laboratory of von Euler (1) consists of three steps: (1) fractional precipitation of the crude, aqueous tissue extract with alcohol, (2) complete denaturation of the hemoglobin by treatment with alcohol-chloroform mixtures (Tsuchihashi), (3) adsorption of the enzyme on calcium phosphate or aluminum hydroxide and elution with secondary phosphate. The activity of the resulting catalase solution depends on the enzyme content of the starting material, on the extent of its re- moval by the adsorbent, and on the volume and pH of the eluent. Under favorable conditions horse liver, which is the richest of all known sources of catalase, is said to have provided samples with an activity, lc (monomolecular), as high as 40,000. In our hands, this procedure of Zeile and Hellstrom (l), with minor modifica- tions, has yielded enzyme solutions of an average activity, k, of 4000; sometimes Ic has been as low as 1500 and as high as 8000. Such preparations are brown in color and show the enzyme spec- trum in layers of from 1 to 5 cm. Besides catalase, they contain several inactive proteins and small amounts of pigments like verdohemochromogen (2) and riboflavin (3). They may be further purified by ultrafiltration (4) or they may be concentrated without appreciable gain in purity by acetone-CO2 precipitation (5). Recently ammonium sulfate fractionation has been used with good results (6).

Sumner and Dounce (7) have described the crystallization of beef liver catalase by a procedure based on extraction and frac- tionation with dioxane followed by ammonium sulfate treatment. The activity of the crystals was found unchanged after repeated

573

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574 Ultracentrifugal Study of Catalase

recrystallizations. It was lower (k = 25,000) than that of several amorphous horse liver preparations reported in the literature (1, 8). Since protoferriheme IX (protohematin IX) is the colored prosthetic group in both horse (5) and beef (7) liver catalase, this may be due either to species differences in the protein part of the enzyme complex or to the presence of inactive protein in the crystalline preparations. This point can be decided only by per- forming suitable tests for homogeneity. Recent experiments in which crystalline pepsin and carboxypolypeptidase preparations have been shown by electrophoretic analysis t,o consist of several proteins (9) indicate that the second possibility cannot safely be ign0red.l

The present experiments were undertaken (1) to attempt a purification of catalase solutions by quantity ultracentrifugation, (2) to correlate enzymatic activity and the content of porphyrin- bound iron at different levels of the molecular “distribution spec- trum” set up by the intense centrifugal field, and (3) to measure the rate of sedimentation of the purified enzyme in the analytical ultracentrifuge as an index of its molecular size. Some of the results of this study have already been briefly reported (11).

The first point was of particular interest in showing whether the quantity ultracentrifuge would be as useful for purifying enzymes as it has been with virus proteins. The second point should fur- nish additional evidence bearing on the identity of the enzyme with the hematin-protein complex responsible for the characteristic absorption spectrum of catalase preparations (1); heretofore proof of such identity has been based on the correlation between catalytic activity, the hematin content, and the extinction coefli- cient at the absorption maxima of purified catalase solutions (1,4, 12) and on spectroscopic phenomena observed after certain inhibitors (1, 13) and substrates (14) were added. The third point, finally, is important because it permits a comparison be- tween methemoglobin and catalase. Both have protoferriheme as prosthetic group but the carrier proteins are very different.

EXPERIMENTAL

Experiments with the Quantity Ultracentrifuge-Catalase solu- tions for ultracentrifugation were prepared from horse liver, and

1 See also the solubility measurements of Steinhardt (10) on crystalline

pepsin material.

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K. G. Stern and R. W. G. Wyckoff 575

in one instance from beef liver, by the method of Zeile and Hell- Strom (1) with aluminum hydroxide (Eimer and Amend, gela- tinous) as adsorbent. These solutions were spun in the previously described ultracentrifuge (15) for periods of time varying from 2 to 10 hours, the field at the bottom of the tube being about 70,000 times gravity. The quantity heads were chilled in ice before starting and remained cool throughout the experiment owing to the high vacuum maintained in the rotor chamber. At the end of a run the centrifuge was stopped in the usual fashion, by applying air pressure to the reversing jets of the turbine. If reasonable care is exercised in this stopping, there need be no appreciable re- mixing of the contents of the tube. It was invariably found that the color of the solutions after centrifuging was deepest at the bottom of the tubes, while the upper layers were either lightly colored or colorless; in the later centrifugation of concentrates the tubes sometimes contained clearly defined layers separated by sharp boundaries. Suitable amounts of liquid were successively withdrawn from each tube and combined with the corresponding layers of the other tubes. These fractions were analyzed for total solids, porphyrin-bound iron, and catalytic activity by methods already described (12). The results are compiled in Table I. After removal of the brown-black lowest layers solid pellets were sometimes found at the bottom of the tubes. They consisted of a reddish, seemingly microcrystalline material that dissolved readily in water to give clear red solutions. The nature of this substance will be discussed below. In some instances the fractions obtained in the first run were recentrifuged once or twice for fur- ther concentration and purification of the pigmented materials.

It is seen from Table I that after ultracentrifugation both the enzyme (k, and Kat. f.) and the hemin-containing pigment (hemin, Fe,) are strongly concentrated in the bottom layers. In the first series the proportionality between activity and content of porphyrin-bound iron (k/Fe,) was excellent; in succeeding experiments it was less satisfactorily maintained. This may perhaps be due to a partial inactivation of the enzyme solutions in transit between Princeton, where they were centrifuged, and New Haven, where they were analyzed; preservatives were not used in order to avoid protein denaturation. In spite of this lack of proportionality it is apparent that the fractions exhibiting the highest enzymatic activity invariably were also richest in hemin.

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576 Ultracentrifugal Study of Catalase

Several of the lowest fractions compare favorably in activity and purity (Kcct. f.) of enzyme with the best preparations reported in the literature, including the crystalline beef liver catalase of Sumner and Dounce (7). These authors determined the total

TABLE I

Analytical Data jor Catalase Fractions Obtained with Ultracentrifuge

?F fs 0

I

II

III

IV

V

Fraction

Catalase 43

TOP Middle Bottom Catalase 44

Supernatant Bottom Catalase 42

TOP Bottom (1 X)

“ (3 Xl

Catalase 41 Top (1st run)

“ (2nd “ )

Bottom (2 X) Pellet solution Catalase 47

Top (2nd run) Bottom (2 X) Pellet solution

-

Hemin Fe,

p%.

26 3.5:

11.7: 187

9.6

3.7 257

16.8

4.2 99.4

402.5

35 3 3

906.5 44 25.7

-

.-

7

3

7

TJ. per 1.

2.2 0.31

1.0 16.1

0.8:

0.3: 22

1.41

0.3: 8.5;

34.6

3.0 0.2( 0.21

77.9 3.7: 2.2

700 60.2 20.5 1.7(

5,390 12,000 8,983 2439 461 750 8,000 1,857 24302241

2,125 9,500 4,474 2125 810

34,750 32,400 21,385 2158 173 2,725 10,100 5,450 33231052

545 8,200 1,330 17032216

28,000 54,000 10,370 1272 210 2,122 10,000 4,245 1474 595 1,024 7,000 2,926 27701640

11,750 27,800 8,545 1374 279 64,450 57,000 22,614 1863 141

6,466 14,500 8,917 2155 414

296 5,800 1,020 1381933 200 6,200 666 770 2066

161,505 96,000 33,448 2073 105.9 4,542 11,000 8,259 1202 250 2,310 11,400 4,052 1045 444 1,097 12,000 1,829

79,500 70,000 22,714 1320 100 1,312 6,000 4,367 745 292

Fe=, porphyrin-bound iron; k, catalytic activity, expressed in terms of

the monomolecular rate constant, determined with hydrogen peroxide under standard conditions (pH 6.8, at 0”, 0.02 N HzOt); Rat.. j., ratio of activity, k, to total solids contained in 50 cc. of the reaction mixture.

iron in their crystals rather than the hematin content, a procedure which affords no information concerning the presence of extraneous iron. To our knowledge, the lowest fraction of the second run of Series IV, with an activity of k = 161,000 and a hemin content of 906 mg. per liter, represents the most concentrated catalase

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K. G. Stern and R. W. G. Wyckoff 577

preparation on record. On the basis of the content in solids (see Table I) this solut,ion contained 96 gm. of enzyme per liter; it probably was saturated with catalase.

In order to test the correlation between light absorption, hemin content, and catalytic activity, the bottom fraction of the Series I (Table I) and the original enzyme solution were compared visually in a spectral calorimeter (12). The point of matching was ob- tained at an intensityoratio of 7.5: 1 when the absorption band of the enzyme at 6220 A. was chosen for comparison. It will be seen from Table I that the ratio of porphyrin-bound iron in the two solutions was 7.2:l and the ratio of the catalytic activities 6.4: 1. The highest Kat. f. values and the lowest rat.ios of dry residue to hemin, both of which may be taken as indices of the purity of the enzyme, were found in the recentrifuged lowest fractions of Series IV and V. These fractions were also highest in absolute hemin content and in absolute activity. Nearly 1 per cent of the total dry matter was hemin in both instances.

Experiments with the Analytical Ultracentrifuge-A series of studies was made of the sedimentation diagrams obtained by photographing the original solutions and the fractions of Table I in an air-driven analytical ultracentrifuge (15, 16) arranged for absorption measurements according to the original method of Svedberg. Sedimentation constants of the substances producing the boundaries seen in these diagrams were determined from photographs made with ultraviolet light filtered through chlorine and bromine cells. With the light source and films used the photographicaloly active light had wave-lengths in the region between 2700 A. and 2300 A.

A typical sedimentation diagram of a catalase solution made according to the procedure of Zeile and Hellstrom (1) is repro- duced in Fig. 1. The principal feature is the sharp boundary (a) that sediments with the constant ~200 = 11 X lo-l3 cm. sec.-’ dynes-‘. A good preparation, such as that used for this experi- ment, contained practically no light-unsedimentable material but there could usually be seen a faint boundary (6) coming down with

-920” = about 65 X lo-l3 cm. sec.? dynes-‘. The substances responsible for these two boundaries were

strongly concentrated in the lowest fraction from the first quantity ultracentrifugation (Fig. 2). An aqueous solution of the clear

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Ultracentrifugal Study of Catalase

FIG. 1. Sedimentation diagram of a partly purified catalase solution

prepared by the chemical method of Zeile and Hellstriim. The boundary

(a) in this and succeeding figures is due to catalase; the faint and diffuse boundary (5) is caused by the heavy component with szoo = 65 X lo+.

Mean centrifugal field = 94,400 times gravity. Interval between expo- sures = 5 minutes.

FIG. 2. Sedimentation diagram of the lowest fraction from the first

quantity ultracentrifugation of such a solution as that giving Fig. 1. The boundary (5) is more apparent. Mean centrifugal field = 94,900 times

gravity. FIG. 3. Sedimentation diagram of an aqueous solution of the clear red

pellets found in the centrifuge tubes after quantity ultracentrifugation.

As the picture shows, this solution contains the heavy component in purified form. Mean centrifugal field = 67,000 times gravity.

FIG. 4. Sedimentation diagram of the lowest fraction after successive

quantity ultracentrifugations. This solution clearly contains catalase in

purified form. Mean centrifugal field = 109,000 times gravity.

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K. G. Stern and R. W. G. Wyckoff 579

red pellets gave only the boundary corresponding to the heavy material (Fig. 3). After two or three quantity sedimentations of the pooled lower fractions all detectable amounts of the heavier substance had separated out in pellet form. The bottom layers from such successive ultracentrifugations contained the catalase activity in most concentrated and purified form; they showed only the boundary (a) of the lighter material with SW = 11 X lo-l3 cm. sec.-’ dynes-l (Fig. 4). From evidence such as this it follows that boundary (a) is due to the enzyme molecules. SedimeFtation photographs have been made pith blue light (4300 A.) and long wave ultraviolet light (3160 A.). The same boundaries are present in the pictures obtained at these wave- lengths; evidently the heavy molecules that absorb in the ultra- violet region are also responsible for the red and brown colors of these solutions.

The concentrations required to give good contrast with the blue and the near ultraviolet radiations were about 10 times as high as those required for photography in the short wave ultraviolet region. Molecular sedimentation is retarded at these concen- trations, so that all measurements of sedimentation constants have been based on pictures of the dilute solutions photographed with the short wave ultraviolet light. A series of such measure- ments on the more slowly sedimenting boundary due to catalase has yielded szoO = 11.1 X lo-r3 cm. sec.-’ dynes-l. The molec- ular weight of horse liver catalase cannot be accurately calculated from this constant in the absence of determinations of density and diffusion rate. Nevertheless, comparison with the results of Svedberg and his students (17) indicates that unless these proper- ties are highly abnormal for catalase, its molecular weight is somewhere between 250,000 and 300,000.

A beef liver concentrate prepared according to the method of Zeile and Hellstrom (1) was also examined. Its sedimentation diagram2 showed a catalase boundary with ~200 = 12.3 X 10-r3. One attempt to obtain crystalline beef liver catalase by the pro- cedure of Sumner and Dounce (7) failed.

* In a note just published Sumner and GralCm (18) have found szoo = 12.0 X lo-l3 for a solution of crystalline beef liver catalase (7). They have also determined the partial specific volume of this form of catalase as 0.73 and its diffusion constant as 4.1 X lo-‘. From these values they have deduced a molecular weight of 263,000.

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Ultracentrifugal Study of Catalase

The heavier substance corresponding to the boundary (6) in Figs. 1 to 3 has only a low degree of molecular homogeneity. This heterogeneity may be either a natural property of the sub- stance or the consequence of chemical changes introduced during its purification. Our experiments do not settle this point but some preliminary observations on a partly purified sample indicate that the second alternative may be correct. It is interesting to note that if this heavier pigmented substance is a protein with the usual density, its sedimentation constant ~200 = about 65 X lo-l3 suggests that it should have the extraordinarily high molec- ular weight of 3 to 4 millions.

Further Properties of the Heavier Pigmented Substance-In the present experiments too little of this heavy material was available to permit a detailed study of its nature; but certain preliminary observations have been made. At first it was suspected that it might be an association product arising through concentration of catalase. This was disproved when the two materials were ob- tained separately in concentrated and moderately pure states. The heavier substance in the form of redissolved pellets still con- tained a considerable amount of hemin and had a very appreciable catalase activity (Table I). These could, however, be due to enzyme contamination, since extreme solubility prevented washing and since too little of the material was available for the successive resedimentations that should have given greater purity. The following observations were made on a solution prepared by pooling the various pellet fractions, resedimenting once in the ultracentrifuge, and dissolving the pellet in water. Its total solid content was 5.6 mg. per cc.

The abosorption spectrum in the visible region from 4000 A. to 7000 A. was measured with Hardy’s recording photoelectric spectrophotometer at the Massachusetts Institute of Technology. The undiluted sample and portions diluted with 1 and 3 volumes of distilled water were examined, both the density and the trans- mission cams being used. Two of the resulting curves are repro- duced in Fig. 5. It will be seen that the pigment has no pro- nounced maximum in this range. Its transmission decreases gradually towards the short wave-lengths. In this it contras$s sharply witjf catalase which has maxima of absorption at 6220 A. and 4090 A. The absorption in the ultraviolet region from

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K. G. Stern and R. W. G. Wyckoff 581

4000 A. to 2200 A. was photographed with a quartz spectrograph and a hydrogen discharge tube as light source.3 In this experi- ment the thickness of the solution was varied by means of a quartz Baly tube. It showed no bands on this region either but had a continuous absorption that increased gradually towards the short wave-lengths and was practically complete below 2350 A. This absence of absorption maxima is not necessarily evidence

tm tm I I 980 980 560 560 640 640 700 700

!!P’ !!P’ FIG. 5. Absorption spectrum of red macromolecular liver pigment in

the visible region, as recorded by Hardy’s automatic photoelectric spectro- photometer. Abscissa, wave-length in rnpc; ordinate, transmission in per cent. Curve A, 2.8 mg. of dry residue in 1 cc. of solution; Curve B, 1.4 mg. of dry residue in 1 cc. of solution.

against the protein nature of the pigment but it does suggest that if it is a protein it is probably deficient in aromatic amino acid constituents (19).

Several qualitative chemical tests have been made. Thus sulfosalicylic acid produced a precipitate; the supernatant fluid assumed a pink-violet color indicative of the presence of iron.

3 The authors wish to thank Dr. G. I. Lavin of The Rockefeller Insti- tute for Medical Research, New York, for kindly performing this ex- periment.

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582 Ultracentrifugal Study of Catalase

Heating to the boiling point after adding sodium acetate likewise caused precipitation. The red color of the solution faded on addition of sodium hyposulfite (Na&OJ ; it was not restored upon shaking with air or adding 3 per cent hydrogen peroxide. The color was also discharged by 33 per cent sulfuric acid but not by 0.1 N NaOH. Tests for the presence of a flavin group, e.g. irra- diation with filtered ultraviolet light, were negative. Further experiments must await the preparation of more material.

DISCUSSION

Protoferroheme and protoferriheme4 are combined in nature with a variety of proteins to form important complexes. The specific nature of the conjugated protein determines whether the resulting compound is suitable for oxygen transport, as in hemoglobin and the erythrocruorins, for regulating the intracellular oxygen tension, as in myoglobin, or for special catalytic functions in biological oxidation, as in catalase and peroxidase. The molecular weight, which depends mainly on the protein component, varies within wide limits even for one group of these protoheme proteins. Thus the respiratory transport pigments have molecular weights ranging from about 17,000 to 3,000,000, the lighter ones being enclosed in individual cells, the heavier ones freely dissolved in the plasma (Svedberg (17)). Even in the same organism there exist protoheme complexes of different dimensions; in the mam- mal there are hemoglobin (molecular weight 68,000), myoglobin (molecular weight 17,000), and catalase (molecular weight 250,000 to 300,000). The molecular weight of peroxidase is as yet un- determined.

1 hemoglobin molecule contains 4 heme groupings. It is not known for certain whether four small globin units form the macro- molecule or whether the four prosthetic groups are combined with a single protein molecule. The reversible dissociation (21) of hemoglobin into molecules of half size in the presence of certain amides (urea, acetamide, formamide) would suggest that hemo- globin is at least a dimer. Assuming that the resedimented lowest fractions in Series IV and V (Table I) represent nearly pure catalase, the enzyme would contain 1 per cent hemin. The iron

4 These terms are substituted for the older names, protoheme and protohematin respectively (20).

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K. G. Stern and R. W. G. Wyckoff 583

content is of the order of 0.1 per cent, in agreement with the figures for crystalline beef liver catalase (7). If the molecular weight is taken as about 250,000, this result indicates that catalase, like hemoglobin, has four hemin residues per molecule.

One of the authors (K. G. S.) desires to thank the Alexander Brown Coxe Memorial Fellowship Fund and the Elizabeth Thomp- son Science Fund for their generous support of this investigation.

BIBLIOGRAPHY

1. Zeile, K., and Hellstrom, H., 2. physiol. Chem., 192, 171 (1930). 2. Lemberg, R., and Wyndham, R. A., J. and Proc. Roy. Sot. N. S. Wales,

70, 343 (1937). 3. Stern, K. G., Ber. them. Ges., 66, 555 (1933). 4. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 121,

173 (1936). 5. Stern, K. G., J. Biol. Chem., 112, 661 (1935-36). 6. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 124,

397 (1938). 7. Sumner, J. B., and Dounce, A. L., J. Biol. Chem., 121, 417 (1937). 8. yen Euler, H., and Josephson, K., Ann. Chem., 462, 158 (1927). 9. Agren, G., and Hammarsten, E., Enzymologia, 4,49 (1937); J. Physiol.,

90, 330 (1937). 10. Steinhardt, J., Proc. Am. Sot. BioZ. Chem., J. BioZ. Chem., 123, p. cxv

(1938). 11. Stern, K. G., and Wyckoff, R. W. G., Science, 87, 18 (1938). 12. Stern, K. G., J. BioZ. Chem., 121, 561 (1937). 13. Stern, K. G., J. Gen. Physiol., 20, 631 (1937). 14. Stern, K. G., J. BioZ. Chem., 114, 473 (1936). 15. Wyckoff, R. W. G., and Lagsdin, J. B., Rev. Scient. Instruments, 8, 74,

427 (1937). 16. Biscoe, J., Pickels, E. G., and Wyckoff, R. W. G., J. Exp. Med., 84, 39

(1936). 17. Svedberg, T., Nature, 139, 1051 (1937). 18. Sumner, J. B., and Gralen, N., Science, 87, 284 (1938). 19. Coulter, C. B., Stone, F. M., and Kabat, E. A., J. Gen. Physiol., 19,

739 (1936). 20. Pauling, L., and Coryell, C. D., Proc. Nat. Acad. SC., 22, 210 (1936). 21. Steinhardt, J., Nature, 138, 800 (1936).

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Kurt G. Stern and Ralph W. G. WyckoffCATALASE

AN ULTRACENTRIFUGAL STUDY OF

1938, 124:573-583.J. Biol. Chem. 

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