A convenient method for isolation of murine erythrocyte catalase by gel filtration and DEAE cellulose chromatography

ANALYTICAL

60,

BIOCHEMISTRY

A Convenient

537-544

Method Catalase

(1974)

for

Isolation

by Gel

Cellulose

Filtration

of

Biology,

Received

DEAE

H. A. HOFFMAN

AND

Cancer

Institute,

12, 1973;

accepted

National December

and

Erythrocyte

Chromatog;raphy

C. K. GRIESHABER Laboratory

of Murine

Bethesda, March

Maryland

20014

6, 1974

A method for isolation of catalase from small volumes of murine red blood cells is described. The purified enzyme had a specific activity of 25,699 IU/mg protein and was judged to be homogeneous by polyacrylamide and sodium dodecyl sulphate electrophoresis. The specific activity of the isolated murine erythrocyte catalase is approximately 14-25% that of human erythrocyte catalase isolated by the method described.

The genetics of catalase (H,Oz: H,O, oxidoreductase: EC 1.11.1.6) expression in murine erythrocytes and liver has been reported in which activity of the enzyme was measured as the inherited trait (l-3). Widely differing levels of catalase activity in red blood cells of inbred strains of mice (4), illustrate the need for quantitative determination of catalase protein concentration. Isolation of murine erythrocyte catalase is essential as the first step in answering the question of whether the above disparity in enzyme activity observed between strains of mice is due to the presence of different concentrations of the enzyme or to different levels of enzymatic activity. Established procedures for isolation of catalase from mammalian erythrocytes require large volumes of blood as the source (5-7). To our knowledge, catalase has not been isolated from murine erythrocytes presumably due to insufficient red blood cell volumes as well as to the lability of the enzyme following lysis of the red blood cells. We have devised a method for the isolation of catalase in high yield from relatively small volumes of packed erythrocytes. Catalase from human red blood cells was also isolated by this method as a reference standard. MATERIALS

AND

METHODS

Erythrocytes from 20 to 45 inbred BALB/cHf mice were washed twice in cold phosphate buffered saline by centrifugation at 7509, and once at 20009. The packed red blood cell pellet (5-10 ml) was lysed by freezeCopyright All rights

@ 1974 by Academic Press, of reproduction in any form

537 Inc. reserved.

538

GRIESHABER

AND

HOFFMAN

thawing three times with 2 vol of 0.01 M phosphate buffer, pH 7.1 containing 0.5 M ethanol (PBE) and centrifuged at 10,OOOg for 20 min. The lysate supernatant solution was saved, the stroma washed with an additional 2 vol of PBE, and the supernatant solution combined with the first resulting in a 1: 4 lysate dilution with respect to packed RBCs. The lysate was pipetted onto a Sephadex G-100 column (K 25JlOO; Pharmacia Fine Chemicals) equilibrated with PBE at room temperature. Fractions were eluted with PBE and assayed for catalase activity. Catalase active fractions from the G-100 column were pooled and pumped onto a precycled (8) DEAE cellulose column (Whatman DE-52) equilibrated with PBE at room temperature. Fractions were eluted consecutively with 0, 0.017, 0.05, 0.125, and 0.5 M NaCl steps in PBE, assayed for catalase activity and absorbance at 540, 405, 280, and 260 nm measured. Fractions containing catalase activity were pooled and concentrated on a UM-10 membrane (Amicon Corporation) under nitrogen. Catalase activity was measured spectrophotometrically (Cary Spectrosystem 100) at 230 nm (9) and International Units (IU) calculated from the first order reaction rate; 1 IU is that amount of enzyme capable of consuming 1 ,umole H,OJmin. Protein content was estimated by absorbance ratios at 280/260 nm (10). Polyacrylamide gel electrophoresis was carried out for 60 min at 400 V in 7% gels with Tris-citrate (0.074 M Tris, 0.005 M citric acid), pH 8.65 gel buffer and sodium borate (0.03 M boric acid, 0.009 M NaOH) , pH 8.65 electrode buffer. Proteins were stained with Coomassie Blue, catalase activity was negatively stained by the method of Woodbury et al. (11). Sodium dodecyl sulfate gel electrophoresis was carried out after reduction of samples with mercaptoethanol by the method of Shapiro et al. (12). Catalase (Peak B, Fig. 1) from DEAE cellulose and G-100 catalase fraction was injected into individual rabbits (New Zealand White) for production of anti-catalase serum. Immunoelectrophoresis was carried out in 1% agarose buffered with Tris-EDTA borate (0.09 M Tris, 0.0025 M EDTA, 0.009 M boric acid), pH 9.1 for 60 min at 40 V/cm with sodium-borate (0.075 M boric acid, 0.0125 M NaOH), pH 8.65 in the electrode chambers. RESULTS

AND DISCUSSION

Gel filtration through Sephadex G-100 removed 90-95% of the hemoglobin from the catalase active fraction which appeared in the excluded volume (100 ml). The elution volume of hemoglobin should be greater than that of catalase since the molecular size of hemoglobin is approximately one-fourth that of catalase. Molecular aggregation of hemo-

ISOLATION

OF

MURINE

ERYTHROCYTE

539

CATALASE

globin through sulfhydryl oxidation between adjacent P-chains has been demonstrated in BALB/c RBC lysates (13j which precludes complete separation of catalase and hemoglobin on Sephadex G-100. The specific activity of the pooled catalase after gel filtration was 800-1100 IU/mg protein which is 20- to 30-fold greater than in the RBC lysate (Table I), Three major peaks of catalase activity were eluted from DEAE cellulose by 0.017, 0.05, and 0.125 M NaCl, respectively (Fig. l), with peak B containing most of the enzyme activity. Since these peaks correspond well with those reported by others for human erythrocytes catalase following elution from anion exchange columns by increasing buffer molarity (7), we have designated them A, B, and C with respect to increasing negative charge. The first protein peak from the DEAE cellulose column contained hemoglobin, all others were hemoglobin free. The absorption ratio 405/280 nm for fractions A and B were 0.9 to 1.3 with negligible absorbance at 540 nm indicating a pure preparation of catalase. A linear NaCl gradient (O-O.5 M) gave similar results except that catalase was eluted as one broad peak. This result suggests that the three peaks of catalase activity eluted by the above step gradient may not represent three distinct catalase proteins but are likely artifacts of abrupt changes in ionic strength. However, the second peak (Peak B, Fig. I) of catalase eluted by 0.05 M NaCl is a purer preparation than can be isolated by a linear gradient; thus, the stepwise elution scheme was judged to be a more desirable procedure for obtaining catalase protein of antigenie purity. The highest specific activity obtained was 25,000 IU/mg protein from peak B, corresponding to a Ic,’ of 1.7 X lo7 ~-l set-l (14) which, although low with respect to RBC catalase from other mammalian species, is not significantly less than that from a value of 2.8 X lo7 M-I set-l reported for murine liver catalase (3). The k,’ of 6.8 X lo7 M-I set-l for human erythrocyte catalase isolated according to the above conditions is con-

Comparison

TABLE 1 Activity, Purification, and Yield BALB/c and Human RBC Catalase

of Specific

Between Sp act (JU/mg

X 18)

Purification

Factor

Fraction

BALB/c”

Human

BALB/c*

Human

RBC lysate G-100 pool Peak Ba

0.04-0.05 0.8-1.1 15-25

0.11-0.12 3.04.0 118-122

23-25 425550

25-28 1000-1200

a Concentrated peak B from DEAE-cellulose. b Range of values from three isolations. o Range of values from two isolations.

of Enzyme

r. Yield BALBIcb

Human

100 S&90 3@40

100 90-95 75-80

540

GRIESHABER

AND

HOFFMAN

__, .-.-...,... ..t.....

0.8 ._.....

:

I

2--10.*

F0.7 P , 0.6

E

2 .o x

205 if P P 0.4

n III :’’ I: /: ’1 i\

0.3

-22

5 *

7 -5.4 , -3.6

0.2

-1.8

0.1 L I r

’ ii

2'0

$0

4'0

FRACTION

5'0

6'0

\

‘\/-,

7'0

. 8d

'!$O

NUMBER

FIG. 1. DEAE cellulose chromatogram of pooled catalase fractions from Sephadex G-100. Elution gradient consisted of 0, 0.017, 0.05, 0.125, and 0.5 M NaCl steps in 0.01 M phosphate buffer, pH 7.1, containing 0.5 M ethanol. Fraction volume = 4.0 ml.

with previously reported values (7,14). It is noteworthy that the presence of ethanol in the column buffers is essential for maximum recovery of catalase activity. Initial attempts at isolation of murine red blood cell catalase resulted in substantial lossesof enzyme activity with low yields; however, it was noted that rodent liver catalase had been purified using high ethanol concentrations in the homogenization buffers (3,9). Upon addition of ethanol (0.05-1.0 M) to the column buffers we observed the high yield and recovery reported here. The optimum ethanol concentration was determined to be 0.5 M. A comparison between catalase from BALB/c and human erythrocytes with respect to specific activity, purification factor, and percent yield is presented in Table 1. The specific activity of human erythrocyte catalase from the lysate and G-100 catalase fractions is 2.5- to 3.5-fold greater than the murine enzyme with essentially the same purification and yield. However, the specific activity of the peak B human enzyme is approximately seven-fold greater than the analogous murine fraction. The large difference in yield in catalase activity between peak B from human and

sistent

1SOLATION

OF

MURINE

ERYTHROCYTE

CATALASE

541

murine RBC lysates is most likely due to differencse in binding characteristics of the two enzymes, since peaks A (10%) and C (20% j from the murine lysate contain a greater portion of the total enzyme activity than those from the human lysate where peaks A and C contain less than 5% of the original activity. These results demonstrate that the most active catalase fraction (peak Bj from human red cells has a greater absolute specific activity than that from murine erythrocytes, although, more rapid deterioration of the murine enzyme during fractionation could account for part of the difference in specific activities. Polyacrylamide gel electrophoresis (Fig. 2.1 shows that peaks A and B from DEAE cellulose are homogeneous with one protein band corresponding to the region specifically stained for catalase activity, fraction C appears to have an additional diffuse component of faster anodic mobility. The G-100 catalase fraction has several other protein bands which are eluted from DEAE cellulose after the catalase peaks by 0.5 M NaCl (Fraction D, Fig. 1). It is noteworthy that fraction D has a protein of similar mobility to catalase, yet a specific staining reaction

FIG. 2. Polyacrylamide gel electrophoresis of Sephadex G-100 azd DEAE cellulose isolated fractions. G = pooled G-100 catalase fraction; A-D = DE.4E cellulose fractions (see Fig. 1); a = stained for protein; h = stained for catalase.

542

GRIESHABER

FIQ. 3

AND

HOFFMAN

FIG. 4

FIG. 3. Sodium dodecyl sulfate gel electrophoresis. BLC = bovine liver catalase; G = pooled G-100 catalase fraction; B, D = DEAE cellulosefractions (seeFig. 1).

Proteins stainedwith CoomassieBlue. FIG. 4. Immunoelectrophoresis of red blood cell lysate and isolated fractions against rabbit anti-G-100 catalase fraction (RAS 26) and anti-peak B (RAS 32). Ly = red blood cell lysate; G = pooled G-100 catalasefraction; B, D = DEAE cellulose fractions (see Fig. 1).

for catalase activity was not observed in this region. In addition, no enzyme activity was detected spectrophotometrically in this fraction. Electrophoresis in SDS gels was carried out on the G-100 catalase fraction and peaks B and D (Fig. 3). Bovine liver catalase (BLC: Sigma, St. Louis) was used as a reference. The single stained band of the same mobility in the BLC and peak B gels indicate that the murine red cell catalase eluted in peak B is homogeneous and has subunits similar in molecular size to those of highly purified BLC. The G-100 eluate and peak D appear to contain several stained regions of similar mobility including one in the C-100 eluate which had the same mobility as BLC and peak B. The intensity of the stained region in peak D is greatly reduced compared to the other fractions.

ISOLATION

OF

MURINE

ERYTHROCYTE

543

CATALASE

The results of immunoelectrophoresis of red blood cell lysate and isolated fractions against rabbit anti-G-100 catalase fraction and peak B are presented in Fig. 4. Rabbit anti-catalase (Peak B) serum (RAS 32) was reactive to a single component in the red blood cell lysate, G-100 catalase fraction and peak B, as well as in peaks A and C. The single precipitation arc in peak B coincided with catalase activity as determined by the presence of oxygen bubbles after flooding a coelectrophoresed plate with H,Oz. Precipit,ation arcs in comparable regions in red blood cell lysate, G-100 fraction and peaks A and C also exhibited a catalactic reaction when flooded with H,O,. Rabbit anti-G-100 catalase fraction (RAS 26) was reactive to active catalase as well as to a faster migrating region in the lysate, G-100 fraction and fraction D (Fig. 4). Rabbit anti-peak B catalase (RAS 32) was specifically reactive to enzymatically active catalase protein. Since double diffusion of RAS 26 and RAS 32 with RBC lysate and peak B catalase showed a pattern of nonidentity between peak B and peak D proteins, these regions represent either unrelated proteins or inactivated or precursor forms of catalase which are antigenically dissimilar to active catalase. This same region (peak D) is also present in human RBC preparations and can be eluted from DEAE cellulose by 0.5 M NaCl. The 405/280 nm absorption ratios, polyacrylamide and SDS electrophoresis indicate that the material in peak B has been purified to homogeneity. This conclusion is substantiated by the above immunoelectrophoretic studies. Direct measurements of enzyme concentration and specific activity in red blood cells of several strains of mice are currently being investigated using the rabbit anti-murine red blood cell catalase serum as an immunochemical reagent as well as the isolation method outlined here. ACKNOWLEDGMENTS We expert

thank W. C. Miller, technical assistance.

W. D.

Levillain,

B. H.

Clipper,

and

J. Dorsey

for

their

REFERENCES 1. FEINSTEIN, R. N., HOWARD, J. B., BXAUN, J. T., AND SEAHOLM, J. E. (1966) Genetics 53, 923-933. 2. HESTON, W. E., HOFFMAN, H. A., AND ~~ECHCIGL, M., Ja. (1965) Genet. Res. 6, 387-397. 3. GANSCHOW, R. E. AND SCHIMKE, R. T. (1969) J. Viol. Chem. 244, 46494658. 4. HOFFMAN, H. A. AND RECHCIGL, M., JR. (1971) Enzyme 12, 219-225. 5. STANSELL, M. J. AND DEUTSCH, H. F. (1965) J. Viol. Chem. 240, 4299-4305. 6. HIGASHI, T., SHIBATA, Y., YAGI, M., AND HIRAI, H. (1966) J. Biochem. 59, 115-121.

544

GRIESHABER

AND

HOFFMAN

7. MORIKOFER-ZWEZ, S., CANTZ, M., KAUFMANN, H., VON WARTBURG, J. P., AND AEBI, H. (1969) Eur. J. Biochem. 11, 49-57. 8. HIMMELHOCH, S. R. AND PETERSON, E. A. (1966) Anal. Biochem. 17, 383-389. 9. PRICE, V. E., STERLING, W. R., TARANTOLA, V. A., HARTLEY, R. W., JR., AND RECHCIGL, M., Jr., (1962) J. Biol. Chem. 237, 3468-3475. 10. LAYNE, E. (19571, in Methods Enzymol., (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3 pp. 447454, Academic Press, New York. 11. WOODBURY, W., SPENCER, A. K., AND STAHMANN, M. A. (1971) Anal. Biochem. 44, 301-305. 12. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V. (1967) Biochem. Biophys. Res. Commun. 28, 815-820. 13. RIGGS, A. (1965) Science 147, 621-622. 14. CHANCE, B. AND MAEHLY, A. C. (1955), in Methods Enzymol. (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 2, pp. 764-776, Academic Press, New York.