ANALYTICAL BIOCHEMISTRY 76, 561-567 (1976)

Electrophoretic Separation and Quantitation of Creatine Kinase lsozymes

NORMAN HALL AND MARLENE DELUCA’

’ Depwtrnenf.s of’ Mrdicitre cud Chemistry. Clniversify of‘ Ctrlifim itr. Sun Diego. LN Jo//n, C~tlijiwnitr 92093

Received June 4. 1976: accepted August IO. 1976

A method is described for separating and quantitating the isozymes of creatine

kinase in various tissue extracts. We have tested the sensitivity and precision of

the method over a wide range of mixtures of isozymes. The quantitation is linear

in the range of O-5 mlU of CK analyzed, and the lower limit of quantitation

of an isozyme in a mixture is IO mIU/ml. For certain tissues the presence

of dithiothreitol is necessary to prevent inactivation of the brain-type isozyme.

Creatine Kinase (CK; adenosine-5’ triphosphate-creatine phospho- transferase; (EC 2.7.3.2.) catalyzes the reaction

creatine + ATP Mg+

& creatine phosphate + ADP.

The enzyme is widely distributed (1) and its physiological role is generally associated with the energy metabolism of contractile or transport systems. Several CK isozymes are known. the best characterized being commonly referred to as BB (CK-I, “brain”). MB (CK-2, “hybrid”), and MM (CK-3, “muscle”). These isozymes are all dimers. molecular weight about 82,000 daltons. The isozyme composition differs in various tissues and is also dependent upon the stage of development (2.3). Methods which have been used for quantitation of creatine kinase isozymes have either involved physical separation by electrophoresis (4-9) or column chromatography (lo-13), or have been based on the immunological, kinetic, or stability properties of the isozymes (14-16). Since the work in our laboratory involves developmental studies using several different animal models, we need a method for quantitating the isozymes which combines speed and convenience with sensitivity and precision.

Immunological methods require that the isozymes to be assayed first be purified for use as antigens. Methods based on kinetic or stability properties are not applicable to mixtures of more than two iso- zymes. Column separations require either the assay of many fractions from a gradient elution or the use of step gradients, which can give incomplete separations if great care is not taken in their design.

562 HALL AND DELUCA

Electrophoretic separation methods have been reported which employ starch. agar, or agarose gels (4-6) and cellulose acetate strips (7-9). The cellulose acetate strips are convenient since they are commercially available. After electrophoretic separation, quantitation is achieved either by elution of the portions of the strip or gel containing the different isozymes followed by assays (4,7.9) or by incubating the strips or gels with an overlay of the same support medtum containing the sub- strates and enzymes for the standard Rosalki assay (17).

creatine phosphate + ADP Mg’

& creatine + ATP. [II

ATP + glucose Mg++

+ G-6-P + ADP. hexokinase

c21

G-6-P

G-6-P + NADP dehydrogenase - 6-phosphogluconate + NADPH. [31

The NADPH produced is measured by fluorometric scanning (.5,6,8). This scanning method is more convenient than elution. and offers a further advantage in that the entire isozyme profile is recorded.

We report here the details of the method we have used to quantitate CK isozymes, employing cellulose acetate electrophoresis and fluorescent scanning. The experiments were designed to test the reliability of the method over a wide range of mixtures of isozymes in crude extracts of various tissues. The presence of a sulfhydryl reducing agent in the electrophoresis buffer was found to be essential to prevent inactiva- tion of some isozymes.

MATERIALS AND METHODS

Sources of enzymes. Leg muscle and brain tissue from adult mice and heart and pectoral muscle from chick embryos (19 days in OLY~) were homogenized in 5 vol of 0.05 M Tris-HCI. pH 7.4, I x IO-” M EDTA, I x lo-” M ,!3-mercaptoethanol, and diluted togive 1000 mIU/ml of creatine kinase activity, Human MM and MB isozymes partially purified by DEAE-Sephadex chromatography were generously supplied by Dr. Johannes Everse and were diluted with the same buffer to give 1000 mIU/ml.

Assuy.for total creatine kinme. Extracts were assayed by the Rosalki method (Eqs. 1. 2, and 3) using the Calbiochem CPK-Max-Pack reagent. which provides a reaction mix of the following composition: 40 mM creatine phosphate, 1.2 mM ADP, 14 mM glucose, 8.4 mM magnesium salt, 1.7 ItJim of hexokinase, 1.8 mM NADP, 1.7 IU/ml of glucose-6-phosphate dehydrogenase. 3.6 mM AMP (to inhibit adenylate kinase), and 0.05 M buffer at pH 6.5. The formation of NADPH was

CKEATINE KINASE ISOZYMES 563

followed at 340 nm during the first 3 min of the reaction at 30°C. The reaction is linear in the range of 10 to 3000 mlU/ml.

E/~~c~tt.opho~esi.s. The method is essentially that of Klein ef ~1. (8). A volume of tissue extract sufficient to give about 5-10 mIU of total activity was applied to Gelman Sepraphore III cellulose acetate strips (Gelman Instrument Co., Ann Arbor, Mich.) prepared in 0.06 M

Tris-barbital buffer. pH 8.8. supplemented where indicated with 1 mM dithiothreitol (DTT). Volumes between 5 and 40 ~1 were applied by hand, using a 50-~1 microsyringe (Hamilton Co.. Reno, Nev.). The samples were subjected to electrophoresis in a Gelman 51 IO1 chamber for I .5 hr for mouse extracts .2 hr for human, and 4 hr for chicken extracts. at 300 V, 4°C. The damp strips were placed in a plastic box and overlayed with cellulose acetate strips which had been soaked in 3x concentrated Calbiochem CPK Statpack fluid and blotted dry. (Sub- strate concentration: 54 mM creatine phosphate, 3.3 mM ADP. 43.5 mM glucose, 1.5 mM NADP, and 0.15 M buffer at pH 6.8.) Care was taken to ensure that excess buffer clinging to the strips did not reach the region to be stained. The two strips were pressed with a blotter to remove air bubbles, and the closed box was placed in a 37°C bath for I5 to 20 min. When increasing amounts of human enzymes in ~-PI volumes were placed on strips, stained. and scanned without electrophoresis, some departure from linearity was seen when IO-mIU samples were stained for 20 min. When most of the activity is expected to appear in a single band, a maximum of 5 mIU should be applied or the staining time should be reduced. After staining. the top strips were removed and discarded, and the stained strips were then dried on paper towels for IS to 20 min in a 54°C oven. This drying was found to enhance the fluorescence of the stained areas and to greatly slow the reoxidation of NADH, but overdrying must be avoided.

The dried strips were placed between glass slides in a special holder on the tic scanning door of a Turner III fluorometer (G. K. Turner Associates. Palo Alto, Calif.) equipped with a CS 7-83 primary filter and a CS-3-74 secondary filter. Output was recorded on a Photovolt Densicord 541 recorder with an automatic integrator (Photovolt Corp., New York). It is possible to analyze up to 40 samples per day in this way.

RESULTS AND DISCUSSION

In order to assess the reliability of the method in quantitating the isozymes present in mixtures of widely differing compositions. the tissue extracts and the partially purified human enzymes were all adjusted to 1000 ? IO mIU/ml. Mixtures were prepared of known amounts of MM and BB (or MB in the case of the human enzymes) with approximately IO, 20. 40, 60. 80. and 9Oc7r compositions from the two sources from each

564 HALL AND DELUCA

t -t a -t b - a

MOUSE CHICKEN HUMAN

I\;,*:.;:

a c

b b

321012 321012 321012 DISTANCE FROM ORIGIN Icml

FIG. 1. Representative scans of cellulose acetate strips stained for CK after electro- phoresis of tissue extracts. Seven microliter samples of extracts or mixtures containing 1000 mIU/ml were used. Mouse: Mixture of 90% brain extract (BB. peak a) and 10% skeletal muscle extract (MM. peak b). Chicken: Mixture of 90% heart extract (BB. peak a) and 10% skeletal muscle (MM, peak b). Human: Extract of human papillary muscle con- taining 14% MB (peak a). MM (peak b). and the mitochondrial isozyme (peak c).

of the three species tested. Electrophoresis was carried out as de- scribed with samples from each tissue extract and from each mixture. Representative scans of strips following electrophoresis of mixtures of CK isozymes from each species are shown in Fig. 1.

Figure 2 shows the observed compositions of the mixtures plotted against the known compositions in the cases of the mouse and chicken

+ 11’ ,‘: I

/I’ I’

-- 11’ Y : ;‘: / I I I.

ZIOO- c :

*If D I'. .I P'

," tlo- ,':

1

.P ,,:

Y - ", /' 5 60- .I' ;,'

:: /d I I 2 40- ,i'

I I' ,'

*I' . I'

20 - "'

~- I/ I, ,

I/* I I 1 I r' / I 1 I /

0 20 40 60 80 100 0 20 40 60 80 100 %BB

I I t / I J, 1 I I I,

100 80 60 40 20 0 IO0 80 60 40 20 0 %MM

RELATIVE COMPOSITIONS OF MIXTURES

FIG. 3. Quantitation of CK isozymes in mixtures of extracts of mouse tissues (A + B) and of chicken tissues (C + Dl. Dithiothreitol (I .O mrvr) was added to the buffer used in experiments B and D. but not in A and C. The different symbols in A indicate data from two independent experiements. In the other experiments, the points are from duplicate samples from one set of mixtures.

CREATINE KINASE ISOZYMES 565

TABLE I

DETECTABILITY OF MOUSE CPK ISOZYMES IN THE PRESENCE OF DTT

Relative amount

BB added (%)

Total activity (U/liter)

Measured percentage of

BB (mean) s

(n = 4)

1.0 1 .ooo 1.8 0.8 2.5 300 3.7 0.1 5.0 700 5.4 2.7

extracts. The results with the human enzymes were very similar to those with the chicken extracts and are not shown.

The most striking feature of these results is the extreme deviation from linearity in the case of the mouse mixtures and the correction of this deviation when dithiothreitol is present. The data from the two experiments shown in Fig. 2A indicate that approximately 65% of the catalytic activity of the BB isozyme is lost, relative to that of the other isozymes in the mixtures. This preferential loss of the BB isozyme was not prevented by adding DTT to the staining mixture, but was prevented by the addition of I mM DTT to the electrophoresis buffer. This is not surprising, since creatine kinase is known to contain sulfhydryl groups important for the catalytic activity (18). In the presence of DTT, the maximum deviation seen from the nominal values is ?7%, and most values are much closer.

To determine the lower limit at which the method can detect the presence of a minor isozyme, mixtures containing mouse brain extr;!ct at I, 2.5. and 5% and with 1000, 500, and 200 mIU/ml of total activity were subjected to electrophoresis in quadruplicate. The limiting cases, in which the standard deviations were small enough to allow the measured values to be unambiguously distinguished from zero, are shown in Table 1. From these results, it appears that 10 mIU/ml of an isozyme can be quantitated. When the strips were examined under ultraviolet light, as little as 5 mIU/ml could be detected qualitatively. Another series containing 100 mIU/ml of total activity and requiring 50 ~1 or more to be placed on the strips gave less than satisfactory results, with wide, overlapping bands. Still, even in this case, the pres- ence of 10 mIU/ml of BB from the brain extract could be detected qualitatively under ultraviolet light.

The results indicate that the method described can achieve good quantitation of the isozymes present in crude extracts. If greater sensitivity is necessary, the results reported by Roberts rr ul. (9). indicate that some improvement in precision is obtained by cutting out

566 HALL AND DELUCA

and eluting appropriate sections of the strips. They report a limit of detectability of 2 mlU/ml and a disparity from expected values of ?3%.

The column methods described by Yasmineh and Hansen (11) can detect the MB isozyme at 2 mlU/ml. but fail to resolve the mito- chondrial isozyme (Fig. 1 and Ref. 3). Yasmineh and Hansen reported that the electrophoretic method used by them, similar to the one described here, consistently overestimated the MB when it was present in small quantities. We have also observed this when larger amounts of the enzyme mix or when longer staining times than those we recom- mend here are used. We feel that when proper precautions are taken, the electrophoretic method best resolves and quantitates all of the CK isozymes present in tissue extracts.

The question of substrate concentrations deserves comment. We found essentially no difference in the activity of the mouse or chicken enzymes when the phosphocreatine concentration in the Maxpack assay was varied from 15 to 40 mM. However. optimal substrate concentrations are different for each of the isozymes of chicken and human (15,19). The BB isozymes have lower K,,,‘s for both ADP and phosphocreatine than do the MM isozymes. At higher substrate concentrations, the BB isozyme is inhibited while the MM is not. Therefore, it is not possible to assay a mixture of BB and MM using substrate concen- trations which will reflect V,,, for both enzymes. However, as long as the substrate concentrations are kept constant, the relative activities measured are comparable from sample to sample.

ACKNOWLEDGMENTS

This project was supported by National Institutes of Health Research Grant No. HL 17682-01 awarded by the National Heart and Lung Instiute.

REFERENCES

I. Dawson. D. M.. and Fine. I. H.( (1967)Arch. Nerrrol. 16, 175-180. 2. Ziter. F. A. (1974) El-p. Nrrrrol. 43, 539-546. 3. Hall. N.. and DeLuca, M. (1975) Bioclzr,,z. Biophys. Re,s. Conmun. 66, 988-993. 4. Murone. I.. and Ogata. K. (1973) J. Bbxl1en2. 74, 41-48. 5. Roe, C. R.. Limbird, L. E.. Wagner. G. S.. and Nerenberg. S. T. (1972) .I. Lab. Clin.

Med. 80, 577-590. 6. Somer. H.. and Konttinen. A. (1972) C/in. C/h. Ada 40, 133-13X. 7. Keutel, H. J.. Okabe, K.. Jacobs, H. K., Ziter. F.. Maland. L.. and Kuby. S. A. (1971)

,4rcl1. Biocllrm. Biopllys. 150, 648-678. 8. Klein, M. S.. Shell, W. E., and Sobel. B. E. (1973) Cnr&o~usc. Res. 6, 412-438. 9. Roberts. R.. Henry, P. D., Witteveen. S. A. G. J.. and Sobel. B. E. (1974) Amer.

J. Cotdt’ol. 44, 650-654. IO. Mercer. D. W.. and Varat. M. A. (1975) C/in. Clzrw. 21, 108X- 1092. I I. Yasimineh, W. G.. and Hanson. N. Q. (1975) C/il~. Chem. 21, 381-386.

CREATINE KINASE ISOZYMES 567

II. Takahashi. K.. Ushihubo. S.. Oimomi. H.. and Shinko. T. (1971) C/i/r. Chif?l. Ac,frr

38, X5-290. 13. Nealon. D. W.. and Henderson. R. A. t 197.5) Clip. C‘/I~VI. 21, 392-397. IJ. Jockers-Wretou, E.. and Pfleiderer. G. (197.5) Clirl. Cllir~. .4(.ftr 58, 223-232. IS. Witteveen. S. A. G. J.. Sobel. B. E.. and DeLuca. M. (1974) Prrw. ,Ytrr. AWL/.

Sc,i. USA 71, 13X3-1387. 16. Rae. P. S.. Lukes. J. J.. Ayres. S. M.. and Mueller. H. (1975) Clirl. C/ICJ;U. 21, l612-

161X. 17. Rosalki. S. B. (19671 J. LO/J. C/it!. !drd. 69, 696-705. IX. Watts. D. C.. and Rabin. B. R. (1967) Bio&c,rt~. ./. 85, 507-516. 19. Dawson. D. M.. Eppenberger. H. M.. and Kaplan. N. 0. t 1966) ./. Bbjl. C‘hc,rn.

tl2, ‘10-217.