ANA1
YTICAL.
64. 5 17-520
BIOCHEMISTRY
Affinity
Chromatography
An affinity chromatography described. Creatine kinase
from
p-mercuribenzoate-2-aminoethyl-Sepharose. proteins with Tris buffer and is selectively method yields
( 1975)
eluted in X0% a highly purified
of Creatine
technique human
a solution
for hkeietal
purification muscle
Kinase
of creatine kinase is retained on a column
After removal of p-mercuribenzoate.
is of
of contaminating the creatine kinase
yield with a gradient of 2-mercaptoethanol. protein with a specific activity of 300
This units/mg.
Selective adsorption to insoluble agarose, substituted with specific substrates, substrate analogs, or inhibitors, has proved to be an effective method for enzyme isolation and purification (1.2). Creatine kinase (CK; ATP:creatine phosphotransferase, EC 2.7.3.3)’ has in its “essential” sulfhydryl moieties (3-5) a functional group which might be utilized for affinity chromatography. Several molecular species which form covalent bonds with the sulfur atom are available for attachment onto agarose derivatives. We have found that human skeletal muscle CK. in solution with other proteins. is effectively bound to the organomercurial-agarose prepared by attachment of p-mercuribenzoate (PMB) to aminoethylSepharose. The active enzyme can be readily recovered from the substituted agarose by elution with I!-mercaptoethanol. The application of this method for removal of inactive protein species contaminating human skeletal muscle CK during purification is described. METHODS
(PMB-AE-SephaThe PMB derivative of 3 aminoethyl-Sepharose rose) was prepared as described by Cuatrecasas ( 1). One milliliter of the material, suspended in 0.05 M Tris-chloride, pH 7.5. and packed in a column 0.58 cm in diameter, contained about 1.5 pmoles of PMB, estimated by the uptake of [‘JC]cysteine from a solution of 0.001 M DLcysteine. ’ Abbreviations: CK, creatine p-mercuribenroate-2-aminoethyl-Sepharose.
kinase:
PMB.
p-mercuribenzoate:
PMB-AE-Sepharohe.
518
MADELIAN
AND
WARREN
Human skeletal muscle was obtained at autopsy and frozen at -30°C. Creatine kinase was partially purified by homogenization, ethanol fractionation, and ammonium sulfate precipitation, as previously described (6). Enzyme activity was measured by a coupled enzyme spectropho(7). tometric procedure in the presence of 0.03 M ?-mercaptoethanol One unit of CK is that amount which catalyzes transphosphorylation between creatine phosphate and ADP at a rate of 1 pmole/min at 25°C. The protein concentration of CK solutions was determined by absorbance at 280 nm, using a value for E; &, of 10. I (6). Protein electrophoresis on polyacrylamide gels was performed by the method of Davis (8). RESULTS AND
DISCUSSION
Prior to chromatography on PMB-AE-Sepharose, the ammonium fate precipitate was dissolved in and dialyzed against Tris buffer. dialyzed solution, which had a specific activity of I24 units/mg, tained at least five contaminating proteins which were separated CK by electrophoresis on polyacrylamide gels (Fig. 1. gel A). dialyzed solution was applied to the column of PMB-AE-Sepharose
sulThe confrom The
FIG. 1. Polyacrylamide gel electrophorebis of CK solutions. (A) I75 /*g of protein tion before affinity chromatography; (9) 40 fig of protein eluted from affinity column Tris (fraction 8); (Cl IO pg of protein eluted with Tris plus PM9 (fraction 39): (D) of Ck: elutrd with ?-mercaptoethanol (fraction 109).
soluwith 30 pg
AFFINITY
CHROMATOGRAPHY
OF
CREATINE
KINASE
519
FRACTION FIG.
partially toethanol
2.
Afiinity
chromatography
of impure
purified C‘K was disrolved was added to 0.01 M; and
in 0.05 the solution
c‘ti. M
The
ammonium
Tri\-chloride wa\ dialyzed
sulfate
buffer. pH at 4’-C against
precipitate
ol
7.4: 7-mercapthree change\
(2 liters each) of Tris buffer. After dialysis the insoluble mateI-ial has removed hy centrifugation at 2.000 ,g for 70 min. I-ive milliliters of the wluble fraction ( l-2 mg protein/m I) were applied to ;I column (0.5X :I X.0 cm) of PMB-AE-Sepharose at A’(‘. The column M;L\I eluted first with 7 i IO i M PMB (t’ractionh
0.05 M (fraction
il-1.
Tria
buffer 5 I -I 00).
m I of Tri\ Fi\c-minute
buffel-
containing and finally in mixel-: fractions
(I
and eluted as described in Fig . 2. In fractions I-100 the CK in the protein mixture was completely retained on the column, and the contaminating proteins were removed by elution with Tris and PMB (Fig. 1. gels B and C). Creatine kinase was eluted in a single protein-activity peak (fractions 100-l 35) by the 2-mercaptoethanol gradient and had a specific activity of 300 units/mg. The recovery of CK activity from the coumn was 80%. and the enzyme was highly purified (Fig. I. gel D). In an earlier report a method for isolating human CK by isoelectric focusing was described (6). Two closely related CK peaks were obtained. and the difference between these proteins was believed to be due to differences in amide content. The proteins were inseparable by gel electrophoresis. The CK isolated from the PMB-AE-Sepharose exhibited, after isoelectric focusing. the same pattern of distribution as the CK prepared by the earlier method Since CK has several sulfhydryl groups per molecule. it was important to ascertain whether PMB, used to remove contaminating proteins from the Sepharose column. was bound to the enzyme after elution with the ?-mercaptoethanol gradient. By using “C-labeled PMB in the wash solu-
520
MADELIAN
AND
WARREN
tion, it was demonstrated that the CK eluted from the column contained less than 0.3 mole of PMB per mole and that after subsequent dialysis it was completely devoid of PMB. This technique should prove useful for preparing CK from other tissues and species, as well as for studying the structural features of these enzymes. We are currently utilizing the technique to obtain sulfhydryl-containing peptides after proteolytic digestion of CK bound to PMB-AE-Sepharose. ACKNOWLEDGMENTS The authors are grateful to Mrs. Elizabeth Austin. Mrs. Diane Gould, and Mr. Bellantoni for their technical assistance and to the American Heart Association 7 l-635) for support of this project.
Joseph (Grant
REFERENCES I. Cuatrecasas. P. t 1970) J. Biol. c‘he!?~. 245, 3059-3065. 2. Cuatrecasas, P.. and Anfinsen. C. B. t I97 I ) A/III. RcI,. Biochem. 40, 259-278. 3. Mahowald. T. A., Noltmann, E. A.. and Kuby, S. A. (1962) J. Bid. Chrnz. 237, 1535-1548. 4. Kumudavalli. I.. Moreland. B. H., and Watts, D. C. t 1970) Bioc/wm. J. 117, 513-533. 5. Atherton, R. S.. Laws. J. F.. Miles. B. J.. and Thomson. A. R. t 1970) Biochem. J. 120. 5X9-600. 6. Warren. W. A. t 1973) Pwp. Bioclrc~w~. 3, 199-20X. 7. Warren, W. A. (1971) Clitl. C11em. 18. 473-475. 8. Davis, B. J. (1964) A~uI. N. Y. Acod. Sci. 121. 404-417.
YTICAL.
64. 5 17-520
BIOCHEMISTRY
Affinity
Chromatography
An affinity chromatography described. Creatine kinase
from
p-mercuribenzoate-2-aminoethyl-Sepharose. proteins with Tris buffer and is selectively method yields
( 1975)
eluted in X0% a highly purified
of Creatine
technique human
a solution
for hkeietal
purification muscle
Kinase
of creatine kinase is retained on a column
After removal of p-mercuribenzoate.
is of
of contaminating the creatine kinase
yield with a gradient of 2-mercaptoethanol. protein with a specific activity of 300
This units/mg.
Selective adsorption to insoluble agarose, substituted with specific substrates, substrate analogs, or inhibitors, has proved to be an effective method for enzyme isolation and purification (1.2). Creatine kinase (CK; ATP:creatine phosphotransferase, EC 2.7.3.3)’ has in its “essential” sulfhydryl moieties (3-5) a functional group which might be utilized for affinity chromatography. Several molecular species which form covalent bonds with the sulfur atom are available for attachment onto agarose derivatives. We have found that human skeletal muscle CK. in solution with other proteins. is effectively bound to the organomercurial-agarose prepared by attachment of p-mercuribenzoate (PMB) to aminoethylSepharose. The active enzyme can be readily recovered from the substituted agarose by elution with I!-mercaptoethanol. The application of this method for removal of inactive protein species contaminating human skeletal muscle CK during purification is described. METHODS
(PMB-AE-SephaThe PMB derivative of 3 aminoethyl-Sepharose rose) was prepared as described by Cuatrecasas ( 1). One milliliter of the material, suspended in 0.05 M Tris-chloride, pH 7.5. and packed in a column 0.58 cm in diameter, contained about 1.5 pmoles of PMB, estimated by the uptake of [‘JC]cysteine from a solution of 0.001 M DLcysteine. ’ Abbreviations: CK, creatine p-mercuribenroate-2-aminoethyl-Sepharose.
kinase:
PMB.
p-mercuribenzoate:
PMB-AE-Sepharohe.
518
MADELIAN
AND
WARREN
Human skeletal muscle was obtained at autopsy and frozen at -30°C. Creatine kinase was partially purified by homogenization, ethanol fractionation, and ammonium sulfate precipitation, as previously described (6). Enzyme activity was measured by a coupled enzyme spectropho(7). tometric procedure in the presence of 0.03 M ?-mercaptoethanol One unit of CK is that amount which catalyzes transphosphorylation between creatine phosphate and ADP at a rate of 1 pmole/min at 25°C. The protein concentration of CK solutions was determined by absorbance at 280 nm, using a value for E; &, of 10. I (6). Protein electrophoresis on polyacrylamide gels was performed by the method of Davis (8). RESULTS AND
DISCUSSION
Prior to chromatography on PMB-AE-Sepharose, the ammonium fate precipitate was dissolved in and dialyzed against Tris buffer. dialyzed solution, which had a specific activity of I24 units/mg, tained at least five contaminating proteins which were separated CK by electrophoresis on polyacrylamide gels (Fig. 1. gel A). dialyzed solution was applied to the column of PMB-AE-Sepharose
sulThe confrom The
FIG. 1. Polyacrylamide gel electrophorebis of CK solutions. (A) I75 /*g of protein tion before affinity chromatography; (9) 40 fig of protein eluted from affinity column Tris (fraction 8); (Cl IO pg of protein eluted with Tris plus PM9 (fraction 39): (D) of Ck: elutrd with ?-mercaptoethanol (fraction 109).
soluwith 30 pg
AFFINITY
CHROMATOGRAPHY
OF
CREATINE
KINASE
519
FRACTION FIG.
partially toethanol
2.
Afiinity
chromatography
of impure
purified C‘K was disrolved was added to 0.01 M; and
in 0.05 the solution
c‘ti. M
The
ammonium
Tri\-chloride wa\ dialyzed
sulfate
buffer. pH at 4’-C against
precipitate
ol
7.4: 7-mercapthree change\
(2 liters each) of Tris buffer. After dialysis the insoluble mateI-ial has removed hy centrifugation at 2.000 ,g for 70 min. I-ive milliliters of the wluble fraction ( l-2 mg protein/m I) were applied to ;I column (0.5X :I X.0 cm) of PMB-AE-Sepharose at A’(‘. The column M;L\I eluted first with 7 i IO i M PMB (t’ractionh
0.05 M (fraction
il-1.
Tria
buffer 5 I -I 00).
m I of Tri\ Fi\c-minute
buffel-
containing and finally in mixel-: fractions
(I
and eluted as described in Fig . 2. In fractions I-100 the CK in the protein mixture was completely retained on the column, and the contaminating proteins were removed by elution with Tris and PMB (Fig. 1. gels B and C). Creatine kinase was eluted in a single protein-activity peak (fractions 100-l 35) by the 2-mercaptoethanol gradient and had a specific activity of 300 units/mg. The recovery of CK activity from the coumn was 80%. and the enzyme was highly purified (Fig. I. gel D). In an earlier report a method for isolating human CK by isoelectric focusing was described (6). Two closely related CK peaks were obtained. and the difference between these proteins was believed to be due to differences in amide content. The proteins were inseparable by gel electrophoresis. The CK isolated from the PMB-AE-Sepharose exhibited, after isoelectric focusing. the same pattern of distribution as the CK prepared by the earlier method Since CK has several sulfhydryl groups per molecule. it was important to ascertain whether PMB, used to remove contaminating proteins from the Sepharose column. was bound to the enzyme after elution with the ?-mercaptoethanol gradient. By using “C-labeled PMB in the wash solu-
520
MADELIAN
AND
WARREN
tion, it was demonstrated that the CK eluted from the column contained less than 0.3 mole of PMB per mole and that after subsequent dialysis it was completely devoid of PMB. This technique should prove useful for preparing CK from other tissues and species, as well as for studying the structural features of these enzymes. We are currently utilizing the technique to obtain sulfhydryl-containing peptides after proteolytic digestion of CK bound to PMB-AE-Sepharose. ACKNOWLEDGMENTS The authors are grateful to Mrs. Elizabeth Austin. Mrs. Diane Gould, and Mr. Bellantoni for their technical assistance and to the American Heart Association 7 l-635) for support of this project.
Joseph (Grant
REFERENCES I. Cuatrecasas. P. t 1970) J. Biol. c‘he!?~. 245, 3059-3065. 2. Cuatrecasas, P.. and Anfinsen. C. B. t I97 I ) A/III. RcI,. Biochem. 40, 259-278. 3. Mahowald. T. A., Noltmann, E. A.. and Kuby, S. A. (1962) J. Bid. Chrnz. 237, 1535-1548. 4. Kumudavalli. I.. Moreland. B. H., and Watts, D. C. t 1970) Bioc/wm. J. 117, 513-533. 5. Atherton, R. S.. Laws. J. F.. Miles. B. J.. and Thomson. A. R. t 1970) Biochem. J. 120. 5X9-600. 6. Warren. W. A. t 1973) Pwp. Bioclrc~w~. 3, 199-20X. 7. Warren, W. A. (1971) Clitl. C11em. 18. 473-475. 8. Davis, B. J. (1964) A~uI. N. Y. Acod. Sci. 121. 404-417.
