442
EPIMERASES AND ISOMERASES
[94]
and muscle is inhibited by sulfhydryl and alkylating agents and by photooxidation. The following group-selective reagents inhibit the liver and muscle enzyme: p-chloromercuribenzoate, 5,Y-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, o-iodosobenzoate, p-benzoquinone, iodoacetamide, iodoacetate, and 2,4-dinitrofluorobenzene. The liver enzyme contains, as the muscle enzyme, 10 free sulfhydryl groups titratable with 5,5'-dithiobis(2-nitrobenzoic acid). At least one of these groups is essential for the enzyme activity. 1,3
[94] Triosephosphate Isomerase from Human Erythrocytes 1 B y ROBERT W. GRACY D-Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate
Assay Method Principle. Triosephosphate isomerase activity can be measured in either direction by coupling to the appropriate dehydrogenase and following the rate of oxidation or reduction of N A D H or NAD at 340 nm. Glyceraldehyde 3-Phosphate as Substrate. ~,3 The assay mixture contains in a final volume of 1.0 ml, 50 m M triethanolamine buffer, pH 7.6, 1 m M E D T A , 0.15 m M N A D H , 1.5 m M glyceraldehyde 3-phosphate and one unit of crystalline a-glycerophosphate dehydrogenase (EC 1.1.1.8). The reaction mixture is added to a 1-cm light path quartz cuvette and preincubated for 5 min in the sample chamber of a recording spectrophotometer which is thermostatically maintained at 30.0 °. The reaction is initiated by the addition of 25 td of enzyme sample which has been previously prediluted (in 50 m M triethanolamine buffer, pH 7.6) such that the rate of decrease in absorbance at 340 nm is 0.01-0.2 per minute. After correcting for the predilution and the dilution of the enzyme in the assay cuvette, the absorbance change per minute is divided by 6.22 ~D-Glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1. The work described here was supported in part by grants from the U.S. Public Health Service (AM14638), the Robert A. Welch Foundation (B-502), a Career Development Award from the National Institutes of Health (AM70198), and North Texas State University Faculty Research. 2G. Beisenherz, this series, Vol. 1, p. 387. 3E. E. Rozacky, T. H. Sawyer, R. A. Barton, and R. W. Gracy, Arch. Biochem. Biophys. 146, 312 (1971).
[94]
HUMAN TRIOSEPHOSPHATE ISOMERASE
443
(the mM absorbance index for NADH) to give micromoles of dihydroxyacetone phosphate formed per minute per milliliter of enzyme solution. Dihydroxyacetone Phosphate as Substrate2 ,4 The reaction can be monitored in the other direction by coupling to D-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) and following the reduction of NAD. The assay mixture in a final volume of 1.0 ml contains 50 mM triethanolamine buffer, pH 7.6, 1 mM EDTA, 5 mM dihydroxyacetone phosphate, 0.3 mM NAD, 13 mM sodium arsenate, and 1 unit of crystalline glyceraldehyde 3-phosphate dehydrogenase. The solution is brought to 30 °, and the reaction is initiated with prediluted triosephosphate isomerase. The rate of increase in absorbance at 340 nm is recorded, and calculations as abow: yield the mieromoles of D-glyceraldehyde 3-phosphate formed per minute per milliliter of enzyme solution. Arsenate, a necessary component of the above assay, is a competitive inhibitor of triosephosphate isomerase, and corrections for this inhibition must be taken into consideration. This factor plus the higher cost of dihydroxyacetone phosphate make the former assay using glyceraldehyde 3-phosphate preferable for routine studies. In either of the above assays one unit of enzyme is defined as the amount catalyzing the isomerization of 1 ~mole of substrate per minute, and specific activity is defined as units per milligram of protein. Preparation o] Substrates and Reagents. The two coupling enzymes are both commercially available as crystalline suspensions essentially free of triosephosphate isomerase activity. However, since triosephosphate isomerase is inhibited by ammonium sulfate 5 the coupling enzymes are dialyzed prior to use. Thus, a stable baseline and a linear initial velocity are recorded in either assay. The substrates are also available in highly purified form as their acetal or ketal derivatives, which must be hydrolyzed to the free aldehyde or ketone. For Db-glyceraldehyde 3-phosphate (diethylacetal), 100 mg of the barium salt is dissolved in 6.0 ml of water, containing 1.5 g of Dowex 50-X4-200R H-form. The mixture is heated in a boiling water bath for exactly 3 min, followed by cooling in ice and removal of the Dowex by filtration. The solution is adjusted to pH 6.8 with NaOH and decolorized with activated charcoal. The other substrate, dihydroxyacetone phosphate (dimethylketal cyclohexylammonium salt), is hydrolyzed by dissolving 100 mg in 8.0 ml of water and adding 2.0 g of Dowex as above. After stirring for 1 min the Dowex is removed by filtration, and the solution is hydrolyzed at 40 ° for 6 hr. After neutralization and decolorization, the solutions are stored at 0-2 °. It should be 4 p. K. Chiang, Lile Sci. 10, 831 (1971). ' D. H. Turner, E. S. Blanch, M. Gibbs, and J. F. Turner, Plant Physiol. 40, 1146
(1965).
444
EPIMERASES AND ISOMERASES
[94]
pointed out that the lability of the free triosephosphates necessitates that these solutions be prepared fresh weekly and that the concentrations of triosephosphates be determined daily by the coupled assays described above. Protein Determination. Protein concentrations at various stages of the isolation are determined by the method of Lowry et al. 6 The concentration of the pure protein can be estimated from its absorbance at 280 nm using an absorbance index, elc,~,l~of 12.9. 3 Isolation Procedure Fraction I. One unit (450 ml) of human blood is collected into 67.5 ml of standard anticoagulant (acid-citrate-dextrose solution). All subsequent steps are carried out in an ice bath or in a cold room at 0-4 °. The plasma and bully coat are removed after centrifugafion at 8000 g for 60 rain, and the erythrocytes are washed by resuspension in 500 ml of cold 0.145 M NaC1 followed by centrifugation as above. After three washings, the erythrocytes are suspended in cold deionized water, and frozen and thawed several times; the lysate is centrifuged at 8000 g for 90 rain to remove the cell debris. The pH of the supernatant solution is adjusted to 7.0 by the addition of cold 1 M sodium phosphate buffer. Fraction II. Phosphocellulose (0.91 meq/g) which has been previously cleaned and equilibrated ~ in 5 mM sodium phosphate buffer, pH 7.0, is packed into a 20-cm diameter Biichner funnel to a height of 5 era. The hemolysate is applied to the ion exchange cellulose and is eluted with 5 mM phosphate buffer, pH 7.0, under mild vacuum. Fractions of approximately 200 ml each are collected and analyzed for triosephosphate isomerase activity. Under these conditions triosephosphate isomerase passes through the ion exchanger while most of the other proteins, including hemoglobin, are bound to the cellulose. Fractions containing triosephosphate isomerase activity are pooled and simultaneously concentrated and dialyzed against 5 mM triethanolamine buffer, pH 8.0, by ultrafiltration. Fraction III. The dialyzed fraction II is applied to a 3 X 75-cm column of DEAE-cellulose (0.91 meq/g) which has been previously cleaned and equilibrated in 5 mM triethanolamine buffer, pH 8.5. After washing with approximately 600 ml of this same buffer, a linear salt gradient is applied to elute the enzyme. The gradient mixing chamber contains 1 liter of 5 mM triethanolamine buffer, pH 8.5, and is connected by a siphon to the reservoir containing 1 liter of the same buffer which O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). E. A. Peterson and H. A. Sober, this series, Vol. 5, p. 3
[94l
HUMAN TRIOSEPHOSPHATE ISOMERASE
445
is also 0.4 M in NaC1. Under these conditions triosephosphate isomerase is eluted at approximately 0.15 M NaC1. Fractions containing triosephosphate isomerase activity are collected, concentrated and dialyzed against 5 mM triethanolamine buffer, pH 8.0 by ultrafiltration. Fraction IV. The dialyzed fraction III is added to a 2.1 X 22-cm column of DEAE-Sephadex which has been equilibrated with 5 mM triethanolamine buffer, pH 8.0. After washing with 300 ml of the same buffer, a shallow salt gradient is initiated (500 ml of 5 mM triethanolamine buffer, pH 8.0, in the mixing chamber, and 500 ml of the same buffer containing 0.1 M NaC1 in the mixing chamber). The enzyme is eluted at a flow rate of 30-40 ml per hr and the triosephosphate isomerase activity is found to be coincident with the third protein peak. The appropriate fractions are pooled and concentrated by ultrafiltration. Fraction V. The enzyme (5-10 mg/ml) is crystallized by dialyzing against a 0.50 saturated solution of ammonium sulfate containing 50 mM triethanolamine buffer, pH 7.8, 1 mM EDTA, and 1 mM 2-mercaptoethanol. A small amount of amorphous precipitate may be formed and should be removed by centrifugation. The supernatant solution is then returned to the dialysis bag, and the ammonium sulfate concentration of the dialysate slowly increased to 0.76 saturation where crystallization begins. After 48 hr crystals are collected by centrifugation and resuspended in 50 mM triethanolamine buffer, pH 7.8 containing 1 mM EDTA and 10 mM 2-mercaptoethanol. Specific activities of approximately 10,000 ~moles of glyceraldehyde 3-phosphate isomerized per minute per milligram of protein are routinely obtained. The isolation represents an overall purification of 4000-5000fold with 40-60% recovery. Additional recrystallizations, or rechromatography on DEAE-cellulose, phosphocellulose, or Sephadex do not result in further increases in the specific activity. A typical isolation is presented in the table. Resolution o] the Three Forms. Crystalline human triosephosphate isomerase can be resolved into three forms by electrophoresis or isoelectric focusing2 ,~ For preparative purposes the enzyme is electrofocused in 1% narrow range (pH 5-7) Ampholines for 92 hr at 600 V. Under these conditions a component (I) comprising approximately 5-1Q% of the total triosephosphate isomerase activity is found with an apparent isoelectric pH of 6.3. A second component (II) comprising 70-75% of the total activity electrofocuses at pH 6.0, and a third component (III) is observed at pH 5.6, which accounts for 20-25% of the isomerase activity. T. H. Sawyer, B. E. Tilley, and R. W. Gracy, J. Biol. Chem. 247, 6499 (1972).
446
[94]
EPIMERASES AND ISOMERASES
ISOLATION OF TRIOSEPHOSPHATE ISOMERASE FROM HUMAN ERYTHROCYTES
Fraction
Total activity (units)
Total protein (mg)
Specific activity (units/mg protein)
Hemolysate Phosphocellulose First DEAE Second DEAE Crystals
141,700 99,900 94,500 91,800 73,700
59,000 3,000 295 9.2 7.2
2.4 33.3 320 9,978 10,236
Purification Recovery (fold) (%) (1) 13.9 133 4158 4265
(100) 70 67 65 52
Other Tissues. Triosephosphate isomerase has also been isolated by the above method from human skeletal and cardiac muscle. In contrast to the purification from erythrocytes, the enzyme is not homogeneous after the second chromatographic step on DEAE-cellulose and is contaminated with a protein of large molecular weight. However, this contaminant can be easily removed by gel filtration on a 2.5 X 98 cm column of Sephadex G-100. Since muscle tissue is a much better source of the enzyme it is preferred. However, as is the case with most human enzyme studies, one is often limited to erythrocytes as a tissue source. The three electrophoretic forms of human triosephosphate isomerase found in the erythrocytes are found in essentially the same proportions in muscle tissues. 9 Physical Properties. Although the basis for the three electrophoretic forms is at this time still uncertain, a variety of studies 8 are consistent with an AA, AB, and BB distribution of dimers. The two types of subunits possess similar but distinguishable amino acid compositions and tryptic fingerprints. The three electrophoretic forms of human triosephosphate isomerase yield essentially identical molecular weights (56,000 -- 3000), sedimentation coefficients (s~o. . . . 4.1 X l0 -13 sec) and subunit molecular weights (28,000_+ 2000) when subjected to analytical ultracentrifugation. The enzyme is completely dissociated in 2.0 M guanidinium chloride, and can be totally renatured into catalytically active enzyme by removal of the dissociating agent. Catalytic Properties. Normal Michaelis-Menten kinetics are observed in both forward and reverse assays, and Km values for glyceraldehyde 3-phosphate of 0.34, 0.43, and 0.55 m M are obtained for components I, II, and III, respectively. Apparent Km values for dihydroxyacetone phosphate of 1.5, 0.82, and 0.69 m M are found for components I, II, and III, 9R. M. Snapka, T. H. Sawyer, and R. W. Gracy, unpublished experiments.
[9S]
TRIOSEPHOSPHATE ISOMERASE FROM RABBIT MUSCLE
447
respectively. These values are for the total triose phosphates in solution and do not take into consideration the relative distribution of hydrated gem diols or enediols which are in equilibrium with the free aldehyde and ketone. 1°,11 The enzyme exhibits a broad p H optimum between 7.0 and 9.5 and is competitively inhibited 12 by phosphoglycolate (Ki = 0.96 ~M), arsenate (K~ = 8.15 m M ) , a-glycerophosphate (Ki = 0.42 raM), and A T P (Ki = 16 raM). Stability. H u m a n triosephosphate isomerase is stable when stored in 0.80 saturated a m m o n i u m sulfate at 0-4% I t is recommended t h a t the solution also contain 20 m M triethanolamine buffer, p H 7.5, 1 m M E D T A , and 1 m M dithiothreitol. The Active Center. H u m a n triosephosphate isomerase is rapidly inactivated by the substrate analog, chloroacetol phosphate, by the selective esterification of a single essential glutamyl ,/-carboxylate per catalytic subunit. 13 Peptide maps and autoradiograms suggest the active-site peptide to be identical with the hexapeptide Ala-Tyr-Glu-Pro-Val-Trp isolated from rabbit14,15 and chicken TM muscle and y e a s t J 7 10D. R. Trentham, C. H. McMurray, and C. I. Pogson, Biochem. J. 114, 19 (1969), "S. J. Reynolds, D. W. Yates, and C. I. Pogson, Biochem. J. 122, 285 (1971). ~'~K~ values are given for the unresolved mixture of components I-III. ~'~F. C. Hartman and R. W. Gracy, Biochem. Biophys. Res. Commu~. 52, 388 (1973). ~4F. C. Hartman, Biochemistry 10, 146 (1971). ~sj. C. Miller and S. G. Waley, Biochem. J. 123, 163 (1971). ~GS. DeLaMare, A. F. W. Coulson, J. R. Knowles, J. D. Priddle, and R. E. Offord. Biochem. J. 129, 321 (1972). ~ I. L. Norton, and F. C. Hartman, Biochemistry 11, 4435 (1972).
[95] Triosephosphate Isomerase from Rabbit Muscle 1,2 B y FRED C. HARTMAN a n d I. LUCILE NORTON
D-Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate
Assay Method Principle. Triosephosphate isomerase activity is most conveniently measured in the direction of formation of dihydroxyacetone phosphate by coupling with a-glycerophosphate dehydrogenase and monitoring the oxidation of N A D H at 340 n m 2 Alternatively, the enzyme can be assayed
1 Research from the authors' laboratory was sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. 2D-Glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1. 3G. Beisenherz, this series, Vol. 1 [57].
EPIMERASES AND ISOMERASES
[94]
and muscle is inhibited by sulfhydryl and alkylating agents and by photooxidation. The following group-selective reagents inhibit the liver and muscle enzyme: p-chloromercuribenzoate, 5,Y-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, o-iodosobenzoate, p-benzoquinone, iodoacetamide, iodoacetate, and 2,4-dinitrofluorobenzene. The liver enzyme contains, as the muscle enzyme, 10 free sulfhydryl groups titratable with 5,5'-dithiobis(2-nitrobenzoic acid). At least one of these groups is essential for the enzyme activity. 1,3
[94] Triosephosphate Isomerase from Human Erythrocytes 1 B y ROBERT W. GRACY D-Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate
Assay Method Principle. Triosephosphate isomerase activity can be measured in either direction by coupling to the appropriate dehydrogenase and following the rate of oxidation or reduction of N A D H or NAD at 340 nm. Glyceraldehyde 3-Phosphate as Substrate. ~,3 The assay mixture contains in a final volume of 1.0 ml, 50 m M triethanolamine buffer, pH 7.6, 1 m M E D T A , 0.15 m M N A D H , 1.5 m M glyceraldehyde 3-phosphate and one unit of crystalline a-glycerophosphate dehydrogenase (EC 1.1.1.8). The reaction mixture is added to a 1-cm light path quartz cuvette and preincubated for 5 min in the sample chamber of a recording spectrophotometer which is thermostatically maintained at 30.0 °. The reaction is initiated by the addition of 25 td of enzyme sample which has been previously prediluted (in 50 m M triethanolamine buffer, pH 7.6) such that the rate of decrease in absorbance at 340 nm is 0.01-0.2 per minute. After correcting for the predilution and the dilution of the enzyme in the assay cuvette, the absorbance change per minute is divided by 6.22 ~D-Glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1. The work described here was supported in part by grants from the U.S. Public Health Service (AM14638), the Robert A. Welch Foundation (B-502), a Career Development Award from the National Institutes of Health (AM70198), and North Texas State University Faculty Research. 2G. Beisenherz, this series, Vol. 1, p. 387. 3E. E. Rozacky, T. H. Sawyer, R. A. Barton, and R. W. Gracy, Arch. Biochem. Biophys. 146, 312 (1971).
[94]
HUMAN TRIOSEPHOSPHATE ISOMERASE
443
(the mM absorbance index for NADH) to give micromoles of dihydroxyacetone phosphate formed per minute per milliliter of enzyme solution. Dihydroxyacetone Phosphate as Substrate2 ,4 The reaction can be monitored in the other direction by coupling to D-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) and following the reduction of NAD. The assay mixture in a final volume of 1.0 ml contains 50 mM triethanolamine buffer, pH 7.6, 1 mM EDTA, 5 mM dihydroxyacetone phosphate, 0.3 mM NAD, 13 mM sodium arsenate, and 1 unit of crystalline glyceraldehyde 3-phosphate dehydrogenase. The solution is brought to 30 °, and the reaction is initiated with prediluted triosephosphate isomerase. The rate of increase in absorbance at 340 nm is recorded, and calculations as abow: yield the mieromoles of D-glyceraldehyde 3-phosphate formed per minute per milliliter of enzyme solution. Arsenate, a necessary component of the above assay, is a competitive inhibitor of triosephosphate isomerase, and corrections for this inhibition must be taken into consideration. This factor plus the higher cost of dihydroxyacetone phosphate make the former assay using glyceraldehyde 3-phosphate preferable for routine studies. In either of the above assays one unit of enzyme is defined as the amount catalyzing the isomerization of 1 ~mole of substrate per minute, and specific activity is defined as units per milligram of protein. Preparation o] Substrates and Reagents. The two coupling enzymes are both commercially available as crystalline suspensions essentially free of triosephosphate isomerase activity. However, since triosephosphate isomerase is inhibited by ammonium sulfate 5 the coupling enzymes are dialyzed prior to use. Thus, a stable baseline and a linear initial velocity are recorded in either assay. The substrates are also available in highly purified form as their acetal or ketal derivatives, which must be hydrolyzed to the free aldehyde or ketone. For Db-glyceraldehyde 3-phosphate (diethylacetal), 100 mg of the barium salt is dissolved in 6.0 ml of water, containing 1.5 g of Dowex 50-X4-200R H-form. The mixture is heated in a boiling water bath for exactly 3 min, followed by cooling in ice and removal of the Dowex by filtration. The solution is adjusted to pH 6.8 with NaOH and decolorized with activated charcoal. The other substrate, dihydroxyacetone phosphate (dimethylketal cyclohexylammonium salt), is hydrolyzed by dissolving 100 mg in 8.0 ml of water and adding 2.0 g of Dowex as above. After stirring for 1 min the Dowex is removed by filtration, and the solution is hydrolyzed at 40 ° for 6 hr. After neutralization and decolorization, the solutions are stored at 0-2 °. It should be 4 p. K. Chiang, Lile Sci. 10, 831 (1971). ' D. H. Turner, E. S. Blanch, M. Gibbs, and J. F. Turner, Plant Physiol. 40, 1146
(1965).
444
EPIMERASES AND ISOMERASES
[94]
pointed out that the lability of the free triosephosphates necessitates that these solutions be prepared fresh weekly and that the concentrations of triosephosphates be determined daily by the coupled assays described above. Protein Determination. Protein concentrations at various stages of the isolation are determined by the method of Lowry et al. 6 The concentration of the pure protein can be estimated from its absorbance at 280 nm using an absorbance index, elc,~,l~of 12.9. 3 Isolation Procedure Fraction I. One unit (450 ml) of human blood is collected into 67.5 ml of standard anticoagulant (acid-citrate-dextrose solution). All subsequent steps are carried out in an ice bath or in a cold room at 0-4 °. The plasma and bully coat are removed after centrifugafion at 8000 g for 60 rain, and the erythrocytes are washed by resuspension in 500 ml of cold 0.145 M NaC1 followed by centrifugation as above. After three washings, the erythrocytes are suspended in cold deionized water, and frozen and thawed several times; the lysate is centrifuged at 8000 g for 90 rain to remove the cell debris. The pH of the supernatant solution is adjusted to 7.0 by the addition of cold 1 M sodium phosphate buffer. Fraction II. Phosphocellulose (0.91 meq/g) which has been previously cleaned and equilibrated ~ in 5 mM sodium phosphate buffer, pH 7.0, is packed into a 20-cm diameter Biichner funnel to a height of 5 era. The hemolysate is applied to the ion exchange cellulose and is eluted with 5 mM phosphate buffer, pH 7.0, under mild vacuum. Fractions of approximately 200 ml each are collected and analyzed for triosephosphate isomerase activity. Under these conditions triosephosphate isomerase passes through the ion exchanger while most of the other proteins, including hemoglobin, are bound to the cellulose. Fractions containing triosephosphate isomerase activity are pooled and simultaneously concentrated and dialyzed against 5 mM triethanolamine buffer, pH 8.0, by ultrafiltration. Fraction III. The dialyzed fraction II is applied to a 3 X 75-cm column of DEAE-cellulose (0.91 meq/g) which has been previously cleaned and equilibrated in 5 mM triethanolamine buffer, pH 8.5. After washing with approximately 600 ml of this same buffer, a linear salt gradient is applied to elute the enzyme. The gradient mixing chamber contains 1 liter of 5 mM triethanolamine buffer, pH 8.5, and is connected by a siphon to the reservoir containing 1 liter of the same buffer which O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). E. A. Peterson and H. A. Sober, this series, Vol. 5, p. 3
[94l
HUMAN TRIOSEPHOSPHATE ISOMERASE
445
is also 0.4 M in NaC1. Under these conditions triosephosphate isomerase is eluted at approximately 0.15 M NaC1. Fractions containing triosephosphate isomerase activity are collected, concentrated and dialyzed against 5 mM triethanolamine buffer, pH 8.0 by ultrafiltration. Fraction IV. The dialyzed fraction III is added to a 2.1 X 22-cm column of DEAE-Sephadex which has been equilibrated with 5 mM triethanolamine buffer, pH 8.0. After washing with 300 ml of the same buffer, a shallow salt gradient is initiated (500 ml of 5 mM triethanolamine buffer, pH 8.0, in the mixing chamber, and 500 ml of the same buffer containing 0.1 M NaC1 in the mixing chamber). The enzyme is eluted at a flow rate of 30-40 ml per hr and the triosephosphate isomerase activity is found to be coincident with the third protein peak. The appropriate fractions are pooled and concentrated by ultrafiltration. Fraction V. The enzyme (5-10 mg/ml) is crystallized by dialyzing against a 0.50 saturated solution of ammonium sulfate containing 50 mM triethanolamine buffer, pH 7.8, 1 mM EDTA, and 1 mM 2-mercaptoethanol. A small amount of amorphous precipitate may be formed and should be removed by centrifugation. The supernatant solution is then returned to the dialysis bag, and the ammonium sulfate concentration of the dialysate slowly increased to 0.76 saturation where crystallization begins. After 48 hr crystals are collected by centrifugation and resuspended in 50 mM triethanolamine buffer, pH 7.8 containing 1 mM EDTA and 10 mM 2-mercaptoethanol. Specific activities of approximately 10,000 ~moles of glyceraldehyde 3-phosphate isomerized per minute per milligram of protein are routinely obtained. The isolation represents an overall purification of 4000-5000fold with 40-60% recovery. Additional recrystallizations, or rechromatography on DEAE-cellulose, phosphocellulose, or Sephadex do not result in further increases in the specific activity. A typical isolation is presented in the table. Resolution o] the Three Forms. Crystalline human triosephosphate isomerase can be resolved into three forms by electrophoresis or isoelectric focusing2 ,~ For preparative purposes the enzyme is electrofocused in 1% narrow range (pH 5-7) Ampholines for 92 hr at 600 V. Under these conditions a component (I) comprising approximately 5-1Q% of the total triosephosphate isomerase activity is found with an apparent isoelectric pH of 6.3. A second component (II) comprising 70-75% of the total activity electrofocuses at pH 6.0, and a third component (III) is observed at pH 5.6, which accounts for 20-25% of the isomerase activity. T. H. Sawyer, B. E. Tilley, and R. W. Gracy, J. Biol. Chem. 247, 6499 (1972).
446
[94]
EPIMERASES AND ISOMERASES
ISOLATION OF TRIOSEPHOSPHATE ISOMERASE FROM HUMAN ERYTHROCYTES
Fraction
Total activity (units)
Total protein (mg)
Specific activity (units/mg protein)
Hemolysate Phosphocellulose First DEAE Second DEAE Crystals
141,700 99,900 94,500 91,800 73,700
59,000 3,000 295 9.2 7.2
2.4 33.3 320 9,978 10,236
Purification Recovery (fold) (%) (1) 13.9 133 4158 4265
(100) 70 67 65 52
Other Tissues. Triosephosphate isomerase has also been isolated by the above method from human skeletal and cardiac muscle. In contrast to the purification from erythrocytes, the enzyme is not homogeneous after the second chromatographic step on DEAE-cellulose and is contaminated with a protein of large molecular weight. However, this contaminant can be easily removed by gel filtration on a 2.5 X 98 cm column of Sephadex G-100. Since muscle tissue is a much better source of the enzyme it is preferred. However, as is the case with most human enzyme studies, one is often limited to erythrocytes as a tissue source. The three electrophoretic forms of human triosephosphate isomerase found in the erythrocytes are found in essentially the same proportions in muscle tissues. 9 Physical Properties. Although the basis for the three electrophoretic forms is at this time still uncertain, a variety of studies 8 are consistent with an AA, AB, and BB distribution of dimers. The two types of subunits possess similar but distinguishable amino acid compositions and tryptic fingerprints. The three electrophoretic forms of human triosephosphate isomerase yield essentially identical molecular weights (56,000 -- 3000), sedimentation coefficients (s~o. . . . 4.1 X l0 -13 sec) and subunit molecular weights (28,000_+ 2000) when subjected to analytical ultracentrifugation. The enzyme is completely dissociated in 2.0 M guanidinium chloride, and can be totally renatured into catalytically active enzyme by removal of the dissociating agent. Catalytic Properties. Normal Michaelis-Menten kinetics are observed in both forward and reverse assays, and Km values for glyceraldehyde 3-phosphate of 0.34, 0.43, and 0.55 m M are obtained for components I, II, and III, respectively. Apparent Km values for dihydroxyacetone phosphate of 1.5, 0.82, and 0.69 m M are found for components I, II, and III, 9R. M. Snapka, T. H. Sawyer, and R. W. Gracy, unpublished experiments.
[9S]
TRIOSEPHOSPHATE ISOMERASE FROM RABBIT MUSCLE
447
respectively. These values are for the total triose phosphates in solution and do not take into consideration the relative distribution of hydrated gem diols or enediols which are in equilibrium with the free aldehyde and ketone. 1°,11 The enzyme exhibits a broad p H optimum between 7.0 and 9.5 and is competitively inhibited 12 by phosphoglycolate (Ki = 0.96 ~M), arsenate (K~ = 8.15 m M ) , a-glycerophosphate (Ki = 0.42 raM), and A T P (Ki = 16 raM). Stability. H u m a n triosephosphate isomerase is stable when stored in 0.80 saturated a m m o n i u m sulfate at 0-4% I t is recommended t h a t the solution also contain 20 m M triethanolamine buffer, p H 7.5, 1 m M E D T A , and 1 m M dithiothreitol. The Active Center. H u m a n triosephosphate isomerase is rapidly inactivated by the substrate analog, chloroacetol phosphate, by the selective esterification of a single essential glutamyl ,/-carboxylate per catalytic subunit. 13 Peptide maps and autoradiograms suggest the active-site peptide to be identical with the hexapeptide Ala-Tyr-Glu-Pro-Val-Trp isolated from rabbit14,15 and chicken TM muscle and y e a s t J 7 10D. R. Trentham, C. H. McMurray, and C. I. Pogson, Biochem. J. 114, 19 (1969), "S. J. Reynolds, D. W. Yates, and C. I. Pogson, Biochem. J. 122, 285 (1971). ~'~K~ values are given for the unresolved mixture of components I-III. ~'~F. C. Hartman and R. W. Gracy, Biochem. Biophys. Res. Commu~. 52, 388 (1973). ~4F. C. Hartman, Biochemistry 10, 146 (1971). ~sj. C. Miller and S. G. Waley, Biochem. J. 123, 163 (1971). ~GS. DeLaMare, A. F. W. Coulson, J. R. Knowles, J. D. Priddle, and R. E. Offord. Biochem. J. 129, 321 (1972). ~ I. L. Norton, and F. C. Hartman, Biochemistry 11, 4435 (1972).
[95] Triosephosphate Isomerase from Rabbit Muscle 1,2 B y FRED C. HARTMAN a n d I. LUCILE NORTON
D-Glyceraldehyde 3-phosphate ~ dihydroxyacetone phosphate
Assay Method Principle. Triosephosphate isomerase activity is most conveniently measured in the direction of formation of dihydroxyacetone phosphate by coupling with a-glycerophosphate dehydrogenase and monitoring the oxidation of N A D H at 340 n m 2 Alternatively, the enzyme can be assayed
1 Research from the authors' laboratory was sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. 2D-Glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1. 3G. Beisenherz, this series, Vol. 1 [57].
