Am J Hum Genet 30:14-18, 1978

Purification and Substrate Specificity of Polymorphic Forms of Esterase D from Human Erythrocytes EDWARD M. SCOTT1 AND RITA C. WRIGHT Esterase D (EsD) of human erythrocytes [1], which is polymorphic in most populations, was not observed in early electrophoretic studies of red cell esterases when naphthyl acetates were used as substrates [2]. Discovery of the enzyme depended on using 4-methylumbelliferyl esters [1], although these esters are not good substrates for other red cell esterases. After electrophoresis of hemolysates, EsD activity occurs in four principal bands [1] which can be numbered 1 to 4 in order of increasing anodal mobility. In EsD 1 homozygotes, about two-thirds of the activity is found in band 1 and the rest in band 2. In EsD 2 homozygotes, about 55% is found in band 3 and the balance in band 4. Esterase D appears to be dimeric, since a form of intermediate electrophoretic mobility is found in heterozygotes [1]. Thus, all four bands are found in the EsD 2-1 phenotype in the approximate proportions: band 1, 25%; band 2, 40%; band 3, 25%-30%; and band 4, 5%- 10%. The ratio of activity of the enzyme forms EsD 1, EsD 2-1, and EsD 2 in heterozygotes appears to be about 2:2:1. The same esterase is found in other human tissues [1]. The human polymorphism may be a random event, but if the heterozygotic form of the enzyme has properties that differ from those of the two homozygotic forms, the heterozygotic person might have an advantage sufficient to stabilize the polymorphism in human populations. To explore this possibility, the three types of EsD of human erythrocytes were purified and their properties compared. METHODS

The enzyme was assayed by measuring hydrolysis of 4-methylumbelliferyl acetate. During hydrolysis, absorbance decreased at 280 nm (AE = 4.89 mM-1 cm-') and increased at 340 nm (AE = 7.27 mM-1 cm-'). A unit of enzyme was the amount which hydrolyzed one ,umol per min in a solution of 0. 1 mM 4-methylumbelliferyl acetate, 50 mM potassium phosphate, and 1 mM EDTA at pH 6.0 and 30°C. Protein was estimated from absorbance at 280 nm and specific activity defined as units per mg protein. Heme protein was estimated as oxyhemoglobin from absorbance at 410 nm [3]. Hydrolysis of other substrates was followed by spectrophotometry as shown in table 2, or with a pHstat. The electrophoretic method of Hopkinson et al. [1] was used to determine phenotype. Relative activity of fluorescent bands on the gel was estimated by fluorometry, but the accuracy of this procedure was no better than + 5% of the total activity. Thermal stability was determined Received June 7, 1977; revised August 8, 1977. 1 Alaska Activity, Center for Disease Control, 225 Eagle Street, Anchorage, Alaska 99501. © 1978 by the American Society of Human Genetics. All rights reserved.

14

15

POLYMORPHIC FORMS OF ESTERASE D

at 4700 in 50 mM potassium phosphate (pH 6.0), containing I mM EDTA and 0.1% 2-mercaptoethanol.

Purification Packed red cells, 1.2 liters, from individuals of known phenotype were treated with 1-butanol and chloroform [4] at pH 7 and 50C in the presence of mM EDTA. After removal of denatured hemoglobin, 0.8 ml 2-mercaptoethanol was added and the solution dialyzed twice (for 16 hr and 8 hr) in 10 liters 10 mM potassium phosphate (pH 7.0). All buffers contained I mM EDTA and 0.1% 2-mercaptoethanol. Ammonium sulfate (28 g per 100 ml) was added and a precipitate removed by centrifuging and discarded. Additional ammonium sulfate (10.5 g per 100 ml) gave a precipitate that was removed by centrifuging and dissolved in 15 ml 0. 1 M potassium phosphate at pH 6.5. After dialysis in 5 mM potassium phosphate (pH 6.5), the enzyme solution was passed through a column of CM-cellulose which had been washed with the same buffer. For each gram of heme protein, 80 g CM-cellulose was used. EsD was not retained by the column, but much of the colored protein was. The pH of the enzyme solution was adjusted to 8.0 and passed through a column of DEAE-cellulose which had been washed with 10 mM Tris chloride (pH 8.0). The EsD 1 enzyme was not retained by the column; the EsD 2 enzyme was retained and eluted with 10 mM Tris chloride (pH 8.0) containing 0.5% ammonium sulfate. The EsD 2-1 enzyme separated into three fractions of which two were retained and eluted as above. Only the intermediate fraction, containing primarily the EsD 2-1 hybrid, was saved. Three of the above preparations were combined and passed through a column of polyacrylamide (P-100) gel. The fractions containing esterase were again concentrated by precipitation with ammonium sulfate and the enzyme dissolved in 0.5 ml 0.1 M potassium phosphate (pH 7.0). After dialysis in 5 mM potassium phosphate (pH 7.0), the solution was passed through a column of hydroxyapatite gel (100 g gel/g protein) which had been washed with the same buffer. EsD was retained and eluted with 0.5% ammonium sulfate in 5 mM potassium phosphate (pH 7.0). The purification of the EsD 1 enzyme is summarized in table 1. The EsD 2 phenotype is uncommon (less than 1% in a white population), and the final step in purification of the EsD 2 enzyme was omitted to conserve activity. As expected, the yield of EsD 2-1 hybrid was low. RESULTS AND DISCUSSION

Esterase D cannot be assayed in hemolysates because of the presence of other esterases, but both the EsD 2-1 and EsD 2 phenotypes appeared visually to have less activity after electrophoresis than the EsD 1 phenotype. After electrophoresis, the purified EsD 1 and EsD 2 enzymes gave patterns with 4-methylumbelliferyl acetate TABLE 1 PURIFICATION OF EsD 1 Volume

(ml)

Treatment

Packed red cells

.................

Butanol-CHC13, (NH4)2SO4

1,200 18

CM-cellulose .53

DEAE-cellulose, (NH4)2SO4 P-100, (NH4)2SO4 .0.9 Ca3(PO4)2 gel, (NH4)2SO4 * *

2.7 0.8

Includes esterases other than EsD in the early stages of purification.

Activity* (units)

1,920 620 610 320 155 119

Specific Activity

(U/mg protein) 0.0025

0.25 0.32 1.34 7.2 18.6

16

SCOTT AND WRIGHT

that were indistinguishable from those observed with the corresponding hemolysates. The EsD 2-1 hybrid enzyme had major bands in the 2 and 3 positions and trace amounts of activity in bands 1 and 4. The bands due to other esterases in hemolysates which stain with a-naphthyl acetate [2] were absent in the purified enzymes. Of a large number of esters tested, only those shown in table 2 were hydrolyzed at a rate of more than 0.001 times that found with 4-methylumbelliferyl acetate. The O-acetates of aliphatic alcohols, choline, cholesterol, retinol, benzyl alcohol, serine and hydroxyproline, were not hydrolyzed. The N-acetyl derivatives of amino aCids and aniline were not substrates, nor were the ethyl esters of benzoates and a-N-benzoyl-Larginine. The 4-methylumbelliferyl derivatives of phosphate, sulfate, ,3-D-glucose, ,3-D-galactose, and ,f-D-glucuronate were not attacked. Insoluble substrates were not hydrolyzed. Although tributyrin was hydrolyzed more rapidly than triacetin, increasing the tributyrin concentration to form micelles gave no increase in reaction rate; the low solubility of tributyrin prevented kinetic analysis. Most of the esters in table 2 are esters of an aliphatic acid and a substituted phenol. Ortho substitution of the phenol appeared to decrease the rate of hydrolysis and three such compounds, salicyl acetate, 2-acetoxy-3-naphthoic acid anilide (naphthol AS acetate) and a-tocopherol acetate were not substrates. The last two esters also have a limited solubility. Aryl esters of higher aliphatic acids (e.g., caprylates) were hydrolyzed insofar as they were soluble. EsD activity was optimal at pH 7.5 with 4-methylumbelliferyl acetate but showed little dependence on pH between pH 6 and 8 (table 3). Since many of these substrates hydrolyze spontaneously at higher pH, the kinetic studies were done at pH 6. The apparent activation energy of the enzymatic reaction at pH 6 with 4-methylumbelliferyl acetate was 7,200 kcal per mol. Since Km was not measurably changed between 10°C TABLE 2 SUBSTRATE SPECIFICITY OF EsD 1 ENZYME

Substrate

Kin

(mM)

4-Methylumbelliferyl acetate ... 0.028 4-Methylumbelliferyl butyrate 0.068 2.4 o-Nitrophenyl acetate . 0.96 p-Nitrophenyl acetate . 0.50 p-Nitrophenyl butyrate . a-Naphthyl acetate .2. 1 acetate .2.4 ,l-Naphthyl Indoxyl acetate .3.1 N-Methylindoxyl acetate . 2.9

Phenyl acetate .............. 41.3 p-Tolyl acetate .............. 7.9 ............. 9.4 m-Tolyl acetate o-Tolyl acetate .............. 13.3 Triacetin ................... 9.0 *

V*v (,umol min-' U-1)

1.28 0.86 0.04 0.50 0.20 0.88 0.52 0.33 0.38 0.26 0.10 0.11 0.08 0.08

(nmol min-' U 1)

1,000 510 1.6 47 33 40 21 10 13 0.6 1.2

Maximum selocity per unit of enzyme. Rate ot reaction per unit of enzyme at a substrate concentration ot 0. mM.

1.1

0.6 0.8

Wavelength of Assay (nm)

(mM-, cm-,)

340 340 350 400 400 322 328 375 416 270 272 272 272 pHstat

7.27 7.27 2.24 1.90 1.90 2.07 1.42 1.32 1.52 1.42 1.05 1.49 1.49 *

Af

POLYMORPHIC FORMS OF ESTERASE D

17

TABLE 3

RELATIVE ACTIVITY OF ESTERASE D AT pH 7.0 AND pH 8.0 RELATIVE ACTIVITY*

SLUBSTRATE

4-Methylumbelliferyl acetate ....... ............. a-Naphthyl acetate ............................ Phenyl acetate ................................ p-Tolyl acetate ................................ p-Nitrophenyl acetate .......................... =

pH 8.0

105 101 100

108 109 104 119

102 102

/3-Naphthyl acetate ............................

* Activity at pH 6.0

pH 7.0

71

92

78

100.

and 40TC, the temperature coefficient was independent of substrate concentration. A saturated solution of 4-methylumbelliferone did not inhibit. The EsD 2 enzyme and EsD 2-1 hybrid did not differ from the EsD 1 form with respect to the effect of pH, temperature, and substrate concentration when 4-methylumbelliferyl acetate was the substrate. For the seven substrates in table 4, however, the activity of the three isozymes was not that expected from their activity with 4-methylumbelliferyl acetate. Although maximum rate relative to activity with 4-methylumbelliferyl acetate was the same for the three forms, the Michaelis constant differed as shown in table 4. These differences were of a magnitude such that at concentrations of less than 10-5 M, o-nitrophenyl acetate would be hydrolyzed by the EsD 2-1 hybrid at a rate 20% lower than expected when compared to the EsD 1 enzyme. Similarly, the hydrolysis of 10-5 M f3-naphtyl acetate by the EsD 2 enzyme would be 14% higher than expected. For the other substrates in table 2 (except p-nitrophenyl butyrate which was not studied in detail), the specificity of the three enzyme forms was indistinguishable. The hybrid 2-1 enzyme was intermediate in properties except when o-nitrophenyl acetate was the substrate. Thermal inactivation of these enzymes approximated a first order reaction until it was 70% complete; thereafter, it became slower. Initial rates of inactivation at 47TC were: EsD 1, 0.10 min-'; EsD 2-1, 0.22 minm; and EsD 2, 0.53 min-'. Two other possible differences between the phenotypes that could not be determined are a TABLE 4

MICHAELIS CONSTANTS OF ISOZYMES OF ESTERASE D Substrate

a-Naphthyl acetate ...........

3-Naphthyl acetate ........... o-Nitrophenyl acetate ......... p-Nitrophenyl acetate ......... Indoxyl acetate .............

N-Methylindoxyl acetate ........ o-Tolyl acetate .............

................ ................ ................ ................ ................. .............. .................

EsD (mM)

EsD 2-1 (mM)

EsD 2 (mM)

2. 1 2.4 2.4 0.96

2.2 2.2 3.0 0.88 3.3 3.1 14

2.3 2. 1 2.6 0.85 3.4 3. 1

3.1 2.9 13

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18

SCOTT AND WRIGHT

difference in maximum velocity and a difference in the amount of enzyme protein present in the red cell. The data in table 2 show clearly why this enzyme can be demonstrated in hemolysates after electrophoresis only with 4-methylumbelliferyl esters. None of the results suggest the nature of the physiologic substrate and the effects of the differences in properties of the isozymes cannot be evaluated. However, differences that have been found suggest that the EsD polymorphism need not be neutral in its effect. SUMMARY

Esterase D (EsD), purified from human erythrocytes and tested with a variety of substrates, hydrolyzed only triacetin, tributyrin, and certain soluble aryl esters of aliphatic acids. Esters of 4-methylumbelliferone were easily the best substrates. When the three genetically different isozymes were compared, the less common forms, EsD 2 and EsD 2-1, were less stable than EsD 1. With some substrates, the Michaelis constant of the EsD 2 form differed from that of the EsD 1 form. The EsD 2-1 hybrid form was usually, but not invariably, intermediate in properties. The physiologic significance of the genetic variability of this enzyme is unknown. 1. 2.

3. 4.

REFERENCES HOPKINSON DA, MESTRINER MA, CORTNER J, HARRIS H: Esterase D: a new human polymorphism. Ann Hum Genet 37:119- 137, 1973 TASHIAN RE: Multiple forms of esterases from human erythrocytes. Proc Soc Exp Biol Med 108:364-366, 1961 SCOTT EM, McGRAW JC: Purification and properties of disphosphopyridine nucleotide diaphorase of human erythrocytes. J Biol Chem 237:249-252, 1962 SCOTT EM: Purification of red cell enzymes by treatment with n-butanol and chloroform. Prep Biochem 6:147- 152, 1976