Purification of glyoxalase I from human erythrocytes by the use of affinity chromatography and separation of the three isoenzymes

AhAL.YTICAL

92,

HIOCII~MISTR\

390-393

(1979)

Purification of Glyoxalase Affinity Chromatography

Glyoxalase part

in the

I has

heen

purification

purified procedure

bound to epoxy-activated separated by ion-exchange

I from Human and Separation

to homogeneity involved

Sepharose 6B. chromatography

from

affinity Three

Erythrocytes by the Use of of the Three lsoenzymes

human

erythrocytes.

chromatography

isoenzymes Some of the

on

An

essential

S-hexylglutathione

were found. which could properties of the enzyme

he are

reported.

Glyoxalase I (EC 4.4.1.5) has been purified and characterized from many sources [see (1) for a review]. In most of the species studied the enzyme is present in only one genetically determined form. However, the human glyoxalase I (2,3) as well as the mouse enzyme (4) can exist in three separable forms which originate from two alleles in the genome of each species. The successful use of affinity chromatography on S-hexylglutathione bound to Sepharose 4B in the purification of porcine glyoxalase I (1) prompted an attempt to isolate the human glyoxalase I and to separate the isoenzymes. The present report shows that affinity chromatography is even more efficient for the purification of the human than for the porcine enzyme and that large quantities can be prepared readily. Furthermore, the three isoenzymes could be isolated after ion-exchange chromatography. MATERIALS

AND

described for the porcine erythrocyte zyme (I). RESULTS

METHODS

Outdated red blood cells were obtained from Kabi AB. Stockholm, Sweden. SHexylglutathione was coupled to epoxy-activated Sepharose 6B according to instructions of the manufacturer. Protein concentration was determined from the absorbance at 260 and 280 nm and glyoxalase I activity was measured spectrophotometrically as 0003-26971791020390.04$02,00/O CopyrIght k~ 1979 by Academic Pre\\. Inc. All right< of reproduction m any Corm rcxrved.

en-

390

The first part of the purification procedure essentially followed a method described for purification of glutathione reductase (5). In our laboratory glutathione reductase is prepared by this procedure and it is therefore an advantage to perform the first steps in the purification of glutathione reductase and glyoxalase I simultaneously. Step I. To I8 liters of packed erythrocytes 108 pmol of flavin-adenine dinucleotide (FAD)’ and 900 pmol of PMSF were added. After adjusting the suspension to pH 8.3 with a solution of 13.3 M ammonia, an ice cold mixture of tz-butanol (2.16 liters) and chloroform (0.86 liter) was added and the resultant mixture was stirred for 60 min at room temperature (22°C) before centrifugation at 7000x for 30 min. The sediment was discarded. Step 2. To the supernatant liquid. I .5 vol ’ Abbreviations used: FAD. cleotide; PMSF. phenylmethanesulfonyl N-tris[hydroxymethyl]methyl-?-aminoeth~~nesulfonic acid: GSH. glutathione.

flavin-adenine fluoride:

dinuTes.

PURIFICATIION

Volume tmll

Fraction

OF HUMAN

Total

GLYOXALASE

protein (mgJ

Total activity (pmolimin)

391

I

Specific activity (pm01 < min-’ )i rng-‘1

~__ Erythrocytes Butanol-chloroform precipitation Acetone fractionation Sephadex G-25 77? (NH,)?SO, fraction .T-Hexylglutathione-Sepharose Sephadex G-75 .T-Hexylglutathione-Sepharose ” Butanol

and chloroform

18.000 5.780 1.770 2.070 49.5 40 IO? 26 interfer-e

7.990.000 159.000” I lY.000 90.000 6 1,900 107 It.0 68. I

in thle protein

of cold acetone was added at room temperature. After 30 min the precipita.te was packed by centrifugation at 7000,q for 5 min. The sediment was extracted for 5 h with 10 mM Tris-chloride, pH 7.8, containing 50 PM PMSF and 0.1 mM dithioerythritol. After 5 h the mixture was centrifuged and the supematant fluid saved as extract A. The procedure was repeated to give extract B (5). Srcp 3. Extracts A and B were combined and, in order to remove acetone, applied to a column (12 x 63 cm) of Sephadex G-25 (coarse grade), equilibrated with IO mM Tris-chloride. pH 7.8, containing 50 PM PMSF and 0.1 mM dithioerythritol. This and subsequent steps were carried out at 4°C. Src~p 4. Solid ammonium sulfate (298 g/ liter) was added to the pooled fractions from step 3. After centrifugation, glutathione reductase was found in the precipitate whereas most of the glyoxalase I activity was in the supernatant fluid. To precipitate glyoxalase I, additional ammonium sulfate was added (170 g/liter) to the supernatant fluid. Step 5. The precipitate from step 4 was dissolved in 10 mM Tris-chloride. pH 7.8, and charged onto an S-hexylglutathioneSepharose column (4 x 4.5 cm). After sample application, the column was washed with 10 IIIM Tris-chloride, pH 7.8. clontaining 0.5 M NaCl, and then with 10 mhl Trischloride at the same pH. The column was

679,000 468,000 126.000 167.000 I I I .600 93.000 91.700 69.420

0.085 1.93 I.90 I .80 1.80 860 873 l.OlY

measurements

eluted with IO mM Tris-chloride. pH 7.8, containing IO mM glutathione, 6 mM Shexylglutathione, and about 0.05 M NaCI. Step 6. The pooled fractions from step 5 were concentrated by use of a molecular filter (Millipore immersible separator PTGC) to about 10 ml and added to a column (4 x 26 cm) of Sephadex G-75 (Fine) equilibrated with IO mM Tris-chloride at pH 7.8 and eluted with the same buffer. Srclp 7. The active pooled material from step 6 was made 0.5 M with respect to NaCl before application to a second S-hexylglutathione-Sepharose column (2 x 9 cm), which was washed and eluted in the same manner as in step 5. The results of purification are summarized in Table I. After step 7 the enzyme is at least 95%’ pure as judged by disc electrophoresis. The remaining impurities can easily be removed, after concentration of the sample. by passage through a column of Sephadex G-75 (4 x 26 cm). However. when isoenzymes are to be separated. the pooled fractions from step 7 are applied directly to a column of DEAESepharose.

A sample from step 7 was incubated with 10 mM dithioerythritol for 1 h and applied to a column (I x 5 cm) of DEAE-Sepharose equilibrated with 10 mM Tris/Tes at pH 6.8 (obtained by titrating 10 mM Tes with 10 mM

ARONSSON.TIBBEI,IN.

392

Tris base) containing 2 mM dithioerythritol. The enzyme was eluted with a linear gradient of 0 to 0.1 M NaCl in 10 mM TrisiTes containing 2 mM dithioerythritol (pH 6.8). The elution profile (Fig. I) showed three peaks, each of which contained one protein band when analyzed by disc electrophoresis. A mixture of the separated components gave three bands. The clear separation of the isoenzymes shown in Fig. 1 and after gel electrophoresis was obtained only in the presence of sulfhydryl-containing reagents. Before electrophoresis, the enzyme was usually incubated with 0.1 M GSH and stored at - 18°C overnight. In the absence of thiols. the human enzyme, as is the case for the porcine ( 1) and the rat liver enzymes (Marmstal, E., and Mannervik, B., unpublished results), shows many bands after electrophoresis, which are thought to represent different oxidized forms of glyoxalase I. With glyoxalase I from other mammalian sources it was previously shown that the enzyme consists of two subunits of similar sizes [cf. (l)]. When the human isoenzymes were treated with 6 M guanidinium chloride for 6 h. dialyzed to remove the denaturant, and analyzed by disc electrophoresis. isoenzyme 2 showed three bands whereas isoTABL.E 9 PH’I SISAL. PROPERTIES or GI YOYAL ASE 1 FROM HUMAN ERYT-HROCYI ts Molecular weight [determined according to Ref. (911 Subunit molecular weight [determined according to Ref. (IO)] Metal content (6)

Apparent K ,, for methylglyoxal (at 2 mM free GSH) pH Optimum of activity

Stability of purified Iat -20°C)

5 1,000

26.000 I mol of Zn”/mol of enzyme subunit 0.13 rnM 7.0 (Constant between 6.57.5)

enzyme Several

weeks

AND MANNERVIK

200 fraction

250 number

300

FIG. I. Separation of the three isoenzyme\ of glyoxalase I from human erythrocytes by chromatography on DEAE-Sepharose CL-6B. A column (I x 5 cm) equilibrated with IO mM Tris/Tes at pH 6.8 containing 2 rnM dithioerythritol was used. The sample applied (about 14,000 units) was preincubated with IO rnM dithioerythritol for 1 h. Elution was carried out with a linear gradient of NaCl (O-O.1 b1, total volume 1000 ml) in the buffer used for equilibration.

enzymes 1 and 3 each had one band. We propose that isoenzymes 1 and 3 consist of identical subunits (CQ and ,&) whereas isoenzyme 2 is composed of two nonidentical subunits in an cup dimeric structure. The purified enzyme has previously been shown to be a zinc protein (6) containing about 2 Zn per dimeric molecule. Table 2 lists some additional properties of glyoxalase I purified from human erythrocytes. No significant differences between the three forms of the enzyme have been noted except for the electrophoretic and chromatographic properties. A more detailed characterization of glyoxalase 1 from human erythrocytes will be published elsewhere. DISCUSSION A complete purification of human glyoxalase I has to our knowledge not been published before, but an attempt to purify and characterize the enzyme from human liver has been made (7). The separation of the three isoenzymes supports the genetic evidence for two loci in the genome coding for tht enzyme. In the purification of the

PURIFICATION

OF

HUMAN

human enzyme it was found that tihe chromatogrdphy on S-hexylglutathione -Sepharose was more efficient both regarding purification and capacity than in the previous application of the gel. The adsorption appears to depend on both hydrophobic interactions and a specific recognition of the glutathione moiety of the ligand. Within limits, high ionic strength promotes the binding as expected for hydrophobic interactions (8). The efficient elution by free S-hexylglutathione and GSH is evidence for the specific affinity. The described procedure allows tlhe preparation of relatively large quantities of enzyme (100 mg to 1 g) with reasonable ease.

GLYOXALASE

an early supported ral

,, 2. 3. 4, 5. 6. ,,

393

I

phase of the investigation. by a grant (to B. M.) from

Science

Research

This work was the Swedish Natu-

Council.

REFERENCES Aronsson. ch(./rt.

A.-C., J. 165,

and Mannervik. 503-509.

B. (1977)

Bio-

Kdmpf. J.. Bissbort. S., Gussmann. S.. and H. ( 1975) H/lrlfrrl~yc~rrc~/iX 27, 141- 143. Bagster. 256,

I. A.. 56-57P.

and

Parr.

C. W.

Meo. T., Douglas. T.. and .sc~ierrc~r 198. 31 l-313.

( 1975)

Rijnbeek.

Ritter,

.I. /‘h~.~i~~/. A.-M.

I 1977)

Krohne-Ehrich, G.. Schirmer. R. H.. and UntuchtGrau. R. (1977) Ellu. J. Liioc/zcl)~. 80, 65-71. Aronsson. ( 1978) 1235-

A.-C.. Bicv 1240.

Marmst&l,

h<,/n.

E. and

Rioph.vs.

Uolila. L.. and Koivusalo. (/lCVI. 52, 493-503.

Mannervik.

B.

Kc\.

(‘o~~~m//~i.

8 I,

M. (1975)

El,r.

.I. Hi<>-

.I. Rio/.

C‘h~~/rr.

8.

ACKNOWLEDGMENTS

9. We

thank

Mr.

Henrik

for providing erythrocytes valuable discussions. Mr.

B.jdrling

and

Ms.

and Ms. Ewa Peter Nystr(im

Elsa

Lycke

MarmstPl for took part in

10.

Weber. 244,

K..

and

4406-4412.

Osborn.

M. (19691