Assay of glyoxalase I in blood

BIOCHEMICAL

MEDICINE

30,

305-312

(1983)

Assay of Glyoxalase I in Blood RICHARD B. BRANDT,

MICHAEL

G. WATERS, AND JEROME E. LAUX

Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Box 614 MCV Station, Richmond, Virginia 23298 Received

November

23, 1982

In 1913, the catalytic formation of n-lactic acid in biological materials from methylglyoxal (MeG) was reported (1,2). Eventually it was established that a combined enzyme system was involved, which was composed of glyoxalase I and II (3) with glutathione (4) as a cofactor. Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1 S) in the presence of reduced glutathione (GSH) converts MeG to a thiolester, S-lactoyl glutathione. The second enzyme, glyoxalase II (S-2 hydroxyacylglutathione hydrolase, EC 3.1.2.6), catalyzes the conversion of Slactoyl glutathione to p-lactate (5), rather than to L-lactate, the usual product of glycolysis. This is an enzyme system found in many varied tissues and organisms, while its specific function is unknown. Interest in the enzyme system and the substrate has been stimulated by SzentGyorgyi er al. (6) who suggested its involvement in the inhibition of growth, possibly by reaction with tRNA (7). Recently Gillespie has presented strong evidence for S-lactoyl glutathione (SLG) being related to tumor inhibition (8). Previous short reports from this laboratory (9,lO) have used a modification of the 2,4-dinitrophenylhydrazine (DNPH) method for spectrophotometric determination of MeG (11). The procedure utilizes the reaction of alcoholic, acidic DNPH with MeG under conditions that inactivate glyoxalase I (Glo I) and allow spectrophotometric assay of the bis-2,CDNP hydrazone of MeG in the visible spectrum. This method is reported in detail here to determine Glo I activity and K,,, values from yeast and from blood lysates of rabbit, rat, and humans.

MATERIALS AND METHODS Materials

A Beckman modernization

DU modified with a Gilford Model 252 spectrophotometer system equipped with a strip chart recorder, a Lauda K305 0006-2944/83

$3.00

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

306

BRANDT,

WATERS.

AND

LAUX

2/R constant temperature bath, and an automatic cuvette positioner was used for absorbance measurements. Quartz cuvettes with lo-mm path length and 3.5-ml maximum vol were used for the measurements. Gilson Pipetman adjustable digital microliter pipets were used for small volume transfers. An Eberbach water bath shaker was used for incubations. A Texas Instruments programmable 59 calculator was used for kinetic calculations and statistical analysis. All biochemicals were from Sigma Chemical Company. Aqueous 40% MeG was purified by steam distillation and its concentration determined using the Friedemann method (12). GSH was prepared in 0.1 bi solution with the pH adjusted to 6.8. GSH and MeG solutions were stored at -40°C. Potassium phosphate buffer was 0. I M, pH 6.8. Yeast Glo 1 in 50% glycerol-O.4 M (NH,J2S04-2 mM KH,PO,. pH 6.5. was diluted by adding 10 ~1 of the purified enzyme to 2.4 ml of 5 mM phosphate buffer, pH 6.8, containing 0.1% bovine albumin (fraction V). Stock solutions of 20 mM DNPH in absolute ethanol were diluted to 200 PM daily in an ethanol-HCI solution containing 12 ml of concentrated HCl/lOO ml of absolute ethanol as previously described (11). Human blood was collected in Becton-Dickenson heparinized Vacutainers or in Fenwal citrate-phosphate-dextrose blood pack units. Rat blood was collected after decapitation into 29 x 103-mm polypropylene centrifuge tubes containing 100 USP units of sodium heparin (0. IO ml of 5.9 mg/ml). Blood was collected by heart puncture into polypropylene tubes with heparin, 15 USP units/ml blood. from rabbits anesthetized with sodium pentobarbital(25 mg/kg). Hematocrits were measured using Dade microhematocrit capillary tubes and an International clinical centrifuge. Protein was determined by the biuret method (13). Blood Preparation Blood from rats or humans was lysed by adding 0.3 ml whole blood to 2.7 ml water, mixed, and centrifuged at 27,000~ x 20 min in a Sorvall RC2-B. The cell-free l/10 hemolysate was used for analysis. Rabbit blood was centrifuged at 15OOg x 10 min. The plasma and buffy coat were removed and the erythrocytes (RBC) were washed three times with 3 vol of 0.15 M NaCl centrifuged as above and the supernatant fluid was removed each time. The cells were lysed by mixing with 5 vol of water. Cell membranes were removed by centrifugation at 27,000~ x 20 min and the cell-free l/6 RBC lysate was used for analysis. Routine Assay of Glo I Activity Into two culture tubes (13 x 100 mm) 2.79 ml phosphate buffer and 0. I ml GSH were added, one for the reaction and one for the blank. To the reaction tube, 0.1 ml of 0.105 M MeG was added with mixing. The

ASSAY

OF

GLYOXALASE

307

I

tubes were incubated at 30°C for 30 min to allow the formation of the MeG-GSH hemimercaptal. Ten 13 x loo-mm culture tubes, each containing 5.0 ml of ethanolic, acidic DNPH, were prepared. After incubation of the blank and reaction tubes, a 0.020-ml aliquot was taken from the blank tube and added to an analysis tube. The tube was immediately covered with a polypropylene cap and mixed vigorously for 30 sec. Two reagent blanks were prepared. Two t = 0 standards were prepared by adding 0.020-ml aliquots from the reaction tube and mixing as above. The enzymatic reaction was initiated by adding 0.2 ml. of cell-free sample to the reaction tube with gentle mixing. At about 4, 8, and 12 min (with the exact time recorded), two 0.050-ml aliquots were taken from the reaction tube and delivered to two of the analysis tubes with immediate mixing as above. After all aliquots had been taken, the capped tubes were incubated at 42°C for 40 min and allowed to cool for 5 min, and the absorbance at 432 nm was measured against a water blank. The absorbance data was converted to micromoles using the appropriate volume corrections and a molar absorptivity of 3.4 x 104*rK1cm(II). Enzyme activity was reported as the micromoles of SLG formed per minute per milligram of protein or per milliliter of RBC. Calculations A linear-regression equation from a standard curve for MeG from 0.193 to 2.51 x lo-’ M was used to determine the absorbance in the t = 0 samples: Absorbance

= (3.4 x I04)[MeG]

+ 0.005.

This was corrected for the different aliquot sizes of the t = 0 samples and for the further dilution with addition of the enzyme preparation. The concentration of MeG at t = 0 in the reaction tube was 3.51 mM or 1.40 x 10d5 M in DNPH solution (3.51 x 0.02 m1/5.02 ml). The absorbance (A) at t = 0 (AtJ was 0.481. The absorbance at t = 0, adjusted for dilution and aliquot size (At,, adj), was 2.95 ml At,, x x 2.5, and equals 1.13. 3.15 ml The number of micromoles MeG/A and was 0.105~

at each time was calculated

X

O.lOml At,, adj

X

2.95 ml 2.99 ml

using pmole

308

BRANDT,

For calculation of pmole (A -blank) were used

WATERS.

AND LAUX

MeG at t = .Y. the pmole

pmole MeG,,

MeG/A

and At,

= Ar., x (pmole MeGjAb.

The Glo I activity (~2) was calculated from a linear-regression mination using MeG (micromoles) vs time (minutes). An example data is shown in Table 1.

deterof the

Kinetics Reaction velocities at 3.51, 2.22, 1.40, and 0.884 mM MeG with 3.34 mM GSH in each case were used for calculation of kinetic constants. Equilibrium hemimercaptal (HMC) concentrations were calculated using a kdlss of 3.0 mM as determined by VanderJagt et ul. (14). where Kdi,~ =: [MeG][GSH]/[HMC]. K, and V,,, were calculated using the doublereciprocal method of Lineweaver and Burk (15). Kinetics on human samples were also determined using equimolar MeG and GSH concentrations from 0.884 to 3.34 mM. The assay procedures were identical to that used for the routine activity determination except that the velocities were measured about 9, 7, and 5 min for 2.22. 1.40, and 0.884 mM MeG. respectively.

TABLE DATA FOR ABSORBANCE

Absorbance” 0.534 0.552 0.837 0.830 0.721 0.671 0.595 0.563

I

vs TIME OF REACTION

At,”

0.462 0.480 0.765 0.758 0.649 0.599 0.523 0.491

FOR GLYOXALASE

I Ac IWI

r>

Time cmin)

MeG’ t ymole )

0 I) 3.75 4.35 7.77 x.45 11.75 12.37

10.36” 10.36d 7.02 Cl.95 5.95 5.49 4.80 4.55

’ Determined as described in the text for absorbance at 432 nm for the 2.4-DNP hydrazone of MeG. Blank absorbance of 0.072. b At,, as defined in the text, is the absorbance of the sample ~~ blank absorbance. ’ Glo I activity (v) found from the linear regression of 0.420 pmoleimin. r = 0.95. P < 0.05.

’ Using calculated At,, adj.

ASSAY OF GLYOXALASE

I

309

RESULTS

Activity measurements for rat, human, and rabbit blood preparations are shown in Table 2. Results are expressed per milliliter of RBCs. No enzyme activity was detectable in plasma (less than 0.04 pmoles min-‘emg-’ protein). The rabbit l/6 RBC lysates are three to four times more concentrated, in terms of erythrocytes, than the l/10 hemolysates. Kinetic parameters for the purified yeast and lysed whole blood preparations are shown in Table 3. All velocity measurements were determined at constant initial GSH concentrations (3.34 mM>. Glo I K, was calculated using both MeG and HMC as the substrate. V,,,,, was calculated using MeG as the substrate. For human samples, kinetics were determined under conditions where MeG and GSH were equimolar. These values are compared to those for constant GSH conditions in Table 4. V,,, and K,,, were calculated using both MeG and HMC as the substrate. The double-reciprocal plots used in determining K,,, and V,,,,, were linear (Y = 0.95, P <: 0.05). DISCUSSION

Interpretation of the kinetic data can be complex due to the nonenzymatic reaction of MeG with GSH to form the HMC substrate. The HMC has been shown to be the substrate under conditions where its formation becomes the rate-limiting step in the Glo I reaction (16). Although it has been reported that free GSH inhibits the reaction, this appears to occur only where [HMC] was less than a tenth of the [GSH] (14,17). Results for K, and V,, depend on whether the substrate concentration is expressed as MeG or HMC and on [GSH] (Table 4). Reaction velocities are proportional to [GSH] (16,18). We chose to do our kinetic evaluation under conditions where [GSH] was constant in an attempt to eliminate the effects of [GSH] on the measured velocity of the glyoxalase reaction. The kinetics were simplest when [GSH] was held constant (17). Moreover, at any particular [GSH], the reaction velocities increased with increasing [MeG], whereas the converse was not true (18). TABLE ERYTHROCYTE

GLYOXALASE

2 I

ACTIVITY

Sample

Glyoxalase I activity @mole . min-’ . ml-‘)

Rat l/IO hemolysate Human 1110 hemolysate Rabbit l/6 RBC lysate

63.7 2 1.1 (48) 41.4 2 0.58 (75) 17.6 of: 2.4 (3)

0 Mean f SEM (N = number of samples). The activity is for SLG formed . ml-’ of red blood cells.

Rat l/l0 hemolysate ~~~~~ .~.___~ 63.7 !I 1.1 (48) 83.4 rt 2.7 (42) 1.12 t- 0.13 (43) 0.74 k 0.13 (42)

~

~__~

43.9 54.0 0.77 0.44

+ t i +

1.1 (8) 1.3 (8) 0.05 (8) 0.03 (8)

Human l/IO hemolysate

Rabbit l/6 RBC lysate ~~ __ -~ 17.6 f 2.4 (3) 25.0 2 7.0 (3) 0.96 2 0.32 (3) 0.58 Tt 0.22 (3)

” All parameters are mean 2 SEM ( N). ” Routine activity measurement. Yeast LJand V,.,,, expressed in pmole SLG formed min ’ mg- ’ protein using Sigma Grade III Glo I ” V,,, calculated from double-reciprocal plot using IMeG] as substrate.

~~~-_-~ v@mole min-’ ml ‘)b V ,n,ax(fimole min-’ ml-‘)d K,,, (mM MeG) K,,, (mM HMC)

Kinetic parameter”

TABLE 3 Gr YOXALASE 1 KINETIC PARAMETERS

_~

174 256 1.39 0.82

__

” i -t zt

14 (8)’ 21 (8)' 0.11 (8) 0.07 (8)

Yeast ~-.__

5

:: 2

5

Fi ">

r 5

ASSAY

HUMAN

OF GLYOXALASE TABLE 4 GLYOXALASE I KINETICS

Kinetic

parametef

Constant

K,,, (mM Km (mM V maxd (u V maxd (u

MeG) HMC) vs MeG) vs HMC)

0.77 0.44 54.0 57.8

” b ’ d

All parameters are [MeG] = (0.884 to [MeG] = GSH = V,., in pmole SLG

311

I

f + 2 2

[GSHlb 0.05 (8) 0.03 (8) 1.3 (8) 1.6 (8)

Equimolar[GSH]’ 3.08 1.97 84.1 103

+ k 2 k

0.48 (7) 0.60 (7) 9 (7) 21 (7)

means k SEM (N). 3.34 mM), GSH = 3.34 mM. (0.884 to 3.34 mM). formed min -’ . ml-‘.

Comparison of our velocity data with that of others showed that our routine assay gave results similar to those using the assay of Racker (3). For human RBC, an activity of 38 pmoles . min-‘*ml-’ RBC has been reported (19) using 0.66 mM GSH and 2.0 mM MeG in imidazole buffer, pH 7. As we have shown previously, the assays for the yeast and rat erythrocyte enzymes also agreed well with the ultraviolet method (11,20). Our data for the K,,, was difficult to compare since others used quite different conditions for kinetic determinations. However, the apparent Km (at 2 mM free GSH) was 0.13 mM HMC for the human erythrocyte enzyme in 50 mM sodium phosphate, pH 7.0, or 25 mM imidazole (19,21). Using these same conditions, the yeast enzyme had a K,,, of 0.53 mM (21). Under conditions where [HMC] exceeded free [GSH], a K,,, of 0.30 mM was reported for the yeast enzyme (14). Again, using HMC in excess of [GSH], the K,,, for the rat erythrocyte enzyme was reported as 0.09 rnM (22). In measuring enzyme activity in tissue homogenates, care should be taken to use a sufficiently dilute tissue preparation as Glo I activity is high. This could lead to errors in comparing velocities between tissues. For example, in Table 2, the velocities for the l/10 hemolysates were not strictly comparable with the l/6 RBC lysates, although the activity for the rabbit RBC lysates was, at most, 50% higher when the preparation was diluted fourfold to give a red cell dilution similar to the hemolysates. Solid tissue homogenates should be assayed at an even greater dilution than used for the blood. The procedure reported here is suitable for the routine assay of Glo I in crude cell-free preparations such as lysed blood and tissue and tumor homogenates, as well as for assay of the purified enzyme. The ethanolicacidic reagent stops the enzyme activity and the bis-2,CDNP hydrazone absorbs light in the visible region of the spectrum away from biological interference. The advantage of the method is the ability to determine

312

BRANDT,

WATERS.

AND

LAUX

relative amounts of the enzyme in various tissues in rapid and quantitative fashion. SUMMARY Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) was assayed using alcoholic, acidic 2,6dinitrophenylhydrazine to follow the disappearance of methylglyoxal over time, with the absorbance of formed methylglyoxal bis-hydrazone measured at 432 nm. Erythrocyte glyoxalase I activities were found to be 64, 41, and 18 pmole of S-lactoyl of red blood cells in rat, human, and glutathione formed min -‘m-’ rabbit blood and 174 pmole * min.- ‘amg -’ of protein for yeast. The K,, values found in millimolar hemimercaptal were about 0.5. Glyoxalase I activity can be determined in crude tissue preparations without interference from biological materials. ACKNOWLEDGMENT This work

was supported

by funds

from

the National

Foundation

for Cancer

Research.

REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 13.

Dakin, H. 0.. and Dudley. H. W., J. &I/. C/~rm. 14, 423 ( 1913). Neuberg, C. Biochem. 2. 51. 484 (1913). Racker. E., .I. Biol. Chem. 190, 685 (1951). Lohmann, K., Biochcm. Z. 254, 332 (1932). Brandt, R. B.. Siegel. S. A.. Waters. M. G.. and Bloch. M. H. A,~rc/ Biwhem. 102, 39 (1980). Szent-Gyorgyi. A., Hegyeli, A., and McLaughlin. J. A.. Sc.ience 155, 53Y (19671. Litt, M., Biochemistry 8, 3249 (1969). Gillespie, E., Biochem. Biophys. ReA. Commute. 98. 463 (198li. Waters, M. G.. Brandt. R. B., Muron, D. J.. and Bloch. M. H.. l’tr. J. Sci. 31, 137 (1980). Brandt. R. B.. Waters, M. G.. Muron, D. J.. and Bloch. M. H.. Fed. Pnw. 40, 555. (1981). Gilbert. R. P., and Brandt. R. B.. Anal. Chem. 47, 2418 (1975). Friedemann. T. E. J. Biol. Chem. 73, 331 (1927). Layne, E., in “Methods in Enzymology” IS. P. Colowick. N. 0. Kaplan. Eds.). Vol. 3, pp. 450-451. Academic Press. New York. 1957. VanderJagt, D. L.. Han, L. B., and Lehman, C. H. Biochcmisrr~~ 11, 3735 (1972~. Lineweaver. H., and Burk. D., J. Amer. Chem. SW. 56, 658 (1934). Cliffe. E. E.. and Waley, S. G., Biochem. .I. 79, 475 (1961). Mannervik. B.. in “Glutathione” (L. Flohe. H. C.. Benohr, H. Sies. H. D. Waller. and A. Wendel, Eds.). pp. 78-89. Thieme Verlag, Stuttgart. 1974. Kermack. W. 0.. and Matheson. N. A.. Biochem. J. 65, 48 (1957). Aronsson. A. -C., Tibbelin, G., and Mannervik, B.. Anal. Biochem. 92, 390 (1979). Brandt, R. B., Waters, M. G., Muron, D. J., and Bloch, M. H.. Proc. .Soc. Exp. Bio/. Med. 169, 463 (1982). Marmstal, E., Aronsson, A. -C.. and Mannervkik. B.. Biochem. J. 183. 23 ( 1979). Han. L. B.. Davidson. L. M.. and VanderJagt. D. L.. Biochim. Biophy.\. ~cf~r 445. 486 (1976).