Glyoxalase I catalyses the isomerization of the hemithioacetal formed non-enzymatically from methylglyoxal and glutathione to S-D-lactoylglutathione. The activity of glyoxalase I is conventionally measured spectrophotometrically by following the increase in A240 for which the change in molar absorption coefficient Δε240=2.86 mM−1·cm−1. The hemithioacetal is pre-formed in situ by incubation of methylglyoxal and glutathione in 50 mM sodium phosphate buffer (pH 6.6) at 37°C for 10 min. The cell extract is then added, the A240 is monitored over 5 min, and the initial rate of increase in A240 and hence glyoxalase I activity deduced with correction for blank. Glyoxalase I activity is given in units per mg of protein or cell number where one unit is the amount of enzyme that catalyses the formation of 1 μmol of S-D-lactoylglutathione per min under assay conditions. Glyoxalase II catalyses the hydrolysis of S-D-lactoylglutathione to D-lactate and glutathione. Glyoxalase II activity is also measured spectrophotometrically by following the decrease in A240 for which the change in molar absorption coefficient Δε240=−3.10 mM−1·cm−1. It is given in units per mg of protein or cell number where one unit is the amount of enzyme that catalyses the hydrolysis of 1 μmol of S-D-lactoylglutathione per min under assay conditions. Glyoxalase I and glyoxalase II activity measurements have been modified for use with a UV-transparent microplate for higher sample throughput.

Introduction

MG (methylglyoxal) is a reactive α-oxoaldehyde metabolite formed by the degradation of the triosephosphates glyceraldehyde 3-phosphate and dihydroxyacetone phosphate by MG synthase in bacteria, oxidation of acetone formed in ketone body metabolism, catabolism of threonine and degradation of glycated proteins [1]. MG is a reactive glycating agent and precursor of major quantitative AGE (advanced glycation end-product) adducts formed from protein and DNA. MG accumulation causes inactivation and increased degradation of proteins, DNA strand breaks, mutagenesis and cytotoxicity [2,3]. Protein and DNA damage is prevented by the efficient metabolism and detoxification of MG. The major pathway of MG metabolism is usually the glyoxalase pathway, supported by aldo–keto reductases (including aldose reductase) and aldehyde dehydrogenases [4,5].

The glyoxalase system is present in the cytosol of all mammalian cells and most organisms. Where it is absent, MG metabolism is achieved by aldo–keto reductases and aldehyde dehydrogenases. In the glyoxalase pathway, in the first step Glo1 (glyoxalase I) catalyses the isomerization of the HA (hemithioacetal) formed non-enzymatically from MG and reduced glutathione (GSH) to SLG (S-D-lactoylglutathione). In the second step, Glo2 (glyoxalase II) catalyses the hydrolysis of SLG to D-lactate and GSH, reforming GSH consumed in the Glo1-catalysed reaction.

Recent studies implicate the glyoxalase pathway in human aging and disease, plant and microbial growth, resistance to drug therapy and environmental stress. Increased activity of Glo1 through gene copy number variation, regulation of transcriptional control and small-molecule inducers is now being investigated for improved understanding of mechanisms and pharmacological interventions to sustain healthy aging and for treatment of disease. Cell-permeant Glo1 inhibitors are also in development for treatment of multidrug resistance in cancer chemotherapy [6,7]. A one-step high-throughput microplate assay will be useful for screening of bioactives and drugs to improve and correct dysfunction of glyoxalase activity. This rapid and simple method will be also useful in elucidating the cellular and molecular mechanisms of glyoxalase metabolism.

Measurement of Glo1 and Glo2 activity using spectrophotometric assays

Sample preparation

The activities of Glo1 and Glo2 are made on cytosolic extracts of animal and plant tissues and extracts of animal, plant and microbial cells (Table 1). Samples should be stored at −80°C if analysis cannot be performed immediately. Sample storage validation was reported previously [8]. The extract is conveniently prepared by homogenizing tissue in 10 mM sodium phosphate buffer (pH 7.0) or lysing washed cells in the same buffer by sonication. The homogenate or cell lysate is centrifuged to sediment membranes and the supernatant is the cytosolic extract used for assay of Glo1 and Glo2 activities. With appropriate subcellular fractionation, activity of Glo2 in mitochondria may also be measured [9]. The cell number of cultured cells is counted before sonication if the activities of Glo1 and Glo2 are to be normalized to cell number (per 106 cells). Also, cells must be washed three times in PBS before sonication to remove extracellular protein from culture medium to allow for assay of cell protein and normalization of the activities of Glo1 and Glo2 to cell protein (mg). Total cell protein content is determined by Bradford assay or other method. Tissue and cells may also be lysed in a commercial lysis buffer containing protease inhibitors in accordance with the manufacturer's procedures. For example, Lysis-M buffer (Roche) containing a Complete™ mini EDTA-free protease inhibitor cocktail tablet and phosphatase inhibitor cocktail tablet.

Table 1
Protocol for assay of activities of Glo1 and Glo2

For microplate assay, decrease reagent and cell extract volumes by a factor of 4 or 5 for total well volumes of 250 μl or 200 μl respectively.

Step Description Procedure 
Preparation of biological samples Animal and plant tissue: tissue (~5–10 mg of wet weight) is homogenized in 200 μl of 10 mM sodium phosphate buffer (pH 7.0) at 4°C. Cultured cells: cell pellets (~1×106) are washed three times in PBS, suspended in 200 μl of 10 mM sodium phosphate buffer (pH 7.0) at 4°C and sonicated at 100 W for 30 s. Cell fibrils and membranes are sedimented by centrifugation at 20000 g for 30 min at 4°C and the supernatant is removed and kept on ice for activity assays or stored at −80°C until analysis 
Substrate solutions Glo1 activity assay solutions required: 100 mM sodium phosphate buffer (pH 6.6) at 37°C, 20 mM GSH (hydrochloride salt) in water on ice, 20 mM MG in water on ice (calibrated by the AG method, see above) and water at 37°C. Glo2 activity assay solutions required: 100 mM Tris/HCl (pH 7.4) at 37°C, 3 mM SLG (calibrated spectrophotometrically) and water at 37°C 
Assay of Glo1 activity In a 1 ml cuvette, add 500 μl of 100 mM sodium phosphate buffer (pH 6.6) at 37°C, 100 μl of 20 mM GSH solution, 100 μl of 20 mM MG and 280 μl of water. The mixture is then incubated for 10 min at 37°C. After 10 min, 20 μl of cytosolic tissue/cell extract is added and the A240 is monitored immediately for 5 min. The initial rate of change (increases) in A240 is deduced, (dA240/dt)0, and the activity of Glo1 (units), aGlo1, is deduced, aGlo1=(dA240/dt)0/2.86 
Assay of Glo2 activity In a 1 ml cuvette, add 500 μl of 100 mM Tris/HCl (pH 7.4) at 37°C, 100 μl of 3 mM SLG solution and 380 μl of water. Then, 20 μl of cytosolic tissue/cell extract is added, and the A240 is monitored immediately for 5 min. The initial rate of change (decreases) in A240 is deduced, (dA240/dt)0, and the activity of Glo2 (units), aGlo2, is deduced, aGlo2=−(dA240/dt)0/3.10 
Normalization aGlo1 and aGlo2 are reported normalized to cell protein, assayed in the cytosolic extract or cell number (counted before cell lysis), as units/mg of protein or units/106 cells 
Step Description Procedure 
Preparation of biological samples Animal and plant tissue: tissue (~5–10 mg of wet weight) is homogenized in 200 μl of 10 mM sodium phosphate buffer (pH 7.0) at 4°C. Cultured cells: cell pellets (~1×106) are washed three times in PBS, suspended in 200 μl of 10 mM sodium phosphate buffer (pH 7.0) at 4°C and sonicated at 100 W for 30 s. Cell fibrils and membranes are sedimented by centrifugation at 20000 g for 30 min at 4°C and the supernatant is removed and kept on ice for activity assays or stored at −80°C until analysis 
Substrate solutions Glo1 activity assay solutions required: 100 mM sodium phosphate buffer (pH 6.6) at 37°C, 20 mM GSH (hydrochloride salt) in water on ice, 20 mM MG in water on ice (calibrated by the AG method, see above) and water at 37°C. Glo2 activity assay solutions required: 100 mM Tris/HCl (pH 7.4) at 37°C, 3 mM SLG (calibrated spectrophotometrically) and water at 37°C 
Assay of Glo1 activity In a 1 ml cuvette, add 500 μl of 100 mM sodium phosphate buffer (pH 6.6) at 37°C, 100 μl of 20 mM GSH solution, 100 μl of 20 mM MG and 280 μl of water. The mixture is then incubated for 10 min at 37°C. After 10 min, 20 μl of cytosolic tissue/cell extract is added and the A240 is monitored immediately for 5 min. The initial rate of change (increases) in A240 is deduced, (dA240/dt)0, and the activity of Glo1 (units), aGlo1, is deduced, aGlo1=(dA240/dt)0/2.86 
Assay of Glo2 activity In a 1 ml cuvette, add 500 μl of 100 mM Tris/HCl (pH 7.4) at 37°C, 100 μl of 3 mM SLG solution and 380 μl of water. Then, 20 μl of cytosolic tissue/cell extract is added, and the A240 is monitored immediately for 5 min. The initial rate of change (decreases) in A240 is deduced, (dA240/dt)0, and the activity of Glo2 (units), aGlo2, is deduced, aGlo2=−(dA240/dt)0/3.10 
Normalization aGlo1 and aGlo2 are reported normalized to cell protein, assayed in the cytosolic extract or cell number (counted before cell lysis), as units/mg of protein or units/106 cells 

Assay of activity of Glo1

The activity of Glo1 is measured spectrophotometrically using 1 ml quartz cuvettes by following the initial rate of increase in A240 for which the change in molar absorption coefficient Δε240 (Δε240240[SLG]−ε240[HA])=2.86 mM−1·cm−1 [10]. A blank correction is required. This should give a very low rate of increase in A240 (approximately <0.001 absorbance units per min). If the blank is higher, it may indicate contamination of the cuvette with Glo1 from a previous assay. In this case, clean the cuvettes with concentrated nitric acid, wash with 10 volumes of water and repeat the assay.

HA is pre-formed by incubation of 2 mM MG and 2 mM GSH in 50 mM sodium phosphate buffer (pH 6.6) at 37°C for 10 min ([HA]=0.63 mM; K=333 M−1 [11]). It must be pre-formed or the activity assay may be rate-limited by the rate of the HA formation and not Glo1 in the assay mixture. The HA solution should be freshly prepared and not be prepared in large volumes beforehand as it will slowly isomerize to S-lactoylglutathione non-enzymatically [12].

Δε240 for the Glo1 activity assay relates to the ε240 of SLG and HA and not to that of GSH and MG because, under the initial rate conditions, HA is converted into SLG before the equilibrium involving GSH and GSH relaxes under the new concentration conditions. The activity of Glo1 may be assayed by the accumulation of SLG even with cell extracts containing Glo2 because, under the assay conditions, the high concentration of HA used is a competitive inhibitor of Glo2 [13].

High-purity aqueous solution of MG, prepared and purified as described in [14], should preferably be used in the assay. MG stock solutions must be calibrated before use. This can be conveniently done by derivatization with AG (aminoguanidine). A small aliquot of MG solution is diluted to approximately 50–100 μM in a solution containing 1 mM AG hydrochloride in 50 mM sodium phosphate buffer (pH 7.4), with incubation at 37°C for 4 h and spectrophotometric measurement of the triazine adduct at 320 nm from which the concentration of MG is deduced (ε320=2411 mM−1·cm−1) [15].

Assay of activity of Glo2

The activity of Glo2 is measured spectrophotometrically using 1 ml quartz cuvettes by following the initial rate of decrease in A240 for which the change in molar absorption coefficient Δε240 (Δε240240[D-lactic acid]+ε240[GSH]−ε240[SLG])=−3.10 mM−1·cm−1 [10]. A blank correction is required. This should give a very low rate of decrease in A240 (approximately <0.001 absorbance units per min). If the blank is higher, it may indicate contamination of the cuvette with Glo2 from a previous assay. In this case, clean the cuvettes with concentrated nitric acid, wash with 10 volumes of water and repeat the assay.

SLG stock solution should be calibrated before use as described in [16]. The concentration of SLG is determined by end point assay involving hydrolysis to D-lactate and GSH, catalysed by authentic Glo2. An aliquot of the stock solution of SLG (100 μl), Tris/HCl (pH 7.4) (100 mM, 500 μl) and water (390 μl) is added to a 1 ml quartz cuvette and the A240 is measured against a reference cuvette containing Tris/HCl (pH 7.4) (100 mM, 500 μl) and water (490 μl) at 37°C. Glo2 (100 units/ml, 10 μl) is added to the assay and reference cuvettes and the A240 is monitored until a steady minimum is attained. The concentration of SLG is deduced from the decrease in A240 on addition of Glo2; Δε240=−3.10 mM−1·cm−1 [10].

Sample preparation of cell lysates and measurement of enzymatic activity by high-throughput microplate assay

Samples are analysed as above except absorbance measurements are made with UV-transparent 96-well microplates and reagent and cell extract volumes decreased 4- or 5-fold for 250 μl or 200 μl final assay mixture volumes. The change in absorption coefficients no longer apply as they are for 1 nm bandwidth and measurements made with 1 cm light pathlength. In the microplate reader, the bandwidth is typically 10 nm and the light pathlength approximately 0.4–0.5 cm. The change in absorption coefficients can be deduced by determined specific absorbance for reaction reagents and products in the Glo1 and Glo2 reactions in the microplate readers for wavelength-specific filter or monochromator bandwidths and pathlengths used. We have used 96-well flat-bottomed UV-transparent microplates (Corning), multichannel pipette and plate reader (FLUOstar OPTIMA, BMG Labtech) for measuring enzymatic activity of cell lysates for high-throughput. The A240 was followed by reading the absorbance of samples every 1 min for 20 min at 37°C. For optimal assay conditions of Glo1 activity, we found that 4 μg of sample proteins per well gives a measurable and linear increase in absorbance over 10 min of incubation, although this depends on the specific Glo1 activity of the sample.

Comparison with older protocols

In early periods of glyoxalase research, imidazole buffer, often supplemented with Mg2+ ions, was used for the measurement of Glo1 activity; for example, 7.9 mM MG, 1 mM GSH, 14.6 mM MgSO4 and 182 mM imidazole/HCl (pH 7.0) [17]. Imidazole buffer should not be used as it catalyses the isomerization of HA to S-lactoylglutathione [12]. Magnesium salts are not required and very high concentrations of MG are to be avoided as they will produce some inhibition of Glo1. When kinetic constants of Glo1 are to be determined, for example Michaelis–Menten constant Km and enzyme turnover kcat, the concentration of the HA substrate should be computed from the equilibrium constant of HA formation (333 M−1 [11]), maintaining a relative low and constant concentration of GSH at equilibrium, for example 1 mM, as GSH is a competitive inhibitor of Glo1 (Ki=8 mM) [1].

It has been noted in some studies that phosphate buffer may be a weak inhibitor of Glo1 and approximately 10% higher activity was obtained by using Mops buffer instead [18,19]. We did not find this in lysates of human cells in culture, but it may be worth trying in particular samples of interest.

The activity of Glo1 and Glo2 may be measured with other substrates, such as glyoxal, hydroxypyruvaldehyde and phenylglyoxal. When this is desired, appropriate values for Δε240 may be deduced from the Δε240 of S-glycolylglutathione, S-L-glyceroylglutathione and S-D-mandelylglutathione [10].

Assessment for effect of bioactives and drugs on Glo1 activity by microplate assay

For an in vitro bioassay, it is important to establish a more simple, rapid and quantitative high-throughput method. The microplate-based method provides this (Table 2). It is a suitable method for simply and rapidly measuring large numbers of samples in screening bioactive and drug responses.

Table 2
Comparison of two bioassay techniques for assessing glyoxalase activity

For Glo1 and Glo2 assay, avoid imidazole buffer and high concentrations of MG that inhibit glyoxalases.

Characteristic Spectrophotometer-based assay Microplate reader assay 
Sample throughput Low-throughput Medium/high-throughput 
Simplicity Yes Yes 
Time required 15 min per sample 15 min per plate 
Sensitivity Moderate Moderate 
Quantitative Yes Yes 
Consistency Excellent (‘gold standard’) Good 
Characteristic Spectrophotometer-based assay Microplate reader assay 
Sample throughput Low-throughput Medium/high-throughput 
Simplicity Yes Yes 
Time required 15 min per sample 15 min per plate 
Sensitivity Moderate Moderate 
Quantitative Yes Yes 
Consistency Excellent (‘gold standard’) Good 

A one-step microplate assay was established for simple and rapid measurements of glyoxalase enzymatic activities in cell lysates. In addition, the assay in a large number of samples is possible and it will allow screening of bioactives that may up-regulate glyoxalase in the cells. The microplate method will be useful in elucidating the cellular and molecular mechanisms of glyoxalase metabolism, such as in metabolic function in cell and clinical samples [6,20].

Glyoxalase Centennial: 100 Years of Glyoxalase Research and Emergence of Dicarbonyl Stress: A Biochemical Society Focused Meeting held at the University of Warwick, U.K., 27–29 November 2013. Organized and Edited by Naila Rabbani and Paul Thornalley (University of Warwick, U.K.).

Abbreviations

     
  • AG

    aminoguanidine

  •  
  • Glo

    glyoxalase

  •  
  • HA

    hemithioacetal

  •  
  • MG

    methylglyoxal

  •  
  • SLG

    S-D-lactoylglutathione

We thank other members of host research teams for technical assistance: Dr Mingzhan Xue, Dr Jinit Masania, Dr Fozia Shaheen and Dr Attia Anwar.

Funding

This study was supported by a grant from Young Researcher Overseas Visits Program for Accelerating Brain Circulation by Japan Society for the Promotion of Science (JSPS) fellowship: the collaborative study between Japan and the U.K. of molecular mechanisms of glycation and oxidative stress in psychiatric disorders.

References

References
1
Thornalley
P.J.
The glyoxalase system in health and disease
Mol. Aspects Med.
1993
, vol. 
14
 (pg. 
287
-
371
)
2
Rabbani
N.
Thornalley
P.J.
Methylglyoxal, glyoxalase 1 and the dicarbonyl proteome
Amino Acids
2012
, vol. 
42
 (pg. 
1133
-
1142
)
3
Thornalley
P.J.
Waris
S.
Fleming
T.
Santarius
T.
Larkin
S.J.
Winklhofer-Roob
B.M.
Stratton
M.R.
Rabbani
N.
Imidazopurinones are markers of physiological genomic damage linked to DNA instability and glyoxalase 1-associated tumour multidrug resistance
Nucleic Acids Res.
2010
, vol. 
138
 (pg. 
5432
-
5442
)
4
Rabbani
N.
Thornalley
P.J.
Niwa
T.
Dicarbonyls (glyoxal, methylglyoxal, and 3-deoxyglucosone)
Uremic Toxins
2012
Chichester
John Wiley & Sons
(pg. 
177
-
192
)
5
Rabbani
N.
Xue
M.
Thornalley
P.J.
Activity, regulation, copy number and function in the glyoxalase system
Biochem. Soc. Trans.
2014
, vol. 
42
 (pg. 
419
-
424
)
6
Xue
M.
Rabbani
N.
Momiji
H.
Imbasi
P.
Anwar
M.M.
Kitteringham
N.R.
Park
B.K.
Souma
T.
Moriguchi
T.
Yamamoto
M.
Thornalley
P.J.
Transcriptional control of glyoxalase 1 by Nrf2 provides a stress responsive defence against dicarbonyl glycation
Biochem. J.
2012
, vol. 
443
 (pg. 
213
-
222
)
7
Thornalley
P.J.
Rabbani
N.
Glyoxalase in tumourigenesis and multidrug resistance
Semin. Cell Dev. Biol.
2011
, vol. 
22
 (pg. 
318
-
325
)
8
McLellan
A.C.
Thornalley
P.J.
Sample storage conditions for the assay of glyoxalase activities in whole blood samples
Ann. Clin. Biochem.
1992
, vol. 
29
 (pg. 
222
-
223
)
9
Talesa
V.
Uotila
L.
Koivusalo
M.
Principato
G.B.
Giovanni
E.
Rosi
G.
Demonstration of glyoxalase II in rat liver mitochondria: partial purification and occurrence in multiple forms
Biochim. Biophys. Acta
1988
, vol. 
955
 (pg. 
103
-
110
)
10
Clelland
J.D.
Thornalley
P.J.
S-2-hydroxyacylglutathione derivatives: enzymatic preparation, purification and characterisation
J. Chem. Soc. Perkin Trans. 1
1991
, vol. 
1991
 (pg. 
3009
-
3015
)
11
Vander Jagt
D.L.
Daub
F.
Krohn
J.A.
Han
L.P. B.
Effects of pH and thiols on the kinetics of yeast glyoxalase I: an evaluation of the random pathway mechanism
Biochemistry
1975
, vol. 
14
 (pg. 
3669
-
3675
)
12
Hall
S.S.
Doweyko
A.M.
Jordan
F.
Glyoxalase I enzyme studies. 4. General base catalysed enediol proton transfer rearrangement of methylglyoxal and phenylglyoxal-glutathionylhemithiol acetal to S-lactoyl-S-mandeloyl-glutathione followed by hydrolysis: a model for the glyoxalase system
J. Am. Chem. Soc.
1978
, vol. 
100
 (pg. 
5934
-
5939
)
13
Uotila
L.
Purification and characterization of S-2-hydroxyacylglutathione from human liver
Biochemistry
1973
, vol. 
12
 (pg. 
3944
-
3951
)
14
McLellan
A.C.
Thornalley
P.J.
Synthesis and chromatography of 1,2-diamino-4,5-dimethoxybenzene, 6,7-dimethoxy-2-methylquinoxaline and 6,7-dimethoxy-2,3-dimethylquinoxaline for use in a liquid chromatographic fluorimetric assay of methylglyoxal
Anal. Chim. Acta
1992
, vol. 
263
 (pg. 
137
-
142
)
15
Thornalley
P.J.
Yurek-George
A.
Argirov
O.K.
Kinetics and mechanism of the reaction of aminoguanidine with the α-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone under physiological conditions
Biochem. Pharmacol.
2000
, vol. 
60
 (pg. 
55
-
65
)
16
Allen
R.E.
Lo
T.W.C.
Thomalley
P.J.
Purification and characterisation of glyoxalase II from human red blood cells
Eur. J. Biochem.
1993
, vol. 
213
 (pg. 
1261
-
1267
)
17
Oray
B.
Norton
S.J.
Purification and characterization of mouse liver glyoxalase II
Biochim. Biophys. Acta
1980
, vol. 
611
 (pg. 
168
-
173
)
18
Suttisansanee
U.
Lau
K.
Lagishetty
S.
Rao
K.N.
Swaminathan
S.
Sauder
J.M.
Burley
S.K.
Honek
J.F.
Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum
J. Biol. Chem.
2011
, vol. 
286
 (pg. 
38367
-
38374
)
19
Deponte
M.
Sturm
N.
Mittler
S.
Harner
M.
Mack
H.
Becker
K.
Allosteric coupling of two different functional active sites in monomeric Plasmodium falciparum glyoxalase I
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
28419
-
28430
)
20
Arai
M.
Yuzawa
H.
Nohara
I.
Ohnishi
T.
Obata
N.
Iwayama
Y.
Haga
S.
Toyota
T.
Ujike
H.
Arai
M.
, et al. 
Enhanced carbonyl stress in a subpopulation of schizophrenia
Arch. Gen. Psychiatry
2010
, vol. 
67
 (pg. 
589
-
597
)