Analysis of Glyoxalase II Activity by High Performance Liquid Chromatography
A thesis submitted to the Miami University Honors Program in partial fulfillment of the requirements for University Honors
by
Rachel Sidwell
May 2003
Oxford, Ohio Abstract
Glyoxalase II catalyzes the breakdown of S-D-lactoylglutathione into glutathione and lactic acid. The activity of the enzyme is typically analyzed by a continuous spectrophotometric assay, which follows the decomposition of S-D-lactoylglutathione through its absorbance at 240 nm. The new method described here uses an alternative method, high performace liquid chromatography, to monitor S-D-lactoylglutathione concentrations and thus to to analyze the activity of glyoxalase II. Samples of S-D- lactoylglutathione were reacted with the enzyme for varying periods of time, and the enzyme was then denatured with acid. The S-D-lactoylglutathione concentration of these samples was then analyzed by high performance liquid chromatography, and the results were then used to determine the activity of glyoxalase II. This method resulted in kinetic data for glyoxalase II that was similar to that obtained spectrophotometrically.
ii iii
Analysis of Glyoxalase II Activity by High Performance Liquid Chromatography
by Rachel Sidwell
Approved by
______, Advisor Dr. Ann Hagerman
______, Reader Dr. Michael Crowder
______, Reader Dr. Chris Makaroff
iv v Acknowledgements
Thank you to the Miami University Undergraduate Summer Scholars Program, Miami
University Honors Program, Miami University Harrison Scholars Program, Miami
University Department of Chemistry and Biochemistry, Dr. Ann Hagerman, Dr. Michael
Crowder, Dr. Chris Makaroff, Ginger Henson, and Erin McDonough
vi Table of Contents
Introduction 1
Materials and Methods 4
Results 7
Discussion 14
References 16
vii Figures
Figure 1. Reactions of the glyoxalase system
Figure 2. HPLC chromatogram at 233 nm, showing SLG peak at 5.8 min
Figure 3. 300 µM SLG peak area after heating at various temperatures for 10 min
Figure 4. Time course showing partial denaturation of enzyme by heating at 100oC for 1
min
Figure 5. SLG standard curve using HPLC
Figure 6. Spectrophotometric (left) and HPLC (right) time course
Figure 7. Lineweaver-Burke plot of kinetic data obtained spectrophotometrically
Figure 8. Lineweaver-Burke plot of kinetic data obtained by HPLC
viii 1
Introduction
The glyoxalase system consists of two enzymes, glyoxalase 1 (lactoylglutathione lyase) and glyoxalase 2 (hydroxyacylglutathione hydrolase), which together catalyze the conversion of methylglyoxal to D-lactic acid through the intermediate S-D- lactoylglutathione (SLG) [1]. The enzyme system is important for the elimination of mutogenic and cytotoxic compounds formed during carbohydrate and lipid metabolism, as well as in the regulation of cell proliferation [1-3]. Methylglyoxal is a mutagenic toxin produced as a byproduct during carbohydrate and lipid syntheses. It is formed both spontaneously and through enzymatic reactions [1]. Glutathione (GSH) is an essential cellular compound that functions as a reducing agent and as a coenzyme in several important reactions. Due to the toxicity of methylglyoxal, the glyoxalase system is thought to be important in cell detoxification [1]. The glyoxalase system is shown in
Figure 1.
In addition to SLG, glyoxalase II can use a variety of S-2-hydroxyacylglutathione derivatives, producing GSH and the corresponding acid [1]. Many of these different substrates cannot be followed spectrophotometrically due to the fact that they absorb at the same wavelength as GSH or do not absorb at all, thus making it difficult to determine the kinetics of the enzyme with these novel substrates. Analysis by high performance liquid chromatography (HPLC) presents a distinct advantage by separating substrate from other components of the solution due to differing retention times. For example, although
GSH absorbs only weakly at 233 nm, that absorbance is sufficient that its increase over the course of a reaction with glyoxalase II can mask some of the disappearance of SLG in spectrophotometric determinations. Using HPLC prior to detection solves this problem 2
because GSH elutes at a different time from SLG. With HPLC, SLG’s strong absorbance at 233 nm and GSH’s absorbance at 220 nm can be followed simultaneously, allowing both substrate decomposition and product formation to be observed from the same sample. Methods have already been developed to analyze SLG concentration by HPLC
[4], but a glyoxylase activity assay has not been developed using those methods.
HO O O O H H N OH C H2N N H + O O O SH Glyoxalase-1 Methylglyoxal Glutathione HO O O H N OH H N N 2 H O O S OH Glyoxalase-2 O
S-D-Lactoylglutathione HO O O O H N OH H N N OH 2 H O O + OH SH Lactic acid Glutathione
Figure 1. Reactions of the Glyoxalase system There are inherent difficulties in HPLC analysis that must be resolved in order to do kinetic analyses. Many enzyme assays are continuous assays, in which the reaction progress is followed continuously for several minutes. HPLC is not suitable for continuous analysis; the HPLC can only be interpreted if the reaction is no longer 3
occurring during the separation step. In order to follow the enzyme activity, the reaction must be allowed to proceed for a known amount of time and then stopped in some way.
By repeating these analyses, reaction progress determined at multiple time points can then be used to calculate a rate. In addition, the HPLC system is more delicate than a spectrophotometric system, and care must be taken to avoid damaging the instrument; some assay conditions therefore make samples unsuitable for HPLC analysis. In this research I attempted to solve these problems and develop a viable and reliable way to analyze glyoxalase II activity by HPLC.
4
Materials and Methods
A Beckman High Performance Liquid Chromatography system was used with a
507E autosampler, a 168 detector, and a 126 solvent module, with a 5 µ, 4.6 mm x 4.5
cm C-18 reversed phase column. The HPLC was controlled and the data were collected
with Beckman Instruments’ Gold Nouveau Chromatography Data System, Version 1.6.
The mobile phase was 83 mM sodium sulfate, 1.7 mM ammonium formate, and 0.1 mM
sodium cyanide, pH 3.4. Runs were 10 minutes long, with a flow rate of 0.5 mL/min and
no solvent gradient.
SLG, glyoxalase II, GSH, MOPS, and ammonium formate were purchased from
Sigma. Sodium cyanide was purchased from Matheson, Coleman, and Bell. Sodium
sulfate was purchased from Fisher.
SLG in solid form was weighed out, and water was added to make a 10 mM stock
solution. This solution was dispensed in 200 µL aliquots and stored at –4oC. For each
day’s work, a fresh aliquot was removed and thawed, so that no SLG solution was ever
frozen and thawed more than once. Immediately prior to use, the concentration of each
aliquot of SLG was confirmed by mixing a 1 mL sample of 300 µM SLG and 100 mM
MOPS, then determining the spectrophotometric absorbance at 240 nm and calculating
-1 -1 concentration based on the extinction coefficient (ε240 = 3.1 mM cm ).
The concentration of the enzyme was adjusted, using spectrophotometric trials, so
that 10 µL of enzyme added to a 1000 µL sample containg a 300 µM SLG resulted in the conversion of at least half of the SLG to GSH over a five-minute period. For the commercial preparation of enzyme used, the final concentration of the enzyme in the 5
1000 µL sample was 0.3 µg/mL. The spectrophotometric analysis utilized a reaction
volume of 1000 µL, with 100 mM MOPS and 300 µM SLG. The entire sample was
transferred to a cuvette; a spectrophotometer was then used to record an initial
absorbance. A 10 µL aliquot of enzyme was then added, and the absorbance was
followed at 240 nm.
For the chromatographic analysis, the reaction was performed in a 1 mL volume with a known final concentration of SLG, no greater than 300 µM, and with MOPS at a final concentration of 100 mM to buffer the solution at pH 7.2. To obtain a zero time point, a 100 µL aliquot of the reaction mixture was taken prior to enzyme addition, added to a microfuge tubecontaining 10 µL 1.2 M HCl and immediately mixed. The dilute enzyme (10 µL) was added to the solution remaining in the reaction vial, and the reaction was allowed to proceed. At timed intervals, 100 µL aliquots were taken and added to microfuge tubes containing 10 µL 1.2 M HCl, and immedidately mixed. To prepare for
HPLC analysis, each sample was mixed with 110 µL mobile phase, made up of 83 mM sodium sulfate, 1.6 mM ammonium formate, and 0.1 mM sodium cyanide, buffered at pH
3.4. The samples were filtered through a 45 µm filter by centrifuging at 3000 rpm for 10 minutes. An autosampler was programmed to inject 50 µL of each sample onto the
HPLC system.
The standard curve used the same volume of 1000 µL. The solutions contained
100 mM MOPS and SLG in concentrations of 0, 20, 60, 100, 160, 200, 260, and 300 µM.
Aliquots of 100 µL were taken and added to microfuge tubes containing 10 µL 1.2 M 6
HCl and mixed. Each sample was mixed with an equal amount of mobile phase and filtered as above, then analyzed by the HPLC system.
Kinetic data were acquired using the above methods at starting concentrations of
40, 60, 80, 100, 150, 180, 200, 250, and 300 µM. Duplicate samples of each concentration were prepared at the same time, using the same SLG stock. One of these samples was analyzed spectrophotometrically, while the other was used for HPLC, with samples taken once per minute for four minutes. Kinetic data were determined from initial rates.
7
Results
An SLG chromatogram is shown in Figure 2. The peaks between 2 and 3 minutes are solvent peaks. SLG elutes at about 5.7 minutes. The peak areas used to develop the standard curve and those used for the kinetics data were established using the integration software provided with the HPLC. Arbitrary area units were used.
Figure 2. HPLC chromatogram at 233 nm, showing SLG peak at 5.8 min 8
Much of the research period was spent determining a quick and accurate way to
stop the enzymatic reaction by without altering substrate and product concentrations. I
attempted to stop the enzyme by denaturing it using several different methods. At first,
samples were heated at 100oC for 10 minutes, but it was then found that this heating
degraded a significant portion of the SLG. This led to irreproducible results because the
loss of SLG upon heating depended on the time and temperature of the heating step. In
addition, the apparent enzyme activities determined when I attempted to stop the enzyme
by heating were much higher than the actual activities, determined
spectrophotometrically, because I recorded not only the enzymatic destruction of the SLG
but also the heat-induced destruction.
Figure 3. 300 µM SLG peak area after heating at various temperatures for 10 min
In an attempt to prevent this degradation, heating at lower temperatures for 10 minutes was examined. Figure 3 shows the results—heating at temperatures of 70oC and 9
below caused far less SLG to degrade than heating at 100oC. An attempt was made to
denature the enzyme by heating for 10 min at 70oC, but the first sample, heated at 0.25
min after adding enzyme, showed an SLG concentration less than 2 µM; all points taken later similarly showed almost no SLG. This indicates that heating at 70oC was not
effective in denaturing the enzyme. Next, an experiment was performed to determine
how long samples needed to be heated at 100oC. It was determined initially that heating at 1 min was sufficient to denature the enzyme, but later time courses showed that the enzyme was only partially denatured, as in Figure 4. The large decrease in peak area between the sample taken before enzyme was added (the point at Time = 0) and the sample taken immediately after adding enzyme (the point at Time = 0.25) indicates that
the reaction continued rather than immediately stopping upon heating.
Figure 4. Time course showing partial denaturation of enzyme by heating at 100oC for 1
min 10
A new method needed to be found to denature the enzyme. It was hypothesized that adding a small amount of acid would drop the pH of the solution enough to denature the enzyme and stop the reaction. Concentrated HCl was used. HCl successfully denatured the enzyme, providing similar values for SLG concentration before and immediately after the enzyme was added and a linear rate thereafter. The lowest concentration of acid that successfully stopped the reaction was found to be 10 µL of 1.2
M HCl stock added to the 100 µL sample aliquot. A successful time course with this method of stopping the reaction is shown in the right panel of Figure 6. After an appropriate method of stopping the reaction had been found, a standard curve could be developed with HCl, and rates could be compared between data obtained using the spectrophotometer and that obtained using the HPLC system.
Figure 5. SLG standard curve using HPLC
11
An SLG standard curve, shown in Figure 5, was established, using known
concentrations of SLG from 0 to 300 µM. Each point represents the mean of three runs from the same sample; standard deviation bars are too small to be seen. This standard curve was determined with 10 µL of 1.2 M HCl added for every 100 µL of sample. The standard curve was developed in order to have a means of converting peak areas provided by the HPLC, given in arbitrary area units, into SLG concentrations so that the reaction rate may accurately be determined.
Spectrophotometric data were gathered so that it might be compared with data provided by the HPLC system. The left panel in Figure 6 shows the loss of SLG over time at a wavelength of 240 nm. The change in absorbance over the first four minutes is
–0.109 Abs/min. At 240 nm, SLG has an extinction coefficient of 3.1 mM-1cm-1. The reaction was performed with a cell length of 1 cm, and so, using A=εbc, the change in
SLG concentration was –35 µM/min; with a starting concentration of 300 µM, that is equivalent to a loss of 12% of the substrate each minute.
The right panel in Figure 6 shows a sample time course using the acid stop method and analysis with HPLC. The peak areas provided by the HPLC system have been converted to SLG concentrations using the equation provided by the standard curve, shown in Figure 2. The equation of the line gives a change in [SLG] of –29 µM/min.
With a 300 µM starting concentration, this is equivalent to a loss of 10% of the substrate each minute.
12
Figure 6. Spectrophotometric (left) and HPLC (right) time course
0.12
y = 3.7671x + 0.0125 0.1 R2 = 0.996
0.08
0.06 M/min) µ 0.04 1/V (
0.02
0 -0.01 0 0.01 0.02 0.03 -0.02 1/[SLG] (µM)
Figure 7. Lineweaver-Burk plot of kinetic data obtained spectrophotometrically
A Lineweaver-Burk plot of the kinetic data determined by the spectrophotometric
method is shown in Figure 7. Data at 150, 180, and 200 µM concentrations were not determined due to operator error. Absorbance readings were converted to SLG 13
concentration in µM by use of a standard curve equation. The spectrophotometric data
give a Vmax of 80.0 µM/min and a KM of 301 µM.
0.16 y = 5.2243x + 0.0111 0.14 R2 = 0.9868 0.12
0.1
0.08
M/min) 0.06 µ 0.04 1/V ( 0.02
0 -0.01 0 0.01 0.02 0.03 -0.02
-0.04 1/[SLG] (µM)
Figure 8. Lineweaver-Burk plot of kinetic data obtained by HPLC
A Lineweaver-Burke plot of the kinetic data determined by the HPLC method is shown in Figure 8. Peak areas were converted to SLG concentration by use of a standard curve equation. The HPLC data give a Vmax of 90.1 µM/min and a KM of 471 µM. 14
Discussion
The HPLC method seems to work well. The SLG peak is easily identified and
integrated, and peak area increases linearly with SLG concentration, as shown in Figure
2. The method of stopping the reaction, using small quantities of 1.2 M HCl, also seems to work efficiently.
The time points in Figure 6 show that the concentration of SLG falls in a linear fashion over time, as it does in the reaction monitored spectrophotometrically in Figure 6.
Elementary calculations for the reaction rates show that they are very similar for the two methods, with a 12% loss per minute when analyzed spectrophotometrically and a 10% loss per minute when analyzed by the HPLC. All aspects of the method appear to work well to provide accurate SLG concentrations and accurate estimations of glyoxalase II activity.
Comparison of the kinetic data provided by the two methods, spectrophotometric and HPLC, show that the Vmax determined by HPLC is 12.6% higher than that
determined by spectrophotometry, and the KM determined by HPLC is 56.5% higher than
that determined using the spectrophotometric method. These differences are rather large,
but there is a possible reason for them. As mentioned in the introduction, GSH, a product
of the reaction, shows some absorbance at 240 nm, the wavelength at which the solutions
were analyzed spectrophotometrically. This could result in the spectrophotometric rates
appearing slower than they actually are, meaning that the HPLC kinetic data are actually
more accurate.
There is a great deal of future work to be done. GSH, one of the products of the
reaction, must also be followed as it is formed. A standard curve must be established for 15
GSH, and its rate of production must be determined and compared to the rate of SLG loss. Alternate substrates should be examined to determine if this method can be used to analyze the enzyme’s activity with substrates other than SLG. Kinetic experiments should be repeated to determine if the differences in KM can be reduced. This future work should provide a better view of the efficiency and usefulness of the HPLC method of analyzing the activity of Glyoxalase II.
16
References
[1] Thornalley P.J. (1993). The Glyoxalase System in Health and Disease. Molecular. Aspects of Medicine 14, 287-371.
[2] Bito A., Haider M., Briza P., Strasser P., and Breitenbach M. Heterologous Expression, Purification, and Kinetic Comparison of the Cytoplasmic and Mitochondrial Glyoxalase II Enzymes, Glo2p and Glo4p, from Saccharomyces cerevisiae. Protein Expression and Purification 17, 456-464.
[3] Martins A.M., Cordeiro C., and Freire A.P. (1999). Glyoxalase II in Saccharomyces cerevisiae: In Situ Kinetics Using the 5,5’-Dithiobis(2- nitrobenzoic Acid) Assay. Archives of Biochemistry and Biophysics 366:1, 15- 20.
[4] McLellan A.C., Phillips S.A., and Thornalley P.J. (1993). The Assay of S-D- Lactoylglutathione in Biological Systems. Analytical Biochemistry 211, 37-43.