Automated Assays for Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione Reductase Activity’ Conrad R. Wheeler,2 Jhaine A. Salzman, Division
Nabil M. Elsayed, Stanley of Research, Presidio
T. Omaye, and Don W. Korte,
of San Francisco,
Received August 21, 1989
Automated assays for catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase are presented. The assay for catalase is based on the peroxidatic activity of the enzyme. The glutathione peroxidase and reductase assays measure the consumption of NADPH following the reduction of t-butyl hydroperoxide and oxidized glutathione, respectively. The assay for superoxide dismutase is based on the reduction of cytochrome c. All assays utilize the Cobas FARA clinical automated analyzer and provide considerable time savings over the manual assays. o ISSO Academic
Products of oxygen reduction can lead to free radical mediated reactions in the cell with toxic consequences. Under normal circumstances the levels of reduced oxygen products are low enough to be effectively removed by the natural defense mechanisms of the cell. There are many compounds, however, that enhance the production of oxygen radicals to such an extent that cellular defenses are overwhelmed, and the cell is injured. To establish the mechanism of toxicity as oxygen radical mediated, there are a number of direct and indirect methods. Direct methods include the measurement of superoxide, hydrogen peroxide, or hydroxyl radical. These species are very reactive and their quantitation can be difficult. Therefore, indirect methods of study are often used. One
such method is the measurement of changes in endogenous antioxidant enzyme activity (1). The antioxidant enzymes include superoxide dismutase (SOD),3 catalase (CAT), glutathione peroxidase (GP), and indirectly, glutathione reductase (GR). Their roles as protective enzymes are well known and have been investigated extensively both in uiuo and in model systems. The first three enzymes directly catalyze the transformation of peroxides and superoxide to nontoxic species. Glutathione reductase reduces oxidized glutathione (GSSG) to glutathione (GSH), a substrate for glutathione peroxidase. The consequences of oxidative stress are serious and, in many cases, are manifested by increased activity of enzymes involved in oxygen detoxification (2-8). In our laboratory we have been studying the effects of alkylating agents in producing oxidative stress. In the course of a typical experiment a number of parameters are measured, including the activities of SOD, CAT, GP, and GR. Due to the tremendous volume of assays a simple in uivo experiment generates, the manual assays require too much time to be feasible. A review of the literature showed that none of the specific methods we wished to use had been automated. In this paper we present automated methods for the determination of CAT (peroxidatic activity), GP, GR, and SOD. To accomplish this, enzyme assays were adapted to the Cobas FARA clinical automated analyzer. Although the automated assays presented here are specifically designed for the Cobas FARA, they can be adapted to a variety of other clinical analyzers. MATERIALS
’ The opinions, interpretations, and conclusions contained in this report are those of the authors and do not reflect the views of the Department of the Army. In conducting the research described in this report, the investigation adhered to the NIH’s “Guide for the Care and Use of Laboratory Animals.” * To whom correspondence should be addressed. 0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Materials Catalase (EC 126.96.36.199, from bovine liver), glutathione reductase (EC 188.8.131.52, type III from baker’s yeast), gluta3 Abbreviations GP, glutathione serum albumin;
used: SOD, superoxide dismutase; CAT, catalase; peroxidase; GR, glutathione reductase; BSA, bovine t-BuOOH, tert-butyl hydroperoxide. 193
thione peroxidase (EC 184.108.40.206, from bovine [email protected]
), superoxide dismutase (EC 220.127.116.11, from bovine liver), xanthine oxidase (EC 18.104.22.168, from buttermilk), bovine serum albumin (BSA, fraction IV), NADP+, NADPH, GSH, GSSG, hydrogen peroxide (H,O,), tertbutyl hydroperoxide (t-BuOOH), xanthine, and ferricytochrome c were purchased from Sigma Chemical Co. (St. Louis, MO). Purpald (5mercapto-1,2,4-triazole, 4-amino-5-hydrazino-4H-1,2,4-triazole-3-thiol), potassium cyanide, and potassium periodate were obtained from Aldrich Chemical Co. (Milwaukee, WI). All solutions were prepared in deionized, purified water. Buffers were stored at 4°C while enzyme and cofactor/substrate solutions were prepared immediately before use. Catalase solutions were prepared in phosphate buffer (25 mM, pH 7.0) containing 2 mg BSA/ml; solutions of GP, GR, and SOD were prepared in 10 mM Hepes containing BSA (2 mg/ml). The use of BSA in the buffer containing the commercial purified enzymes significantly increased their stability and reduced the variation from assay to assay. Manual Enzyme Assays Manual assays were conducted on a Shimadzu (Shimadzu Scientific Instruments, Columbia, MD) UV-265 spectrophotometer using 1 cm pathlength quartz cuvettes with a total volume of 1.5 ml. Catalase (catalase activity). The catalase activity of catalase was determined at 20°C by following the decomposition of HzOz at 240 nm according to the method of Aebi (9). Using a molar extinction coefficient of 43.6 M-’ cm-‘, the rate for the first 30 s was used to calculate the units of activity. One unit is equal to the decomposition of 1 pmol H,Oz/min/ml. Catalase (peroxidatic activity). The peroxidatic activity of catalase was measured at 20°C using an assay based on the method of Johansson and Borg (10). The following were added to a 13 X loo-mm test tube: 150 ~1 phosphate buffer (250 mM, pH 7.0), 150 ~1 methanol (12 M), and 30 ~1 HzOz (44 mM). The reaction was initiated with 300 ~1 of sample. The reaction was allowed to proceed for lo-20 min and terminated by the addition of 450 ~1 Purpald (22.8 mM in 2 N potassium hydroxide). The incubation mixture was mixed briefly on a vortex mixer and left for 20 min. Potassium periodate (65.2 mM in 0.5 N potassium hydroxide, 150 ~1) was added, and the tube vortexed briefly again. The absorbance of the purple formaldehyde adduct produced was measured at 550 nm. Standard solutions of formaldehyde ranging from 25 to 500 pM were prepared in 25 mM phosphate buffer, pH 7.0. Glutathione peroxidase. GP was assayed at 25°C by the method of Flohe and Gunzler (ll), with one modification. The amount of GR was increased from 0.24 to 0.5 U/ml of incubation mixture. Units of enzyme activity
were calculated using a millimolar extinction coefficient of 6.22 mM-’ cm-’ for NADPH. Glutathione reductase. Activity was determined at 25°C by following the generation of NADP+ from NADPH during the reduction of GSSG according to the method of Goldberg and Spooner (12). Automated
Automated assays were performed using a Cobas FARA clinical analyzer (Roche Diagnostic Systems, Inc., Montclair, NJ). Once the reagents and samples have been placed on this instrument, all reagent additions, mixing, spectrophotometric analyses, and calculations are performed automatically according to the programmed instructions. The essential details of analysis are presented as follows. Assay reagents are placed in a container with compartments for a main reagent (capacity, 20 ml) and a start reagent (capacity, 5 ml). Samples are added to 600~~1 disposable plastic tubes and placed in a rack adjacent to the reagent rack. Incubations are performed in a rotor composed of 30 cuvettes. Each cuvette has a sample well (maximum volume, 400 ~1) and a reagent well (maximum volume, 95 ~1). Two robotic pipetting arms add sample or reagent to the appropriate wells. An optional rinse volume (water) may be programmed for the sample pipettor to ensure complete delivery of the sample or start reagent. The contents of the two wells are combined in the cuvette (maximum volume 400 ~1) by centrifugal action as the rotor is spun. The longitudinal or long axis of the cuvette is parallel to the light path and absorbance is monitored while the cuvette rotor is spinning. With this arrangement the absorbance is independent of the total filling volume of the cuvette (13). The calibration of the sample pipettor was determined using solutions of potassium dichromate in 0.01 N sulfuric acid (14) containing 2 mg BSA/ml. The absorbance at 350 nm was stable for at least 1 h in the presence of the protein. Catalase (peroxidatic activity). The automated assay was conducted using the same proportion of reagents and sample as used in the manual procedure. The buffer, methanol, and HzOz solutions were combined (referred to as Assay reagent and added to the reagent container manually. The Purpald solution was added to the start reagent container. From this point on, the process was performed automatically by the instrument according to the stored program. Assay reagent (66 ~1) and sample (30 ~1, with a 30-111rinse) were added to cuvettes, mixed, and incubated for 10 min. At 10 min, 90 ~1 of Purpald solution (sample probe, no rinse) was added, mixed in the cuvette, and left for 15 min. During the 15-min derivatization period the Purpald container was manually exchanged for one containing potassium periodate solution. After the 15-min derivatization, 30 ~1 of periodate
solution was added with the sample probe (no rinse) and mixed. The color was allowed to develop for 5 min and the absorbance was measured at 550 nm. A series of formaldehyde standards was analyzed each time the assay was performed. Glutathioneperoxidase. The final reagent concentrations were the same as those for the manual assay. To simplify pipetting, the following were combined in the reagent container: 9.25 ml phosphate buffer (100 mM, 1 mM EDTA, pH 7.0), 1.25 ml NADPH (2.25 mM in 0.1% sodium bicarbonate), 0.25 ml GR (25 U/O.25 ml phosphate buffer), and 0.5 ml GSH (37.5 mM in buffer). The start reagent container was filled with t-BuOOH. To begin the analysis, Assay reagent (225 ~1) and sample (40 ~1, with a 50-~1 rinse) were added to the cuvette and mixed by spinning the rotor. t-BuOOH (15 mM in water, 30 ~1 with a 30-~1 rinse) was added to the reagent well. After 2.5 min of temperature equilibration the rotor was spun to mix the start reagent with the cuvette contents. The absorbance was measured at 340 nm every 10 s for 2 min, and the enzyme activity calculated automatically. Glutathione reductase. The final reagent concentrations were the same as those for the manual assay. The following were combined in the reagent container: 20 ml potassium phosphate buffer (147 mM, 0.74 mM EDTA, pH 7.2) containing GSSG (3.7 mM). NADPH (2.0 mM in 0.1% sodium bicarbonate) was added to the start reagent container. The assay was started and performed as described above for GP. Super-oxide dismutase. This assay is based on the method developed by McCord and Fridovich (15) as modified by Elstner et al. (16). Adaptation of the assay to the Cobas necessitated the use of several rinse volumes of water. In order to achieve the same concentration of phosphate buffer as that in the manual assay, all reagents were prepared in 73.5 mM potassium phosphate containing 0.15 mM DETAPAC, pH 7.8. The following were manually combined in the reagent container: 3.750 ml ferricytochrome c (0.05 mM), 0.721 ml xanthine (1.3 mM), 0.360 ml potassium cyanide (520 PM), 0.375 ml catalase (50 units/ml), and 6.044 ml buffer. Xanthine oxidase was diluted with buffer such that a 30-~1 aliquot would catalyze an absorbance change of 0.030 per minute at 550 nm, due to the reduction of cytochrome c (a total volume of 375 ~1 gives a pathlength of 1.5 cm). This solution was then added to a start reagent container and placed on the Cobas. SOD solutions were added to sample vials and placed in the rack. From this point on, the process was performed automatically by the instrument according to the stored program. Minor adjustments in the concentration of xanthine oxidase were made by programming the instrument to vary the amount of the enzyme (start reagent) and rinse volumes on the Cobas (60 ~1 total volume). The assay was initiated by transferring reagent (225 ~1) and sample (40 ~1, with a 50-~1 rinse) to
the cuvette and mixing. After allowing 45 s for temperature equilibration, the absorbance was measured every 15 s for a total of 90 s. Xanthine oxidase (30 ~1, with a 30-~1 rinse) was added and allowed 30 s for temperature equilibration before being mixed with the contents of the cuvette. Ten seconds after mixing, the rate of increase in absorbance was measured every 10 s for 2 min. Samples of enzyme and buffer were analyzed simultaneously to obtain the inhibited/noninhibited rates of cytochrome c reduction. For the determination of SOD activity in tissue the concentration of sample was adjusted to produce a 40 to 60% inhibition of the maximum rate. The amount of enzyme necessary to produce a 50% inhibition (1 unit) was then calculated. Preparation of liver and lung cytosol: Liver and lungs from Sprague-Dawley rats were homogenized in an icecold buffer (w/v, 1:9) containing 0.15 M sucrose, 0.15 M mannitol, and 10 mM Hepes, pH 7.4, using a Polytron homogenizer (Brinkman Instruments, Westbury, NY). Samples of the homogenates were removed for protein analysis according to the Lowry method (17), and the remainder was centrifuged at 30,OOOg for 30 min at 4°C. The supernatants were diluted as indicated for the enzyme assays. Calculations
Activity for all enzymes except SOD was calculated in terms of international units (II). Units of GP and GR activity for the manual assays were calculated in the usual manner using the Beer’s law relationship. The Cobas FARA, however, measures absorbance along a longitudinal light path; thus, the following equation was used (18): Units/ml
= aA)( a
where AA = change in absorbance per minute at 340 nm; a = millimolar extinction coefficient of NADPH (6.22 mM-’
r = the length of each side across the base of the cuvette (0.5 cm); SV = sample volume (ml). CAT activity was determined by linear least-squares regression of the final absorbance of the formaldehyde standards. Standard curves were linear up to 600 pM with the automated assay. SOD activity was expressed as the amount of protein causing 50% inhibition of cytochrome c reduction under the conditions of the assay. The concentration of liver or lung cytosol was varied until the inhibition was between 45 and 55%. The 50% value was calculated by interpolation.
CAT + HzOz -N CAT-H202
+ H202 + CAT + 2Hz0 + O2 (catalase activity)
To validate the automated assays for GP and GR, solutions of the enzymes were analyzed simultaneously using the conventional manual spectrophotometric assay and the automated method. The assays for CAT and SOD were conducted only on the Cobas FARA since standard curves were employed. The enzyme catalase catalyzes two reactions which proceed through a common intermediate, referred to as complex I (Reaction [ 11). This intermediate is formed by the binding of hydrogen peroxide to the enzyme. Reaction of complex I with a second molecule of hydrogen peroxide leads to the formation of water and oxygen (Reaction ). If a hydrogen donor (AH,) such as methanol or ethanol reacts with complex I, the products are water and formaldehyde or acetaldehyde (Reaction ):
5: ; 0.8 -
l Formaldehyde 0 Buffer Blank A Formaldehyde
P 0.4 -
Sample Sample - Blank
8 fj 0.6 $
10 15 20 25 Derivatization Time (min)
Dependence of the final absorbance on derivatization time with Purpald. The assay was performed using the manual method (n = 3). Only the standard deviation for the formaldehyde sample is shown.
+ AH2 --f CAT + 2Hz0 + A (peroxidatic
The relative rates of product formation depend on the concentration of hydrogen peroxide and the hydrogen donor. Catalase activity is usually determined by monitoring the decomposition of hydrogen peroxide at 240 nm. An assay for the second reaction, or peroxidatic activity of catalase, was recently developed by Johanssen and Borg (10). In this assay the formaldehyde produced from methanol is reacted with Purpald to produce a chromophore. Quantitation is accomplished by measuring the absorbance at 540 nm and comparing the result to those obtained with formaldehyde standards. In our laboratory we automated only the assay for peroxidatic activity. The Cobas FARA, like most automated analyzers, uses disposable plastic cuvettes that cannot be used in the uv region, thus only the latter assay was feasible. A number of methods employ Purpald for quantitating formaldehyde and the concentration of base is generally 1 to 2.5 M (19-21). When 2 M potassium hydroxide was substituted for the 7.8 M solution used by Johanssen and Borg, there was no difference after subtraction of the blank values. The use of the higher concentration led to higher absorbance values including buffer blank values. In the original assay of Johanssen and Borg (lo), Purpald was dissolved in dilute acid and added immediately after the addition of 7.8 M KOH. Although Purpald was more stable in the acidic solution, we found it to be stable in base (for approximately 2 h). Thus dissolving Purpald directly in 2 M KOH had no effect on the assay and sim-
plified the procedure. Agitation of the solution during derivatization produced a purple solution due to air oxidation of the initial adduct of formaldehyde with Purpal& without agitation, the color was faint. However, on the addition of the oxidizing agent, potassium periodate, this was reversed. After addition of the potassium periodate, the solution that was not agitated was considerably darker. In order to determine the minimum amount of time required for derivatization in the absence of continuous agitation, a series of timed incubations were performed using the 150 I.IM formaldehyde standard. As shown in Fig. 1, the reaction required approximately 20 min to reach completion. Proteins can react with formaldehyde and thus decrease the amount that could react with the derivatizing agent. To determine this effect we used bovine serum albumin. The assay was conducted with BSA concentrations ranging from 0 to 250 pg/ml in the presence of formaldehyde concentrations ranging from 0 to 300 j&M. The addition of protein always diminished the final absorbance to some extent. The use of a non-protein-containing blank led to errors of 20-40% at low catalase concentrations. However, there was no effect of protein on the assay as long as the appropriate controls were used. A buffer blank should be used with the standards and a protein solution should be used for the protein-containing samples. When these controls were used, the apparent inhibition by protein was 6% or less with the 25,50, and 100 PM samples, and nonexistent with higher concentrations of formaldehyde. For actual experiments a boiled aliquot of the sample would serve as the protein blank.
G g 2 E 2 6
8.1 U/ml Catalase
Linearity of formaldehyde production with time. Samples with catalase activities of 3.6 + 0.2 and 8.1 + 0.8 units/ml were examined using the assay for peroxidatic activity (manual method). Based on the formaldehyde produced after 9 min of incubation the corresponding peroxidatic activities were 8.5 f 0.04 and 19.7 f 0.11 mu/ml, respectively. Each point represents the mean of four determinations; standard deviations are smaller than the symbols plotted.
The linearity of formaldehyde production was examined with purified bovine liver catalase using the manual assay. Timed reactions were conducted up to 15 min. The resulting absorbance after derivatization and oxidation is presented in Fig. 2. At the higher concentration (8.1 units/ml catalase activity) the activity is not quite linear for the full 15 min; however, both samples were linear for 10 min. For the sake of comparison, the catalase activity of catalase is presented in Table 1 along with the peroxidatic activity. Although it has been established that the catalase and peroxidatic activities are related (27) we are just beginning to study the relationship. The results in Table 1 and other unpublished data show that the peroxidatic activity is over a hundred times less than the catalase activity under the conditions of the assays. In fact, the successof the peroxidatic activity assay is due to the high sensitivity of the Purpald assay for formaldehyde. SOD catalyzes the dismutation of superoxide to hydrogen peroxide (Reaction ):
20; + HzOz + Hz0
Most assays measure the enzyme activity indirectly. This is accomplished by including in the assay mixture a source of superoxide and a compound that reacts with superoxide to produce a measurable product. SOD inhibits product formation and one unit of activity is usually
defined as the amount of SOD that inhibits product formation by 50%. The simplest indirect assays use as sources of superoxide the autooxidation of compounds such as epinephrine, hydroxylamine, or pyrogallol. In this case superoxide acts as a chain-propagating species to produce an oxidized product that can be measured spectrophotometritally. The assay using pyrogallol has already been adapted to the Cobas Bio automated analyzer (22). However, several compounds, including BSA, glutathione, and ascorbate, interfere with the autooxidation of pyrogallol. A comparison of seven different SOD assay methods showed that the method utilizing pyrogallol is most sensitive to inhibition by BSA (23). Glutathione and ascorbate at low6 M inhibit the reaction by 20% (24,25). In addition, pyrogallol is a good substrate for peroxidase. We automated the widely used method developed by McCord and Fridovich (15). In this assay the reduction of cytochrome c by superoxide generated from xanthine and xanthine oxidase is followed at 550 nm. Adaptation to the Cobas FARA was accomplished by scaling down the reagent volumes. To investigate the effect of SOD concentration on the reduction of cytochrome c, incubations were conducted with varying amounts of SOD. The results are presented in Fig. 3. The concentration of SOD producing 50% inhibition corresponded to approximately 0.14 pg SOD/ml. This value is very close to reported values ranging from 0.1 to 0.15 pg/ml(10,23,26). GP catalyzes the reduction of hydroperoxides by GSH (Reaction ) and GR catalyzes the NADPH-dependent reduction of GSSG (Reaction ): ROOH + 2GSH 2 ROH + GSSG + Hz0
(R = H, aliphatic or aromatic organic group) GSSG + NADPH
+ H+ 2 2GSH + NADP+
Results for the manual and automated assays of GP and GR activity are presented in Table 2. For both enzymes, the automated analyzer values were initially about 10%
Comparisonof the Catalase(Manual Assay) and Peroxidase (Automated Assay) Activities of Catalase” Manual assay for catalase activity (U/ml) Mean + SD
Automated assay for peroxidatic activity (mu/ml) Mean k SD 5.14 + 0.12 7.81 + 0.34 12.81 XL 0.02
3.62 ic 0.16 4.61 f 0.39 8.09 + 0.84 a Both assays were performed concentrations (n = 3).
SOD Cone @g/ml) FIG. 3. The inhibition of cytochrome c reduction by bovine superoxide dismutase (SOD). Each point represents the mean of three determinations; most standard deviations are smaller than the symbols plotted.
lower than the values determined on the spectrophotometer. When the automated assay was scaled up and conducted manually on the spectrophotometer, the resulting values were about 7-8% higher than those determined by the automated analyzer. Using potassium dichromate solutions (14), it was determined that approximately 5% of this difference was due to the calibration of the pipettor on the automated analyzer. The loss of pipetting accuracy was noticed only with protein-containing solutions and depended on the rinse to sample
volume ratio. The source of the remaining discrepancy is unknown. The automated analyzer results shown in Table 2 have been corrected upward for the total 7-8% discrepancy. A plot of the data from the manual assay against that from the automated assay shows a linear relationship. Linear regression analysis of the GP data gives the following values: slope = 0.971, intercept = 0.002, R2 = 0.999. Similar analysis of the GR data gives: slope = 1.027, intercept = 0.004, R2 = 0.999. The within-run precision for each assay is also shown in Table 2. A comparison shows that the coefficient of variation was usually smaller with the automated assay. To determine the feasibility of applying the automated assays to actual tissue samples, the enzyme activities were determined in rat lung and liver. The activities of catalase, GP, GR, and SOD are presented in Table 3 and show that the automated methods are applicable to tissue samples. Although the peroxidatic activity of catalase is much lower than the catalase activity, it is clear from the dilution volumes that sufficient peroxidatic activity is present for measurements. In conclusion, we have automated assays for catalase, GP, GR, and SOD which require only a fraction of the time necessary for the manual methods. The automated assay for the peroxidatic activity of catalase is much lengthier than the other three enzyme assays and requires approximately 40 min to complete. In spite of this, there are still considerable advantages over the manual assay: Setup time is less, and operator attendance is not required once the samples are placed in the automated analyzer. Although the assays can be transferred to other automated analyzers, there are several advantages to using an analyzer with a longitudinal light path such as the Cobas FARA. With this arrangement, the absorbance is independent of the filling volume.
Precision for a Series of Glutsthione Manual Mean
Peroxidase (GP) and Glutathione Reductase (GR) Dilutions” assay
+ SD (n)
f SD (n)
0.238 0.190 0.159 0.121 0.085 0.034
+ + + f f f
0.007 0.006 0.006 0.002 0.002 0.001
(5) (4) (4) (4) (5) (6)
2.9 3.2 3.8 1.7 2.4 2.9
0.232 0.184 0.159 0.123 0.084 0.033
4 k If: -+ + k
0.001 (5) O.OOl(5) 0.002 (4) 0.001 (4) O.OOl(4) 0.001 (6)
0.4 0.5 1.3 0.8 1.2 3.0
0.233 0.193 0.151 0.103 0.049 0.026
k 0.012 zk 0.000 + 0.003 * 0.002 r 0.002 310.002
(5) (3) (3) (3) (3) (5)
5.2 0 2.0 1.9 4.1 7.7
0.240 0.196 0.149 0.100 0.050 0.026
zk 0.001 (4) + O.OOl(5) k 0.003 (3) * 0.002 (3) + 0.092 (4) AZ 0.001 (5)
0.4 0.4 2.0 2.0 1.3 3.8
’ Each sample
by the manual
Catalase, Glutsthione Peroxidase, Glutathione Reductase, and Superoxide Dismutase Activities in Rat Lung and Liver’
Liver Lung Liver Lung Liver Lung Liver Lung
GP GR SOD
5. 6. 7. 8.
1:600 150 1:50 1:lO 15 1:2 1:lO 1:8
0.387 0.057 0.233 0.095 0.027 0.017 1.775 3.185
f 0.002 + 0.000 -+ 0.001 + 0.000 f 0.000 + 0.000 f 0.042 + 0.025
(3) (3) (4) (5) (5) (5) (5) (5)
’ Activity for all enzymes except SOD is expressed in international units. One unit of SOD activity is the amount of protein required to produce a 50% inhibition in the rate of cytochrome c reduction.
10. 11. 12.
Thus, only the accuracy of sample pipetting will affect the assay. Furthermore, the dimensions of the Cobas cuvette (0.5 cm on each side across the base) means that an incubation volume of 250 ~1 gives a pathlength of 1 cm. Unlike other analyzers which use fixed pathlengths, larger volumes on the Cobas FARA result in a longer pathlength and enhanced sensitivity.
14. 15. 16. 17. 18.
ACKNOWLEDGMENTS We thank Mary Lyons and Paul Waring for help in programming the Cobas FARA and Helen Lew for help in preparing the manuscript.
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