data from which this value was calculated are displayed in the calibration curve of Figure 9. The detection limit can also be given by dl = 3Sb/m where S b is the standard deviation of the background current. From background measurements a t different times and in different supporting electrolytes, S b was estimated to be 3.7 nA, which gives dl = 8.1 X M . Reasonable agreement of these two methods permits some confidence that the calculated detection limit is realistic. We can assume measurement of current due to the analyte has a precision a t least as good as that of the background determination. At concentrations greater than about ten times the detection limit, this inherent precision is generally better than the overall precision of determination in a practical sample. The detection limit for NOHOPro is somewhat higher than for the other two compounds. For NOPro, a detection limit of 8 X M corresponds to about 10 11811. which is adequate for laboratory studies and for many investigations of the prevalence of nitrosamines in the environment (31).
LITERATURE CITED (1) P. N. Magee and J. M. Barnes, Adv. CancerRes., I O , 163 (1967). (2) H. Druckrey, R. Preussmann, S. Ivankovic, and D. Schmahl, Z.Krebsforsch., 69, 103 (1967). (3) A. Wolff and A. E. Wasserman. Science, 177, 15 (1972). (4) T. Aune, Nord. Veterinaermed.,24, 356 (1972). (5) B. Goweniock and W. Luttke, Quart. Rev., 12, 321 (1958). (6) R. H. White, D. C. Havery, E. L. Roseboro, and T. Fazio. J . Assoc. Off. Anal. Chem., 57, 1380 (1974). (7) E. T. Huxel, R. A. Scanlan. and L. M. Libbey. J. Agric. Food Chem., 22, 698 (1974). (8) T. A. Gough and K. S.Webb, J . Chromatogr., 79, 57 (1973). (9) D. D. Bills, K. I. Hildum, R. A. Scanlan, and L. M. Libbey, J. Agric. Food Chem., 21, 876 (1973).
(10) E. T. Huxel, R. A. Scanian, and L. M. Libbey, J . Agric. FoodChem., 22, 698 (1974). (11) C. L. Walters, E. M. Johnson, and N. Ray, Analyst (London), 95, 485 (1970). (12) F. L. English, Anal. Chem., 23, 344(1951). (13) J. G. Osteryoung and R. A. Osteryoung, Am. Lab., 4&(July), 8 (1972). (14) J. H. Christie, J. G. Osteryoung, and R. A. Osteryoung, Anal. Chem., 45, 210 (1973). (15) I. M. Kolthoff and A. Liberti, J. Am. Chem. SOC.,70, 1884 (1948). (16) B. Martin and M. Tashdjian. J . Phys. Chem., 60, 1028 (1956). (17) H. Lund, Acta Chem. Scand., 11, 990(1957). (18) L. Holleck and R. Schindler, Z.Elektrochern., 62, 942 (1958). (19) F. Pulidori. G. Borghesani, C. Bighi, and R. Pedriali, J . Electroanal. Chern.,27, 385 (1970). (20) G. Borghesani, F. Pulidori, R. Pedriali, and C. Bighi, J . Nectroanal. Chem.,-32,303 (1971). (21) R. Zahradnik. E. Svatek, and M. Chvapil, Collect. Czech. Chem. Commun.. 24. 347 119.59). , (22) D.J. Myers and Janet Osteryoung, Anal. Chem., 46, 356 (1974). (23) J. H. Christie and R. A. Osteryoung, J. Nectroanal. Chem., 49, 301 (1974). (24) A. E. Wasserman, Research Leader, Meat Composition and Quality Research, USDA Eastern Regional Research Laboratory, private communication. (25) W. Lijinsky, L. Keefer, and J. Loo, Tetrahedron, 26, 5137 (1970). (26) I. M. Kolthoff and J. J. Lingane, "Polarography", Vol. 1, 2nd ed., Interscience, New York, N.Y., 1952, p 86. (27) Ref. 26, p 52. (28) H. C. Jones, "Conductivity and Viscosity in Mixed Solvents". Carnegie Institutionof Washington, Publ. No. 80, (1907). (29) P. Deiahay, "Double Layer and Electrode Kinetics", Interscience, New York, N.Y., 1965, p 297. (30) R . K. Skogerboe and C. L. Grant, Spectrosc. Lett., 3 , 215 (1970). (31) R. Preussmann, "On the Significance of KNitroso Compounds as Carcinogens and on Problems Related to Their Chemical Analysis", pp 6-9, in NNitroso Compounds Analysis and Formation, IARC Sci. Publ. No. 3, Lyon (1972). I~
.~
~
~~
RECEIVEDfor review July 3, 1975. Accepted September 5, 1975. This work was supported in part by NIH Grant CA 15028-01 and by NSF Grant G P 31491X.
Spectrophotometric Determination of Methyl Glyoxal with 2,4Dinitrophenylhydrazine Robert P. Gilbert and Richard B. Brandt' Department of Biochemistry, Virginia Commonwealth University, Medical College of Virginia, MCV Station-Box
A sensitive method has been developed for the spectrophotometric determination of methyl glyoxal (MeG) uslng an ethanol-HCI solution of 2,4-dinitrophenylhydrazlne (2,4DNPH). Optimal~conditlonsInclude: 2 X 10-4M 2,4-DNPH in 12 ml of concentrated HCI per 100 ml of ethanol, heated 40 min at 42 k 1 OC showed over 99% of reaction completion with MeG when measured at the absorption maximum at 432 nm. The system conforms to Beer's law up to 1.38 X 10-5M. A molar absorptivity of 3.36 X l o 4 cm2/mmol was found. Glutathione, o,L-lactate, pyruvate, and glucose did not interfere with the assay at expected biological levels. Yeast glyoxalase I activlty was measured and found to correspond to the actlvity determined by a standard method. The method will have application to measurement of glyoxalase I activity in tissues.
Methyl glyoxal (MeG), also known as pyruvaldehyde, is an a,j3 dicarbonyl. At one time in the history of biochemistry, it was thought to be the source of lactic acid from the glycolytic pathway ( I ) . MeG is the substrate for the combined enzyme system composed of glyoxalase I and glyoxal
A u t h o r t o w h o m correspondence should be addressed.
2418
727, Richmond, Va. 2 3 2 9 8
lase I1 with glutathione as a cofactor and D-lactate as the product of catalysis. Glyoxalase I is S-lactoyl glutathione methylglyoxal-lyase (isomerizing), E.C. 4.4.1.5, which in the presence of reduced glutathione (GSH) converts MeG to a hemimercaptal, S-lactoyl glutathione. The second enzyme, glyoxalase I1 is S-2 hydroxyacylglutathionehydroxylase, E.C. 3.1.2.6, which catalyzes the conversion of S-lactoyl glutathione to D-lactate, rather than 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 have been stimulated by the finding of SzentGyorgi ( 2 ) who suggested its involvement in inhibition of growth. His observations have led to a number of publications by others on the glyoxalase system. The analysis of MeG has been performed by manometric ( 3 ) ,arsenophosphate colorimetric ( 4 ) , oxidative-titrimetric (5), isotope dilution ( 6 ) , and UV spectrophotometric (7, 8) procedures. The use of a visible spectrophotometric method using 2,4-dinitrophenylhydrazine(2,4-DNPH) has been reported (9, I O ) , but we found the procedures not applicable for multiple analysis of glyoxalase I in biological systems. We report here a spectrophotometric procedure for quantitative assay of MeG applicable to the measurement
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
of glyoxalase I. The procedure utilizes the reaction of 2,4D N P H (shown in Equation 1) with MeG under conditions that inactivate glyoxalase I and allow spectrophotometric assay of the bis-2,4-DNP hydrazone of MeG in the visible spectrum.
R
R
I NH 1
HN 0 R-N-NH, II
0
II II
-t CH,-C-CH
I N1 I II
N CH,-C-CH
(1)
H where R is 24dinitrophenyl.
EXPERIMENTAL Apparatus. Absorbance readings in the 2,4-DNPH analysis of MeG were made on a Bausch and Lomb Spectronic 20 with 1.17cm glass tubes provided by Bausch and Lomb. The ultraviolet spectra were recorded on a Bausch and Lomb 505 with 1.0-cm quartz cells. A Gilford Recording Spectrophotometer equipped with constant temperature compartment and automatic cell changer was used for the enzymatic analysis. An Eberbach watershaker bath was used for incubations. An Olivetti Programmable 100 calculator was used for least square and statistical analysis. Reagents and Chemicals. All chemicals used in this study were reagent grade and used as supplied unless otherwise indicated. Hydrogen peroxide (30%) was diluted daily with distilled water to make a 3% solution and was stored at 4 "C until the time of use. A 1% aqueous solution (w/v) stock solution of crystalline reduced GSH from Sigma Chemical was stored at 4 "C until used. GSH solutions were prepared with the pH adjusted to 6.8 with 0.1N NaOH. MeG, 40% aqueous from Sigma Chemical, was purified by steam distillation and stored in sealed glass vials at 4 "C. The concentration of MeG was determined by the method of Friedemann ( 5 ) .MeG from the vials was diluted in distilled water to the range required for analysis just prior to use. Glyoxalase I (activity 360 units/mg of protein) was obtained from Sigma Chemical and stored at 4 "C. The glyoxalase I was diluted 1/100 in potassium phosphate buffer pH 6.8, 0.005M containing 0.1% bovine albumin (fraction V). Potassium phosphate buffer was 0.1M pH 6.8 except as noted. Stock solution A of 2,4-DNPH (0.02M) was prepared in absolute ethanol (Gold Shield from Commercial Solvents Corp.) and kept at room temperature. Various stock solutions of B were prepared daily by diluting Stock A l / l O O in ethanol ranging from 0.4 ml of concentrated HC1 per 100 ml of ethanol to 40 ml of concentrated HCl per 100 ml of ethanol. Procedure. Preparation of 2,4-DNPH Solutions. After optimal conditions were established, all Stock A and B solutions were made as above except the ethanol-HC1 solution was prepared with 12 ml of concentrated HC1 solution per 100 ml of absolute alcohol. Hydronium Ion Effects. For the determination of the optimal amounts of HC1 used in the 2,4-DNPH-ethanol solutions, 4.0 ml of stock solution B with varying amounts of HCl were added to 13 X 100 mm culture tubes containing 0.025 ml of water. A known amount of MeG (about 3 X mol) was added in a volume of 0.025 ml. The reagent blank was 4.0 ml of Stock B and 0.05 ml of water. All samples and blanks were incubated at 42 "C for 17 minutes and were cooled at room temperature for 5 minutes prior to spectrophotometric measurement to determine the maximum absorbance vs. the reagent blank. The optimal HC1 concentration was determined by choosing the solution that had the lowest absorbance for the blank and the highest absorbance for the sample minus the blank at the wavelength maximum of 432 nm. Kinetics of the 2,4-DNPH Reaction. To determine the optimal reaction time, two different concentrations of MeG were added to stock solution B containing 12 ml of concentrated HC1/100 ml of ethanol. Reagent blanks were also prepared containing H20, and the tubes were covered with polypropylene caps and incubated at 42 "C in a shaker bath. At various time intervals from 5 to 90 minutes, the absorbance of' samples and the corresponding blanks was measured at 432 nm on the Spectronic 20 vs. the HzO blank. Methylglyoxal Standard Curve under Optimal Conditions.
Various samples of dilute MeG (1.15 X -3M) ranging from 5 to 100 p1 were added to 4.0 ml of Stock B solution. Additional distilled water was added to bring the calculated volume of the solution to 4.1 ml. Reagent blanks were prepared by addition of 0.1 ml of distilled water to 4.0 ml of Stock B. The samples and blanks were incubated at 42 "C for 40 minutes. At the end of the incubation, the samples were allowed to cool for 5 minutes at room temperature, the absorption maximum was determined, and the absorbance measured at the maximum of 432 nm in the Spectronic 20 vs. a distilled water blank. Comparison of the 2,4-DNPH Assay with the Racker Method. Samples of MeG solution in distilled water were prepared and standardization for MeG content was done using the method of Friedemann ( 5 ) .The sample was then assayed by the ultraviolet method of Racker (8) and the 2,4-DNPH method was applied to an aliquot of the Racker assay tube after the reaction was complete. The 2,4-DNPH method was conducted as described above. Interfering Substances. Various concentrations of potentially interfering compounds (glutathione, pyruvic acid, D,L-lactic acid, or glucose) were added to the 2,4-DNPH assay mixture both in the presence and absence of MeG. The 2,4-DNPH method was conducted as above. Assay of Glyoxalase I. Activity of yeast glyoxalase I was determined using the spectrophotometric procedure of Racker (8) and the 2,4-DNPH method reported here. Protein was measured by the Lowry method (11). The reaction mixture was prepared by adding 50 fil of 0.074M MeG and 50 pl of 0.074M GSH solution to 3.0 ml of pH 6.8, 0.1M phosphate buffer. The solution was incubated at 30 "C for 10 minutes to allow sufficient time for maximum formation of the addition product between MeG and GSH. Following this period of incubation, 50 p1 of the 1/100 solution of glyoxalase I was added to the reaction mixture and gently mixed. For the Racker assay ( 8 ) , the reaction mixture was placed in a 1.0-cm pathlength cuvette and the increase in the absorbance was determined at 240 nm on a Gilford recording spectrophotometer vs. a reaction tube without enzyme. The rate in absorbance/min was determined for the linear portion of the graph for the first five minutes. Absorbance/min was converted to enzyme activity as the pmoles of MeG condensed per minute per mg of protein using the appropriate volume corrections and a molar absorptivity of 3.37 X lo3 cm2/mmol (8). The 2,4-DNPH method was used to assay the same enzyme reaction mixture. In this procedure, the reaction mixture was placed in a 13 X 100 mm culture tube and incubated in a water bath at 30 "C. At appropriate time intervals (5-20 minutes) after addition of enzyme, 0.030-ml aliquots were taken from the reaction mixture and added to 3.0 ml of Stock B solution in 13 X 100 mm culture tubes and mixed. After all aliquots had been taken, the capped tubes were incubated at 42 "C for 40 minutes, allowed to cool for 5 minutes, and the absorbance at 432 nm was measured in a Gilford Spectrophotometer vs. a water blank. The data in absorbance vs. min was subjected to least square analysis and subsequently converted to enzyme activity as the pmoles of MeG condensed/min/ mg of protein using the appropriate volume corrections and a molar absorptivity of 3.36 X lo4 cm2/mmol which was determined in the standard curve for the reaction between MeG and 2,4DNPH reported here.
RESULTS AND DISCUSSION The visible spectra of the bis-2,4-DNP hydrazone of MeG in ethanol-HC1 solution vs. the reagent blank are shown in Figure 1, demonstrating a broad maximum a t around 432 n m which was the wavelength selected for the MeG assay. The results from the experiment of the variation of the HC1 concentration in ethanol on the reagent blank and the reaction are shown in Figure 2. The reagent blank shows a rapid decrease in absorbance from 0.4 t o 20 ml of HC1/100 ml of ethanol followed by a gradual decrease with increasing amounts of HCl. The reaction absorbance increases rapidly with the addition of acid and reaches a maximum a t about 1 2 ml of concentrated HCl solution per 100 ml of absolute ethanol, and then rapidly decreases above 15 ml of concentrated HCl per 100 ml of absolute ethanol in the stock solutions. The minimum blank and maximum reaction absorbance occurs a t 12 ml of concentrated HC1/100
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
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Table I. 2,4-DNPH Analysis of MeG Using Optimal Conditions
MeG X
106M Mean absorbance
* SEM
No. of samples
Absor. bance minus blankQ
Absorbance minus blank/ MeG X 106M
8 0.110 0.002 0.000 0.000 0.164 0.006 7 0.054 0.046 8 0.208 0.098 0.004 0.042 0.299 8 0.189 0.004 0.041 8 0.356 0.246 0.005 0.042 8 0.487 0.377 0.004 0.040 8 0.566 0.011 0.456 0.039 0.656 0.011 7 0.546 0.039 0.789 7 0.010 0.679 0.039 23.0 0.979 0.010 7 0.869 0.037 Equation for straight line from least square analysis of data to 1.38 X lO-'M is OD = (3.93 X 104)M+ 0.008. 0.0
410
'
4 I
I
430
450
Wavelength
4 70
nm.
Figure 1. Absorbance spectra of the bis-2,4-DNP hydrazone of MeG
in ethanol-HCI vs. reaaent - blank.
Concentration of MeG: ( A ) 1 X
(D)0.2x
(8) 0.5 X IO-'M, (C)0.4 X iO-5M,
i o - 5 ~
1.15 2.30 4.60 5.76 9.22 11.5 13.8 17.2 Q
I
02 5 ~
Table 11. Study of Interfering Species in 2,4-DNPH Analysis of MeG MeG concentration,
.If
Compound
Glutathione D-Glucose
7.11 X 7.11 X lo-" ... 7.11 X 7.11 X
... Pyruvic acid
7.11 7.11
X X
... Flgure 2. Effects of HCI increase on absorbance of bis-2,4-DNP hydrazone of MeG and on reagent blank of 2,4-DNPH -0-0-0-0-
absorbance of the sample minus the blank, absorbance of the blank
05
B
203
I
1 10
30
50
70
90
Time,min
Figure 3. Rate of formation of bis-2.4-DNP hydrazone of MeG under
assay conditions Trace A, concentration of MeG 1.2 X 10-5M: Trace Bconcentration of MeG
0.6x
i o - 5 ~
ml of absolute ethanol. These data defined the assay conditions with respect to HCl concentration for preparation of the 2,4-DNPH stock solutions. Under the reaction conditions of hydronium ion content and temperature, apparently only the MeG bis-2,4-DNP hydrazone is formed (12) with a mechanism of reaction possibly involving protonation of both the hydrazino and carbonyl functions. Reagent solvent prepared by bubbling dry HC1 gas into 95%ethanol gave'similar results when diluted to the same HC1 concentration. Figure 3 shows the increase in absorbance a t 432 2420
D,L-Lactic acid
7.11 X 7.11 X
lod6 ...
Concentration interfering species, ,M
0 3.95 x 10-5 7.90 x 10-5 0 3.43 x 10-5 3.43 x 10-5 6.86 X 0
6.02 X 1.20 x 10-5 1.20 x 10-4 0
2.84 x 10-4 8.17 x 1 0 - 5 8.17 x 10-4
Absorbance minus blank
0.320 0.320 0.000 0.312 0.312 0.000 0.000 0.320 0.325 0.020 0.220 0.318 0.300 0.000 0.000
nm with incubation at 42 O C for 2,4-DNPH and MeG under optimal HCl concentration. The half time of reaction as determined by the time to reach half maximal absorbance was found to be four minutes for different concentrations of MeG (0.6 and 1.2 X 10-5M).The data from Figure 3 were linear on a semilog plot (2.3 log of concentration of MeG a t a given time vs. time) indicating a first-order reaction. Since two compounds are reactants in the formation of the bis-2,4-DNP hydrazone as shown in Equation 1, this reaction would be expected to have its rate dependent on the concentrations of both compounds and display secondorder kinetics, but, by using an excess of 2,4-DNPH, the reaction kinetics are dependent only on the concentration of MeG, giving a typical first-order kinetics plot. Thus, a second-order reaction has been converted to a pseudo-firstorder reaction where the half time of the reaction is independent of MeG concentration. This kinetic information assures that complete reaction of MeG with 2,4-DNPH will occur within an incubation time of 40 minutes, which was chosen to represent 10 half times or over 99.9% completion of reaction, and that this time of incubation will not have to be changed for each concentration of MeG to achieve an equivalent percent completion of reaction. Table I shows the results of analysis of MeG concentration from 1.15 X 10-6M to 2.3 X 10-5M using the 2,4DNPH spectrophotometric method. The data in Table I were subjected to least square analysis. A slight negative deviation from linearity was noted when concentrations
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
Table IV. Various Analytical Methods for Methyl Glyoxal
Table 111. Comparison of Enzymatic Method with 2,4-DNPH Method for Assay of MeG
Method
MeG concentration in vial, M
Trial
1 2
3
Method of Friedemann (5 )
Enzymatic Method (a), A
2,4-DNPH assay of enzymatic assay t u b e , B
1.44 1.44 1.44
1.26 1.26 1.26
0.163 0.163 0.163
Sum A+B
1.42 1.42 1.41
Reference
Arsenophosphatecolorimetric
(4)
Manometric
(3)
UV absorption of S-lactoyl glutathione
(8)
UV absorption of semicarbazone Aqueous 2,4-DNPH
(7) (9, 1 0 )
~~
above 1.38 X 10-jM were used. The use of absorbance values for concentrations below 1.39 X 10-5M for calculation of the slope and molar absorptivity was considered to be more accurate than when the slope was calculated using all the absorbance values determined. Occasionally precipitation occurred a t MeG concentrations above 1.38 X 10-5M. The regression coefficient for least squares determination on values below 1.39 X 10+M was 0.999. The molar absorptivity determined was 3.36 X lo4 cm2/mmol with a standard deviation of f O . O 1 X lo4 cm2/mmol. This molar absorptivity was used to convert absorbance readings a t 432 nm to MeG concentration or amount in all assays of MeG and in kinetic studies using the 2,4-DNPH method. In Table I1 are shown the results from the studies of possible interfering compounds. The substances tested represented not only products and substrates of the enzymatic reaction but also some metabolites which may also react with 2,4-DNPH. Of the compounds examined, only pyruvate in a concentration of 1.20 X 10-5M interfered with an absorbance of 0.020 (reagent blank value subtracted). This concentration of pyruvate represents approximate physiologic concentrations. Higher concentrations of pyruvate interfered proportionally. In liver cytosol preparations that have been reported (7) for the analysis of the glyoxalase enzyme system, an effective 1 to 100 dilution of the tissue and the metabolites was used, making interference from pyruvate an unlikely problem. Also, since a monocarbonyl has only an approximate molar absorptivity of 1.5 X lo3 cm2/mmol (calculated from pyruvate data of Table 111, which is only about 5% of that of the dicarbonyl MeG, the assay conditions favor the noninterference from monocarbonyl functions. Since the 2,4-DNPH method will be used for glyoxalase I measurement, these systems will be supplemented with the substrate (MeG) and the enzyme activity measured by a decrease in initial absorbance. These conditions favor low blank values. The lack of interference also maintains the simplicity of the system, obviating extensive corrections. In Table I11 are the data for the analysis of MeG, using the standardization procedure of Friedemann ( 5 ) , the Racker procedure, and aliquots from the Racker assay tubes for residual MeG by the 2,4-DNPH procedure. The difference between the standard procedure and t h a t of Racker indicates a 12.5% low value for the Racker assay. Further analysis using the 2,4-DNPH value plus the Racker value accounts for 99% of the standard value. Since the MeG analysis described here was to be used for measurement of glyoxalase I activity, an experiment to show the validity of the MeG analysis in enzyme analysis against that of the method of Racker (8) was done. The activities of yeast glyoxalase I i s t d dev expressed as wmoles of MeG utilized/min/mg was 180 f 1 for the Racker procedure and 185 f 36 for the 2,4-DNPH procedure. These values are statistically the same ( p > 0.4). At MeG concentration of 4.4 X 10-3iM, the maximum activity using the 2,4-DNPH procedure was 527 f 30 @molof MeG utilized/
Isotope dilution Oxidative-titrimetric
(6)
(5)
Comments
Nonspecific, many compounds that are oxidized interfere. Tedious and requires the use of both glyoxalase I and 11. High blank values for saturating conditions of MeG required and low molar absorptivity. Possible tissue absorbance at 260 nm. Several steps required, high blank and poor reproducibility at low concentrations. Tedious. Nonspecific and larger amounts of MeG required.
min/mg of yeast glyoxalase I. The higher rate also gave a lower standard deviation which might be expected from this assay type where the rate values are more precise when a greater decrease in absorbance occurs. The true substrate for glyoxalase I is not MeG but rather the non-enzymatically formed adduct between MeG and GSH ( 1 3 ) .This adduct catalytically forms a hemimercaptal which absorbs radiation maximally a t 240 nm. The increase in absorbance from the adduct to that of the hemimercaptal is the basis of the Racker assay (8).Although this is an easy procedure to use since it is a continuous assay and one where there is an increase in absorbance, several problems occur which have led us to developing the work reported here. For enzyme assays in tissues, the presence of glyoxalase I1 will decrease the amount of the hemimercaptal, since the hemimercaptal is both the product of glyoxalase I and the substrate for glyoxalase 11. Another difficulty in measuring glyoxalase I by the Racker procedure ( 8 ) is due to the conditions for assay where the substrate concentration should be about twenty times the K,. The K , for glyoxalase I is about 10-4M ( 1 4 ) . Since the molar absorptivity for the adduct is 440 cm2/mmol ( 1 4 ) , this gives a calculated absorbance of a t least 0.88 for the blank. Thus, saturating conditions of substrate interfere with enzyme analysis because of the high background level. Table IV shows various other procedures for analysis of MeG with comments on the method. The most obvious drawback to many of these methods is in the application to measurement of enzyme activity where multiple samples are taken from biological material. Some of these analytical systems do have strong points for specific purposes. However, for the purposes that we required, none of the methods were completely satisfactory. The 2,4-DNPH method reported here has been used for the determination of K , and V,,, of glyoxalase I from yeast and rat liver cytosol ( 1 5 ) yielding values similar to those reported by others (14, 16). The ethanolic-HC1 reagent stops the enzyme activity and the bis-2,4-DNP hydrazone absorbs light in the visible region of the spectra away from biological interference. In future reports, the 2,4-DNPH method developed here will be used in the measurement of glyoxalase I in various treated and untreated tissues of normal and neoplastic origin.
LITERATURE CITED (1)C.Neuberg and M. Kobel, Biochem. Z.,207,232 (1929).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
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(2) A. Szent-Gyorgyi, A. Hegyeli, and J. A. Mclaughlin, Science, 155, 539 (1967). (3) K. Lohman, Biochem. Z.,254, 332 (1932). (4) N. Ariyama, J. Biol. Chem., 77, 359 (1928). (5) T. E. Friedemann, J. Bid. Chem., 73, 331 (1927). (6) D. J. Walton and S. A. McLean, Anal. Biochem.,43, 472 (1971). (7) N. M. Alexander and J. L. Boyer, Anal. Biochem., 41, 29 (1971). (8)E. Racker, J. Biol. Chem., 190, 685 (1950). (9) R. Cooper and A. Anderson, FEBS Lett., 11, 273 (1970). (10) V. Riddle and F. W. Lorenz, J. Biol. Chem., 243, 2718 (1968). (11) 0.H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Bioi. Chem., 193, 265 (1951). (12) L. A. Jones, C. K. Hancock, and R. B. Seligman, J. Org. Chem., 26, 228 (1961). (13) E. E. Cliffe and S. G. Waley, Biochem. J., 79, 475 (1961). (14) D. L. Vander Jagt. L. B. Han. and C. H. Lehman, Biocbemistry, 11, 3735 (1972). (15) R. P. Gilbert, Unpublished data, Masters Thesis, Medical College of Virginia, Richmond, Va., June 1975. (16) R. A. Striznek and S.J. Norton, Texas J. Sci.. 24, 1 (1972).
RECEIVEDfor review June 26, 1975. Accepted August 21, 1975. This work was supported in part by A. D. Williams Summer Fellowship, Norman Jolliffe Student Fellowship sponsored by the American Society for Clinical Nutrition, 1973 (R.P.G.) and funds from the American Cancer Society Institutional Research Grant IN-105. Preliminary reports of part of this work have been presented a t the Southeastern Regional Meetings of the Society for Experimental Biology and Medicine, Richmond, Va., 1972, and Atlanta, Ga., 1974, and a t the Annual Meeting of the Virginia Academy of Science, Lexington, Va., 1972. This work was in partial fulfillment of the requirements for the degree of Master of Science in the Department of Biochemistry a t the Medical College of Virginia, June 1975, for R.P.G.
Spectrophotometric Determination of Manganese(l1) and Zinc(l1) with 4-(2-Pyridylazo)resorcinol (PAR) Sten Ahrland’ and Richard G. Herman2 Inorganic Chemistry 1, Chemical Center, University of Lund, P.O.B. 740, S-220 07 Lund, Sweden
Convenient procedures for the routine determinations of trace amounts of manganese(i1) and zinc(ll) In aqueous solutions using 4-( 2-pyridyiaro)resorclnol (PAR) as the spectrophotometric reagent are described. Color development of the Mn(ll)-PAR complex was found to vary with time of equilibration and to be a function of PAR concentration, the buffer used, and, in some cases, the presence or absence of a reducing agent, ascorbic acid. When a borate buffer without the reducing agent present is used, nearly a 50-fold molar excess of PAR is required for complete compiex formation. Full color development occurred immediately for the Zn(ll) system in a borate buffer, and a large excess of PAR was not necessary. in borate buffers, the Sandell sensitivities were observed to be 0.71 (pH 10) and 0.81 ng/cm2 (pH 8) for the manganese(l1) and zinc(ll) complexes, respectively.
Spectrophotometric determinations of trace concentrations of metal ions are usually simple, quick, accurate, and precise. In a study of the sorption of divalent transition metal ions on semicrystalline zirconium phosphate ( I 1, it was necessary to routinely analyze very low concentrations (micromolar range) of manganese(I1) and zinc(I1) in aqueous solution. Several spectrophotometric methods have been used to determine manganese in aqueous solutions, but all of these suffer from pronounced drawbacks. The peroxydisulfate and periodate methods are the most commonly used routine procedures (2, 3), but they are often rather tedious to perform, and the presence of reducing substances, such as chloride ion, interferes with the color development of sample solutions (4-10). Of all colorimetric determinations, the most sensitive one is probably the reaction between permanganate and 4,4’-tetramethyldiaminotriphenylmethane (tetrabase), in which the latter rei A u t h o r t o w h o m correspondence should b e addressed. Present address, Center for Surface a n d Coatings Research, Sinclair Laboratory, L e h i g h University, Bethlehem, Pa. 18015.
2422
Table I. Spectrophotometric D a t a Reported for the Manganese(I1)-PAR Complex in Aqueous Solution Amax,
PH
Buffer
nm
Refer-
Eta
M-l
cm-l
10.0 Noneb 9.7-10.7 Ammonia
490 500
38300‘ 78000
10.3
Ammonia
500
85000
9 11.2
Borax Phosphate
490 496
32000 79100
Reducing agent
ence
Noned Ascorbic acid Hydroxylamine None Ascorbic acid
( 25) (26) (27)
( 29) (28)
t = molar absorptivity (determined a t Amax). 0.1N N a O H used t o adjust the pH. Also reported in the reference as 32900 M-1 cm-1. d Ascorbic acid found t o interfere.
agent is oxidized to a yellow product (11).The Sandell sensitivity is less than 0.0001 Fg/cm2 a t 470 nm, but there are apparent deviations from Beer’s law (12). Disadvantages of this method are that the color is stable for only five minutes after developing fully, and that the determination must be carried out in a darkened room. For the spectrophotometric determination of zinc(II), the dithizone (13-16) and Zincon (13, 14, 17) methods have been widely used. The dithizone complex has to be extracted into a nonaqueous phase (13, 1 4 ) , and the same drawback also applies to the PAN (18-20) and a-PAN (21) methods. The Zincon method (17) and the recently reported Arsenazo I11 method (22) are less sensitive (Sandell sensitivities = 0.0034 and 0.0023 pg/cm2, respectively) than is desirable for the determination of trace concentrations. The value of pyridylazo compounds as wide-range spectrophotometric reagents for metal ions is now firmly established (23). The sensitivities of these reagents for many metal ions are higher than those of other reagents, but they are not selective. However, for routine analyses when only one metal ion is present in low concentrations in aqueous systems, e.g., in the study of solution equilibria, the use of
ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 14, DECEMBER 1975
