New Substrates for the Fluorometric Determination of Oxidative Enzymes George G. Guilbault, Paul J. Brignac, Jr., and M a r k Juneau Department of Chemistry, Louisiana State University in New Orleans, Lakefront Campus, New Orleans, La. 70122 A survey of 25 indicator substrates for the fluorometric determination of oxidative enzymes is described. Of these, p-hydroxyphenylacetic acid was judged to be the best substrate. I t is completely stable to autooxidation, and has advantages over homovanillic acid of low cost and a higher fluorescent coefficient (fluorescence/concentration in M). With this substrate, 0.3-30 pg per ml (1-100 pg total) of D-glucose, D-galactose, stachyose, 2-deoxy-~-galactose, methyl-p-D-galactopyranoside, D-raffinose, D-galactosamine, Nacetyl-D-galactosamine, a-D-melibiose, and sucrose can be determined with an accuracy and precision of about 1.5%. Analytical procedures are also described for the determination of p-hydroxyphenylacetic acid, tyramine, tyrosine, homovanillic acid, and 3,4-dihydroxyphenylacetic acid based upon their oxidation by peroxide and peroxidase to highly fluorescent (or colored) products.
REFERENCES for the quantitative analysis of carbohydrates using enzymic colorimetric, manometric, and pH methods are given in recent reviews ( I , 2). Enzymes offer advantages of specificity and sensitivity over other nonenzymic methods for the determination of carbohydrates. However, the lowest detectable concentration of most sugars is still only about 10 pg, and attempts to increase sensitivity have been at the expense of time. Roth (3) was able to determine 20-150 pg of galactose with a spectrophotometric method using a 30minute incubation. In order to analyze from 2-20 pg, a 120-minute incubation time was required. Because of the inherent sensitivity of fluorometric methods, such techniques have found increasing use in analysis of enzymes ( I , 2). Recently Guilbault, Brignac, and Zimmer (4) described the use of homovanillic acid (HVA), 3-methoxy-4hydroxyphenylacetic acid, for the quantitative determination of oxidative enzymes. The compound (I) is nonfluorescent, but is converted upon oxidation to the highly fluorescent com-
(1)
(11)
pound (11), which has a A,,= 315 mp and a A,, = 425 mp. In initial attempts to extend the HVA procedure to other sugars, it immediately became obvious that the desired sensitivity was not being obtained. Methods developed for Dgalactose, stachyose, 2-deoxy-~-galactose, methyl-P-D-galactopyranoside, D-raffinose, D-galactosamine, N-acetyl0 galactosamine, and a-D-melibiose which are catalytically oxidized by galactose oxidase, for 2-deoxy-~-glucosewhich is a substrate for glucose oxidase, and for sucrose which is first (1) G. Guilbault, ANAL.CHEM.,38, 530R (1966). (2) G. Guilbault, ibid., 40, 459 R (1968). (3) H. Roth, S. Segal, and D. Bertoli, Anal. Blochem., 10,32 (1965). (4) G. Guilbault, P. Brignac, and M. Zirnrner, ANAL.CHEM.,40, 190 (1968). 1256
ANALYTICAL CHEMISTRY
converted to glucose by the enzyme invertase, revealed that all the analytical curves were in the range of 50-500 pg per ml. These results are, however, still analytically useful and will be included in this paper.
Sucrose Sugar H20z
-
Invertase
Galactose oxidase or Glucose oxidase
+ HVA
Glucose
’
fluorescent compound (11)
A systematic search was conducted for other substrates which might offer better sensitivity using an initial rate method. Of 25 substrates surveyed, three were found that work well in the range of 0-20 pg : p-hydroxyphenylacetic acid, tyramine, and tyrosine. These compounds are oxidized via a mechanism similar to homovanillic acid (4,yielding fluorescent products with wavelengths similar to 11, but with higher fluorescent coefficients (total fluorescence/molarity). All were stable in aqueous solution, and cost considerably less than HVA. p-Hydroxyphenylacetic acid, which was judged to be the best substrate for oxidative enzymes, costs $0.44 per gram compared to $30 per gram for HVA. Analytical methods were developed for the sugars mentioned above, in the range of 0.3-30 pg per ml (1-100 pg total) with a precision and accuracy of about 1 %. Procedures were also developed for the determination of HVA, p-hydroxyphenylacetic acid, tyramine, tyrosine, and 3,4-dihydroxyphenylaceticacid, based on their oxidation by peroxide and peroxidase to highly fluorescent (or in the case of the last compound, colored) products. EXPERIMENTAL All solutions were prepared using triply distilled water, which is obtained by passing once distilled water over a charcoal column to remove organic impurities, followed by a double distillation from first alkaline then neutral potassium permanganate. Enzymes. Aqueous solutions of all enzymes were prepared as follows: peroxidase, 1 mg/ml solution of horse radish peroxidase, B grade, RZ = 0.3, [Calbiochem. Co., activity from assay ( 5 ) was 125 units/mgJ; galactose oxidase, 2 mg/ml solution from Dactylium Dendroides (Sigma, activity 15 unitslmg); glucose oxidase, 2 mg/ml of fungal glucose oxidase (Calbiochem. Co., activity 20 enzyme units/mg); invertase, 2.3 mg/ml from baker’s yeast Type VI [Sigma, activity 10 units/mg (1 unit catalyzed the hydrolysis of 100 pmole of sucrose to invert sugar per minute at pH 4.5 and 55 “C)]. Buffers. One-tenth normal tris(hydroxymethy1) amino methane, buffer, pH 8.50, was prepared by dissolving the pure compound (Sigma) in triply distilled water and adjusting the pH with concentrated HCl. An acetic acid-sodium acetate buffer was prepared by taking 0.05M sodium acetate and adjusting the pH to 6.00 with glacial acetic acid.
( 5 ) A. Meahly and B. Chance, “Methods of Biochemical Analysis,” Vol. 1, D. Glick, Ed., Interscience, New York, 1954, p 337.
Substrates. All solutions were made by dissolving the substance (homovanillic acid, Calbiochem. Co., 2.5 mg/ml; p-hydroxyphenylacetic acid, Columbia Organic, 3.7 mg/ml; tyramine hydrochloride, Sigma, 2.3 mg/ml; tyrosine, Sigma, 2 mg/ml) in triply distilled water. The carbohydrates, D-galactose, stachyose, 2-deoxy-~galactose, D( +)-raffinose, a-D(+)-melibiose, D( +)-galactosamine HC1, and N-acetyl-D-galactosamine, were obtained from Sigma (St. Louis). Methyl P-D-galactopyranoside was obtained from Mann (New York, N. Y.), 2-deoxy-~-ghcose from Aldrich, and L-altrose and D-talose from Dr. Nelson Richtmeyer of the National Institutes of Health. A mixed reagent solution for the analysis of sugars was prepared by dissolving peroxidase and p-hydroxyphenylacetic acid in tris buffer, pH 8.50, in one solution at the concentrations specified above. This solution is stable for two weeks if stored overnight under refrigeration, and eliminates the need for three separate solutions. T o prepare 50 ml of solution, dissolve 2 mg of peroxidase and 7.4 mg of p-hydroxyphenylacetic acid in tris buffer, O.lM, pH 8.50. Apparatus. SUBSTRATE STUDY. Fluorescent wavelength measurements were made with an Aminco-Bowman spectrophotofluorometer which utilizes grating monochromators and a Xenon arc lamp. Spectra were recorded on a Houston Model No. R-97 X-Y recorder. A constant temperature of 25 "C was maintained with a thermoelectric cooler. SUGARANALYSIS USINGHVA. Fluorescent measurements were made with an Aminco-Fluoromicrophotometer filter instrument. The lamp used was a Turner 110-850 general purpose UV lamp. A Corning 7-60 (4.5 mm) primary filter, a Kodak Wratten 47B and 2A combination secondary filter, a circulating water bath to control the temperature at 30 "C, and a Beckman linear recorder for automatic readout were used. MICROANALYSIS USING HYDROXYP PHENYLACETIC ACID. Fluorescent measurements were made with the filter instrument mentioned previously except that a special Turner 110-855 lamp and a Kodak Wratten 47B secondary filter, were used. Calibration. SUGARANALYSISUSING HVA. The fluorometer was set to 0.70 fluorescent units (70% on the 0.01 scale of the Aminco Bowman) using 0.01 pg/ml solution of quinine sulfate in 0.1N H2S04, using the same filter system: primary CS 7-60 and secondary 47B and 2A. The fluorescent coefficient (fluorescence/molarity) for quinine sulfate under these conditions was 4 X lo6. MICROANALYSIS.The sensitivity of the instrument was set to the limit, using the Turner 110-855 lamp. The quinine sulfate solution using the same filter system as above, gave a fluorescent coefficient of 1.12 x lo8. Procedures. SUGARANALYSES USINGHVA. To 2.7 ml of tris buffer, pH 8.50, are added 0.1 ml of the sugar to be analyzed, 0.1 ml of HVA, and 0.1 ml of peroxidase. The fluorometer is set to zero. At zero time, 0.1 ml of galactose oxidase or glucose oxidase is added, and the initial rate of the reaction (AF/minute) is recorded. From calibration plots of AF/minute cs. sugar concentration, the amount of carbohydrate present can be calculated. MICRO METHODUSING HYDROXYP PHENYLACETIC ACID. T o 2.0 ml of tris 0.1N pH 8.5 are added 0.1 ml of the sugar to be assayed, 0.1 ml of p-hydroxyphenylacetic acid, 0.1 ml of peroxidase, and 0.1 ml of galactose oxidase. The maximum rate is recorded, and calibration plots were made as above. In the case of low sugar concentrations (0.3-3 pg per ml) a 10-15 minute induction period is required before a change in fluorescence is observed, but normal reproducible rates are obtained. At concentrations of 3-100 pg per ml, analysis can be performed within 3-5 minutes based on an initial rate method. SUCROSE ANALYSIS.T o 0.5 ml of acetate buffer, pH 6.0, are added 0.1 ml of sucrose and 0.1 ml of invertase. After 10 minutes of incubation at room temperature, add 2.7 ml of
mixed reagent solution and 0.1 ml of glucose oxidase. The initial rate change of fluorescence is recorded, and calibration plots are made. GALACTOSE OXIDASE ANALYSIS.To 2.7 ml of mixed reagent solution are added 0.3 ml of an 18 mg/ml solution of galactose, and 0.1 ml of galactose oxidase. The initial rate of fluorescence is recorded, and the activity of enzyme calculated from a calibration plot of AF/minute cs. enzyme concentration. ANALYSIS OF HYDROXY PHENYLACETIC ACID, HVA, AND TYRAMINE.T o 2.0 ml of pH 8.5 tris buffer are added 0.1 ml of substrate, 0.1 ml of 0.3% H202,and 0.1 ml of peroxidase. The initial rate of change of fluorescence is recorded and calibration plots were made. With HVA, use a secondary filter 47B and 2A. With others, use a 47B secondary filter. 3,4-DIHYDROXYPHENYLACETIC ACID. This analysis was run using a Beckman DB spectrophotometer with a recorder at 25 "C and 500 mp (blank: 2.0 ml of tris buffer and 0.1 ml of H202). T o 2.0 ml of 0.1N pH 8.0 tris buffer are added 0.1 ml of substrate, 0.1 ml of 0.3% H 2 0 2 ,and 0.1 ml of peroxidase. The initial rate of change of absorbance/minute was recorded and plotted against the concentration, giving a calibration plot from which an unknown concentration of the compound can be calculated. SUBSTRATE STUDY. To 3.00 ml tris pH 8.5 are added 0.1 ml 0.3% H2O2,0.1 ml of a substrate (2 mgiml), and 0.1 ml of peroxidase. The wavelengths of fluorescence were found using the grating fluorometer, and fluorescent coefficients were determined by dividing the total fluorescence obtained by the concentration of the oxidized product formed (assuming 100% conversion). Several concentrations of original substrate were used, in order to obtain accurate values of the fluorescence coefficient. An Aminco filter fluorometer, equipped with a Turner 110-855 UV lamp, a Corning CS 7-60 primary filter, and a Kodak Wratten 47B secondary filter, was used in measurements of fluorescent coefficients. DISCUSSION AND RESULTS
Hornovanillic Acid as Substrate. By using nonlimiting amounts of peroxidase, HVA, and either galactose oxidase or glucose oxidase, the initial rate of change of fluorescence (AFlminute) was found to be directly proportional to the sugar concentration. The following sugars were quantitatively determined using galactose oxidase by an initial rate method within 3-5 minutes (Table I) : D( +)-galactose (50-500 pg/ml), 2-deoxy-~-galactose (50-800 pgiml), N-acetyl-D-galactosamine (50-600 pg/ml), a - ~+)-melibiose ( (100-650 pg/ml), D( +)-galactosamine HCl (50-700 pg/ml), D( +)-raffinose (100-900 pg/ml), methyl-0-D-galactopyranoside (50-400 pg/ml), and stachyose (50-900 pg/ml). An average relative error of about & 2 % was obtained. The tetrasaccharide, stachyose, is quantitatively oxidized immediately by galactose oxidase at pH 8.0 in 0.1M tris buffer. Whether the tetramer is oxidized directly or is immediately hydrolyzed first to galactose is uncertain. The following sugars were determined using glucose oxidase (Table 11): P-D(+)-glucose, 2-deoxy-~-ghcose (10-300 pg/ ml), and sucrose (10-1000 pg/ml) with an average relative error of =k3 %. Sucrose presented a unique problem. The reaction of sucrose with invertase to produce glucose and fructose is rapid at pH 6.0. At a basic pH, the reaction is slow; however, the indicator reaction has to be run under basic conditions, so the following procedure was employed. The sucrose is incubated with invertase for 10 minutes at 25 "C at pH 6.0 with 0.05M acetate buffer. The pH of the solution is then adjusted to a pH of 8.0, and the glucose formed is analyzed, under the same reaction conditions as for the VOL. 40, NO. 8, JULY 1968
1257
Added 41.6 101 214 301 391 Av re1 error
Table I. Determination of Sugars Using Galactose Oxidase, HVA Procedure Stachyose pg/ml D( +)-Galactose, pg/ml 2-Deoxy-~-galactose,pg/ml Founda Re1 error, Added Found= Re1 error, Added Founds Re1 error, 40.0 -3.85 58.4 58.4 0.00 156 156 0.00 97.0 -3.96 117 108 -7.67 260 252 -3.08 224 +4.67 234 238 +1.71 302 292 -3.31 -1.66 296 348 356 +2.30 537 544 +1.30 400 +2.30 467 456 -2.36 732 712 -2.74 13.30 12.80 zt2.08
z
Methyl-P-D-galactopyranoside, pg/ml
Added 25.4 49.1 84.6 164 238 Av re1 error
Found" 25.0 48.0 82.0 168 228
Re1 error, % -1.57 -2.24 -3.07 +2.44 -4.19 12.70
D(+)
Added 95.9 191 369 574 958
Raffinose, p g / d Found4 Re1 error, 104 +7.79 191 0.00 332 -10.0 588 +2.44 958 0.00 14.04
D( +) Galactosamine,
Added 63.2 122 118 316 632
N-Acetyl-D-galactosamine, ,ug/ml
Added 87.0 157 299 456 597 Av re1 error Q
pg/ml Re1 error, -0.32 0.00 0.00 +1.27 $2.06 10.73
WD( +) Melibiose. HzO,
Re1 error, $3.45 +1.91 +O. 33 $4.16 -0.33 12.09
Added 110 163 333 552 644
Founda 110 165 335 530 660
pg/ml Re1 error, 0.00 $1.22 +0.61 -4.00 +2.49 ic1.67
Represents three or more determinations with a relative standard deviation of 1 2 % .
Added 14.9 24.8 49.7 141 248 Av re1 error a
Found" 90.0 160 300 475 595
Founds 63.0 122 178 320 645
Table 11. Determination of Sugars Using Glucose Oxidase (HVA Procedure) Sucrose. ueiml 2-Deoxy-~-glucose,pg/ml Added Founda Founda Re1 error, 35.7 36.0 14.0 -6.05 69.1 71.0 24.8 0.00 336 335 50.0 +0.60 527 525 0.00 141 1,190 1,200 225 -9.26 zk3.18
Re1 error,-% +0.8 $2.7 -0.29 -0.38 +0.84 11.06
Represents three or more determinations with a relative standard deviation of 1.2 %.
glucose analysis. All the sucrose was found t o be quantitatively converted to glucose by invertase in 10 minutes, so that a n assay for glucose can then be used to determine the sucrose concentration. If nonlimiting amounts of HVA, peroxidase, and substrate are used, the enzyme can be quantitatively determined. Glucose oxidase was determined using P-D(+)-glucose. Galactose oxidase (0.05-0.60 unitiml) was determined using
Table 111. Determination of Galactose Oxidase Using D(+) Galactose (HVA Procedure) Galactose oxidase,. Unitsiml units/ml Re1 error
z
0.0982 0.191 0.278 0.437 0.572 Av re1 error
0.0982 0.194 0.290 0.428 0.568
0.00 f1.57 +4.32 -2.06 -0.70 h1.73
4 Represents three or more determinations with a relative standard deviation of ic2Z.
1258
0
ANALYTICAL CHEMISTRY
D( +)-galactose (Table 111). The glucose and glucose oxidase
data have been published earlier (4). Other Substrates. In order t o get the desired sensitivity, another substrate was clearly needed. Table IV lists the compounds studied and their results. These results were obtained using an Aminco Bowman spectrophotofluorometer a t 25 "C. The compounds which seemed to work well are HVA, p-hydroxyphenylacetic acid, tyramine, and tyrosine. As shown in Table IV, considerable variation of the HVA-type structure was tried. It seems that a necessary requirement for this coupling to occur is the following:
n HO
m2-cr'
This further strengthens the mechanism proposed by Guilbault (4, for none of the compounds tested without this essential structure worked. Addition of another hydroxyl group to the benzene ring causes side reactions t o occur that result in nonfluorescent, but highly colored, products. Addition of a methoxy group (HVA) eliminates this side reaction
~~
Table IV. Compound
Characteristics of Oxidized Product A,
Am
Fluorescent coefficienta
N.F.b
...
315
425
1.27 X 106
(p-Hydroxyphenylacetic acid)
317
414
3.28 X 106
(p-Aminophenylacetic acid)
N.F.
(Phenylacetic acid) OCH3
\
HO-~-cHzCooH (Homovanillic acid)
-
OH
(3,4-Dihydroxyphenylacetic acid)
...
N.F.c
-COOH
H O - 0
(p-Hydroxybenzoic acid)
302
418
Weak
354
446
Weak
CH30
\
HO-0-COOH (&Hydroxy-3-methoxy-benzoic acid)
/OH Ho--~-cOoH (2,4Dihydroxybenzoic acid)
...
N.F.
I
(Tyrosine)
326
411
7.8 X 106
(Tyramine)
326
410
3.9
N.F.
...
472
532
x
106
OH
(Dopa) HO
OH (3,4 Dihydroxymandelic acid)
Weak (Continued)
VOL. 40, NO. 8, JULY 1968
0
1259
Table IV. Compound
Characteristics of Oxidized Product (Continued) hex
em
Fluorescent coefficient5
N.F.
...
...
HO
\
HO--O-!=CH
COOH
(3,4-Dihydroxy-cinnamicacid) OH
(Dopamine)
N.F.
R (2 or 3-Methoxy-2,5-dihydroxy-phenyl-
acetic acid)
N.F.
.
I
.
...
p -Aminophenol
N.F. ... ... p-Cyanophenol N.F. ... ... 3J-Dihydroxy benzoic acid N.F. ... ... 3-Hydroxy-mandelicacid Fluorescent coefficient of quinine sulfate = 1.12 X IO4 under conditions described in the text. Although the values of the fluorescent coefficienthave no absolute meaning, they do have significance relative to each other and relative to quinine sulfate for all measurements were made on the same instrument under the same conditions. 21 N.F. = None Found. c Product highly colored,, , ,A = 500 mp. Q
and a fluorescent product is obtained that has a higher A,, as expected (6). Elimination of the methylene group in the side chain results in a large decrease in the rate of oxidation, and the benzoic acid analogs of HVA or p-hydroxyphenylacetic acid yield very small amounts of fluorescent products. Substitution of NH2 for the p-OH group likewise stops the reaction, as does the addition of a double bond to the side chain. Addition of a-CH NH2- group to the side chain (tyrosine) or substitution of a -CH2-NH2 group foi the carboxyl (tyramine) yields an easily oxidizable substrate that gives a highly fluorescent product with approximately the same characteristics as oxidized p-OH-phenylacetic acid. Addition of a second -OH group (dopamine) again leads to complicating side reactions, as does substitution of a -OH for -H in the -CH2 COOH side chain (mandelic acids). Likewise substitution of -OH or -OCHa groups in the ring at any position other than the para position produces a nonoxidizable substrate. In order t o get better sensitivity, a new procedure was developed for the sugar analysis. A new lamp Turner No. 110-855 was used which had a range from 270-350 mp and peak emission at 306 mp. The lamp used in the previous analysis using HVA, Turner No. 110-850, had a range from 300-500 mp with a peak at 360 mp. A combination of the 110-855 light source and the Corning CS 7-60 filter provides light peaking at about 325 mp. This helped considerably, However, with this new lamp and HVA as the substrate, the sensitivity was not significantly improved. A p H os. rate analysis was run on a sample of galactose using the substrate p-hydroxyphenylacetic acid. The filter system was as follows: primary CS 7-60 and secondary 47B. The results are given in Table V, where it can be seen that the (6) E. Wehry in “Fluorescence. Theory, Instrumentation and Practice,” G. Guilbault, Ed., Dekker, New York, 1967, p 60. 1260
ANALYTICAL CHEMISTRY
rate was highest at a p H of 8.5 in tris buffer. Hence this pH was used in the analysis of all sugars. With the micro method, galactose could be determined in concentrations from 0.7-7 pg per ml (2-22 total pg) using either p-hydroxyphenylacetic acid or tyramine as the detecting substrate. The results are given in Table VI. Using p-hydroxyphenylacetic acid, 0.3-4 pg per ml of stachyose (1-12 pg total) were determined, and sucrose was assayed with p-OH PAA using invertase and glucose oxidase in concentrations of 0.2-15 pg per ml (0.6-45 pg total). In all cases analysis was performed with a n average error of less than 1 and a precision of about 1.5 %. As in the assay of any sugar by an initial rate method, the sugar concentration must be well below the K m for best results. Since the Km value for most sugars is about (Table VII), all analyses described are in a region where maximum accuracy and precision can be expected. Results (precision and accuracy) with tyramine and p hydroxyphenylacetic acid are comparable, and both substrates have the advantage over HVA of low cost ($0.44 per gram compared to $30.). All three substrates are extremely
PH 7.00
7.50 8.00 8.50 8 . 50a 8 . SOh
Table V. pH us. Rate (0.1M Tris) Buffer Rate, AF/min Tris 4.50 Tris 15.0 Tris 25.0 Tris 27.0 Glycine 13.0 Phosphate 22.0 Tris 16.0
9.00 Glycine 0.1M. Phosphate 0.1M .
Table VI. Analysis of Sugars Using Various Indicators Re1 error, Galactose, pg total Added Founda8b Foundale p-OH-PAA Tyramine -1.5 +0.9 4.16 4.10 4.20 +0.9 -0.8 8.32 8.40 8.25 -1.6 0.0 12.3 12.5 12.5 +O. 6 t0.6 16.7 16.7 16.6 +1.0 -1.0 21.0 20.6 20.8 -1.2 -1.2 24.7 24.7 25. Od +l.l k0.9 Av re1 error
z
Stachyose, pg total R el ..-.
Added 1.52 2.96 5.70 7.00 9.40 10.5 Av re1 error
Found.,* 1.54 2.95 5.60 7.10 9.46 10.5
error, % $1.3 -0.3 -1.7 +1 4 t0.5 0.0 +l.O
Sucrose, pg total Re1 Added Founda,* error, Z 2.57 0 2.57 4.09 -0.25 4.10 $1.8 5.11 5.20 -1.3 15.5 15.3 25.7 0 25.7 $1.0 41.0 41.4 10.72
a Represents an average of three or more determinations with a relative standard deviation of 1.5 %. * With p-hydroxyphenylacetic acid (p-OH-PAA). Lowest detectable concentration of galactose, l pg. c With tyramine. Lowest detectable concentration of galactose, 4 pg. d 250 pg of glucose added.
stable, and are not air oxidized in aqueous solution. An absolutely zero rate is observed until the oxidase is added with HVA and p-OH PAA. A slight blank rate was observed with tyramine. Blanks were run for 30 minutes with no increase in the fluorescence, and solutions of both p-OH PAA and HVA up t o 3 months old have been used. When mixed with peroxidase in tris buffer, p H 8.50, in a mixed reagent solution, the HVA or p-OH PAA are likewise stable t o autooxidation, but the shelf life of the solution is about two weeks because of the limiting stability of the peroxidase. Another substrate which has the same fluorescent properties as tyramine after oxidation by peroxidase is tyrosine. However as seen in Table IV, the fluorescence coefficient of oxidized tyrosine is 7.8 X lo5, less than that of HVA, 1.27 x 106. The fluorescent coefficient of oxidized tyramine is 3.92 x lo6 and for p-hydroxyphenylacetic acid 3.28 X lo6. These values are relative t o that for quinine sulfate, 1.12 X 108, obtained in the filter photometer, set on maximum sensitivity, with a Corning CS 7-60 primary, a Kodak Wratten 47B secondary filter, and a Turner 110-855 UV light source. It should be realized that the values of the fluorescent coefficient have significance relative to each other and relative to quinine sulfate. Their magnitude will vary from instrument t o instrument, however. The p-hydroxyphenylacetic acid was judged to be the best substrate for peroxidase, because it permits the determination of lower concentrations of sugars caused by its zero blank. The blank rate with tyramine, although slight (about 0.1 fluorescence unit per minute), still prevents the determination of very low concentrations of sugar. With p-OH PAA as substrate, all the sugars listed in Table I can be determined in the concentration range of 0.3-10 pg per ml (1-30 pg total) with a precision and accuracy better than obtained with HVA. Determination of Substrates. By using non-rate limiting concentrations of peroxide and peroxidase, the substrates HVA, p-hydroxyphenylacetic acid, tyramine, and tyrosine
Table VII. Michaelis Constants (Km) for Various Sugars All Km’s measured at pH 8.50 in tris buffer, O.lM, with the peroxidase-homovanillic indicator reaction Sugar Enzyme Km Glucose oxidase D-Glucose 5 x 10-4 Glucose oxidase 2-Deoxy-D-glucose 1.5 x 10-3 Galactose oxidase D-Galactose 2.78 x 10-3 Galactose oxidase Stachyose 6.8 x 10-4 Galactose oxidase 2-Deoxy-~-galactose 3.9 x 10-3 Galactose oxidase D( +)-Raffinose 1.87 x 10-3 CU-D( +)-Melibiose Galactose oxidase 1.62 x 10-3 Galactose oxidase D( +)-Galactosamine 3.6 x 10-3 Galactose oxidase 1.78 x 10-3 N-Acetybgalactosamine Methyl-0-DGalactose oxidase 1.5 x 10-3 galactopyranoside Invertase-glucose Sucrose 3.5 x 10-4 oxidase Table VIII. Analysis of Substrates p-OH PAA, pg total Tyramine, pg total Re1 Re1 Added Founda error, % Added Founda error, % 3.71 3.80 +2.4 4.61 4.60 -0.2 5.57 5.60 $0.5 5.75 5.80 +0.8 7.42 7.30 -1.6 6.90 6.80 -1.4 9.28 9.20 -0.9 10.3 10.2 -1.0 13.0 13.0 0.0 12.6 12.6 0.0 18.5 18.4 -0.5 23.8 24.0 +0.7 Av re1 error + l .o A0.7 HVA, pg total
3,4-diOH PAA, pg total Re1 Re1 Added Found“ error, Added Founda error, 4.60 4.65 +1.1 58.7 59.0 +0.51 8.97 9.00 +0.3 115 112 -2.6 26.8 27.0 10.7 168 173 $3.5 31.9 32.0 $0.3 220 224 $1.8 43.0 -3.1 268 44.4 266 -1.1 55.5 56.0 $0.9 300 306 +2.0 Av re1 error 11.0 11.9 Represents an average of three or more determinations with a relative standard deviation of 1.5%.
z
z
5
could be determined, based on the production of their fluorescent oxidized form a t the excitation and emission wavelengths listed in Table IV. Some typical results for the determination of p-OH PAA (2-20 pg total), tyramine (440 pg total), and HVA (4-60 pg total) are given. An accuracy and precision of about 1 was obtained (Table VIII). The compound 3,4-dihydroxyphenylaceticacid was found to produce an intense color upon oxidation, having a A,, at 500 mp. A spectrophotometric assay was developed for this compound in the 50-300 pg range with a n accuracy of 1.9% and a precision of 1.5 %. It should be possible to assay mixtures of the 3,4-di-OH PAA and either HVA, tyramine, orp-OH PAA, using a colorimetric procedure for the former and a fluorescence technique for one of the latter substances. Temperature Effect. The temperature of the reaction was held constant at 30 “C., because the temperature coefficient of a n enzyme reaction rate is roughly 10% per degree, and a 10 “C rise in temperature can cause a 100% increase in the reaction rate (7). (7) H. Netter, “Theoretische Biochemie,” Springer-Verlag, Berlin, 1959, p 559. VOL. 40, NO. 8, JULY 1968
1261
Inhibitors. In making a n analysis, the inhibitors of the enzyme systems must be absent. A listing of some of the inhibitors of the peroxide-peroxidase system is given below: Mn2+, Sz-, Co2+, Crz012-, “*OH, Pbzf, Fez+, Fe3+, CuZ+, CN-, Cd2+, NiZ+,S032-, cystine, p-amino-benzoic acid, and SeOClz (4). Besides these inhibitors, the ions which inhibit the glucose-glucose oxidase and galactose-galactose oxidase systems should be absent. Specificity of Glucose Oxidase (Table IX). Glucose oxidase is highly specific for P-D(+)-glucose. Any alteration in the molecule enormously reduces the rate of oxidation (8). If the relative rate of oxidation of P-D(+)-glucose = 100, then only five sugars have a relative rate greater than 1 : 2deoxy-D-glucose = 25 ; 6-deoxy-6-fluoro-~-glucose = 3 ; 6-methyl-~-glucose = 1.85 ; and 4,6-dimethyl-~-glucose = 1.22. In reference (8), the alterations of the molecule and the subsequent loss of activity are discussed in detail. Another good reference for a listing of the sugars which are substrates and which are not is given in “Methods of Enzymology” (9). The high specificity of this enzyme makes it one of the most useful reagents for the detection and the quantitative estimation of glucose and 2-deoxy-~-glucosein biological material. Specificity of Galactose Oxidase (Table IX). Galactose oxidase is not so highly specific as glucose oxidase. The CI position need not be free, for galactosides are readily attacked. The P configuration is somewhat favored. However, this is not an important structural requirement. The galactose configuration at position 4 is essential; glucose and its derivatives are completely inert. The configuration a t position 2 is not so critical for D-talose, 2-deoxy-~galactose, and D-galactosamine are good substrates, comparable to D-galactose (10).
CHO
I I HO-C-H I HO-C-H I H-C-OH I
I I HO-C 3-H I
I
H-C ‘-OH
HO-&H
HO-C-H
HO-CeH
H-C-OH
I
I
H-Cj-OH
H-C“OH
I
1
Hz--C‘-OH
Hz--C‘-OH
H 2-C6-0H
D-Talose
D-Galactose
D-Glucose
,
.
n on
n
OH
OH
Stachyose It was found in this study that if the number of carbon atoms is reduced by one, keeping the same configuration as (8) M. Dixon and E. Webb, Eds. “Enzymes,” Academic Press,
New York, 1958, p 285. (9) S. Colowick and N. Kaplan, Eds., “Methods of Enzymology,” Academic Press, New York, 1957, p 107. (10) G. Avigad, D. Amaral, C . Asensio, and B. Horecker, J . Bid. Chem., 237, 2736 (1962).
1262
ANALYTICAL CHEMISTRY
Rate of reaction Glucose Galactose oxidase oxidase
Sugar
P-D( +)-Glucose 2-Deoxy-~-glucose L-Glucose Sucrose D( +)-Galactose 2-Deoxy-D-galactose D( +)-Raffinose Stachyose cu-D-Melibiose
High High None None None None None None None None None None None None
Methyl-6-D-galactopyranoside D( +)-Galactosamine
D-Gulose L- Altrose D-LyXOSe
Table X.
25.0 25.0 2.50 15.0 15.5 15.5 25.7
2.50 25.0 25.0 25.0 25.0 25.0 25.0
Found, c(g Glucose Galactose Sucrose
... ... ... ...
25.1 24.9 2.50 14.8 15.6 15.5 25.4
15.5 25.7 15.5
CHO CHO
1 I
HO-C-H HO--C-H
I
H-C-OH
I
Hz-C-OH D-Lyxose
None None None None High High High High High High High None High None
Analysis of Glucose, Galactose, and Sucrose Mixtures
Present, pg Glucose Galactose Sucrose
CHO
CHO
H-C*-OH
Table IX. Specificity of Sugar Analysis
I I HO--CH I HO--C-H I HO-C-H I
H--C-OH
Ha-C-OH L-Altrose
...
2.49 25.0 25.2 25.0 24.9 25.1 25.0
... ... I
.
.
15.6 25.6 15.3
CHO
I I H-C-OH I HO-CH I H-C-OH I
HX-OH
Hz-C-OH
D-Gulose
D-galactose (for example D-lyxose), the rate of oxidation is reduced drastically. Also L-altrose, which differs from Dgalactose only in the C sposition, gives an appreciable rate compared to D-galactose. Thus the CSposition is not so critical. The C 3 position is critical because D-gulose, which differs from D-galactose only at this position, is not oxidized a t all. From these structural considerations, the enzyme can be very useful as a specific reagent in sugar analyses. Analytical methods for a few more sugars could have been developed if they were commercially available. A listing of these is given in reference (10). In looking at Table IX, it becomes evident that two different sugars can be determined in the same sample by a judicious choice of enzymes. For example, a mixture of P-D(f)-glucose and D(+)-galactose could effectively be analyzed for both sugars using first glucose oxidase, then galactose oxidase.
A three component mixture of 6-D-glucose, D-galactose, and sucrose can be analyzed using three enzymes: glucose oxidase, galactose oxidase, and invertase. Analysis is possible because of the specificity built into these enzymes. Some results obtained in the analysis of such mixtures are given in Table X. It was found that concentrations of galactose up to 100 times that of glucose did not interfere in the determination of the latter, and vice versa. Three component mixtures of glucose, galactose, and sucrose were analyzed for all three components with an accuracy and precision of about 1.5 %. One aliquot (A) was analyzed for glucose using glucose oxidase, another (B) for galactose using galactose oxidase in procedures as described above in the experimental section. A third aliquot
(C) was analyzed for sucrose by addition of invertase to liberate glucose, followed by a determination of total glucose with glucose oxidase. The amount of sucrose present was calculated by subtracting the glucose found in A from that found in C. ACKNOWLEDGMENT
The authors thank Nelson Richtmeyer of the National Institutes of Health for samples of two sugars used in this study. RECEIVED for review February 12, 1968. Accepted April 3, 1968. Work supported by National Science Foundation Grant No. GB 6325.
Use of Metal Ion Catalysis in Detection and Determination of Microamounts of Complexing Agents Autoxidation of 1,-Ascorbic Acid as an “Indicator” Reaction Horacio A. Mottola,’ Martha S. Haro, and Henry Freiser Department of Chemistry, University of Arizona, Tucson, Ariz. 8572I A method for the determination of microamounts of metal complexing agents based on their interaction with metal ion catalysts for oxidation-reduction reactions is presented. The rate of decrease in absorbance of L-ascorbic acid at 265 mp due to atmospheric oxidation catalyzed by Cu(ll) is the basis of a convenient method for the detection and determination of certain Cu(ll)-complexing ligands in aqueous media at pH -6.4. Trace amounts of cysteine, 2-aminoethanethiol, salicylic acid, EDTA, 1,lO-phenanthroline, and ethylenediamine have been determined by this means.
IN THE SEARCH for increasingly sensitive analytical reactions, chemists have employed reagents that would give intensely colored or fluorescent products. This approach, though fruitful, is limited finally by the requirement that the compound of interest be stoichiometrically related to the product. Substances that have catalytic activity or which can modify the action of catalysts, in principle, can be detected at much lower concentrations because the reactants or products of the catalyzed reaction (indicator reaction) will be present at relatively high concentrations, so that changes in these can be readily measured. Provided the uncatalyzed reaction is sufficiently slow, increase of reaction time will further increase the sensitivity of catalyst detection. Both “coordination chain” ( I ) and metal ion-catalyzed oxidation-reduction reactions (2) may be used as indicator reactions (that which provides the time-dependent signal) (3). In both cases the ‘Present address, Department of Chemistry, Oklahoma State University, Stillwater, Okla. 74074 (1) D. W. Margerum and D. K. Steinhaus, ANAL.CHEM., 37, 222 (1965).
(2) H. A. Mottola and H. Freiser, ibid., 39, 1294 (1967). (3) K. B. Yatsimirskii, “Kinetic Methods of Analysis,” Pergamon Press, New York, 1966, p 1.
analytical determination is based on the modification of reaction rate by complexation. A great advantage of such methods is the ease of monitoring the reaction rate. Because the indicator reaction components are in relatively high concentration, a suitable reaction parameter is easily found: absorption or emission of radiant energy, heat of reaction, or any other physical property that would change regularly with the course of the reaction. The application of a metal ion-catalyzed redox reaction to the determination of traces of ethylenediamine-NNN’N’-tetraacetic acid (EDTA) has been reported recently (2), using as indicator system, the oxidation of Malachite Green by periodate ion in which manganese(I1) acts as catalyst. The relatively high values of formation constants of Mn(I1) with EDTA and some of its analogs make the system selective since most other N-containing ligands will not complex Mn(I1) under the experimental conditions employed. This illustrates one of the first steps involved in selecting a suitable reaction system: one in which the metal ion catalyst forms complexes of sufficient stability with the ligand(s) of interest so that formation occurs at low ligand concentrations. If the metal ion concentration required for measurable catalysis is about 10-6M and a change in rate is detectable for a 10% change in metal ion concentration-i.e., [ML]/[M] = 0.1 (assuming the 1 :1 complex to predominate)-it follows that at least lO-7M of the agent is required for formation. Assuming that an additional lO-7M agent is required to ensure complex formation according to mass action considerations, this would give a response whose sensitivity is 2 X 10-7M for the agent. I t can be estimated that the minimum required stability of the metal-agent complex under these conditions would be
Ki’
=
0.1 [ML]/[M][L] = 10-7
=
106
where Kl’ is the conditional stability constant.
Such K values
VOL. 40, NO. 8, JULY 1968
1263
