Elements Impurity, ppm
Table 111. Rare Earth Impurities in High Purity YzO3 Determined by Spectrophotoluminescence Pr Sm Eu Gd Tb DY Ho Er 25 ... 0.56 ... 0.11 1.1 0.38 0.74
same exciting wavelength and which have overlapping emission bands, good analytical results are obtained by determination of the respective excitation spectra which differ markedly. Error. To estimate the error caused ‘Jy sample preparation, nine samples were prepared by three persons (A, B, C ) . Residual rare earth impurities in the Y 2 0 3used in this test mole Dy203and 9 X 10-6 mole Tbz03; the were 4 X mole. The added impurity was dysprosium at 1 X dysprosium concentrations were used to estimate the error. Data obtained are given in Table 11. Accuracy of determination by spectrophotoluminescence was good with an error +lox. The time for one sample preparation was 3 to 4 hours ; operation time for the spectrophotoluminescence determination was 10 to 20 minutes. If several samples are prepared simultaneously, time requirements may be reduced.
Trn
...
In Table I11 is given the result of a quantitative determination of rare earth impurities in 99,999x pure Y203 (Spex Industries, Inc.). Determinations of Dy, Er, Pr, Tb, Eu, Sm, and Tm were made by the emission spectra. Sm, Gd, and Tm were below sensitivity limits. ACKNOWLEDGMENT
The authors thank Satoru Nishikawd for his assistance in sample preparation, and measurements and also Kiyoshi Morita and Hiroshi Sunahara, Government Industrial Research Institute, Nagoya, for their helpful discussions. RECEIVED for review March 1, 1967. Accepted September 22, 1967.
Homovanillic Acid as a Fluorometric Substrate for Oxidative Enzymes Analytical Applications of the Peroxidase, Glucose Oxidase, and Xanthine Oxidase Systems George G. Guilbault, Paul Brignac, Jr., and M a r y Zimmer
Department of Chemistry, Louisiana State Unicersity in New OrIeans, Lakefront Campus, New Orleans, La, 70122 Fluorescence methods are described for the assay of the oxidative enzymes-peroxidase, glucose oxidase, and xanthine oxidase-based on the conversion of the nonfluorescent homovanillic acid to the highly fluorescent 2,2’-dihydroxy-3,3‘-dimethoxybiphenyl-5,5’-diacetic acid. The initial rate of formation of this fluorescent compound is measured and related to the activity of the enzymes. The substrates of these enzyme systems-peroxide, glucose, hypoxanthine and xanthine-can be determined in microgram concentrations in biological samples as blood and urine with a precision and accuracy of less than 3%. Finally, analytical methods are described for various inorganic and organic substances which inhibit these enzyme systems: cyanide, sulfide, dichromate, sulfite, Cu(ll), Fe(ll or Ill), Mn(ll), Pb(lI), Co(ll), Cd(lI), hydroxylamine, and cystine. The reagent homovanillic acid is very stable and can be used for several months.
SEVERAL ARTICLES have described the development of analytical procedures for the determination of various inorganic and organic substances using enzymes as analytical reagents ( I ) . Advantage is taken of the extreme sensitivity (frequently in the nanogram to picogram region) and specificity of the enzyme, thus giving the two factors most desirable in any analytical technique. In general the acceptance of enzymes as useful analytical reagents has been slow, mainly because of the lack of good sensitive analytical methods for their determination. In this (1) G.
1%
Guilbault, ANAL.CHEM., 38, 527R (1966). 0
ANALYTICAL CHEMISTRY
vein Guilbault and Kramer have been particularly active in developing fluorescence methods for enzyme systems based on a measurement of initial reaction rates (2-6). Such analyses resulted in a two- to threefold increase in sensitivity over conventional colorimetric, manometric, or pH techniques, and in a considerable saving in analysis time (inasmuch as a typical reaction rate analysis can be performed in 1 to 3 minutes). Several enzymic methods have been used for the determination of peroxide ( 7 - l l ) , glucose (12), and xanthine (13) based on conventional spectrophotometric procedures. (2) G. Guilbault and D. N. Kramer, ANAL.CHEM., 36, 409 (1964). (3) Ibid., 37, 120 (1965). i4j Ibid.;p. i m . (5) D. N. Kramer and G. Guilbault, Ibid.,35, 588 (1963). (6) Ibid., 36, 1662 (1964). (7) B. Chance and A. C. Maehly, “Methods in Enzymology,” S. Collowick and N. Kaylan, Eds., Vol. 2, Academic Press, New York, 1955, p. 764. (8) B. Chance and A. C. Maehly, “Biochemists Handbook,” C. Long, Ed,, Van Nostrand, Princeton, N. J., 1961, p. 384. (9) N. B. Chapman and B. C. Saunders, J . Chem. SOC.,1941, p. 496. (10) G. G. Guilbault and D. N. Kramer, ANAL.CHEM., 36, 2494 (1964). (11) R. Wilstatter and A. Stoll, Ann., 416, 21 (1927). (12) H. Bergmeyer, “Methods of Enzymic Analysis,” Academic Press, New York, 1963, p. 123. (13) Ibid.,p. 495.
Recently, Guilbault, Kramer, and Hackley described the use of homovanillic acid (HVA) for the quantitative determination of peroxidase (14). The compound (I) is nonfluorescent, but is converted upon oxidation to the highly fluorescent compound (11), which has a A,, of 315 mp and a YHZCOOH
CH,COOH
YH,COOH
OH
OH
“Oxidant”
OMe
MeO
Me0
bH
I1
I
,A, at 425 mp. It was the intent of this study to develop fluorometric methods for a variety of oxidative enzyme systems using homovanillic acid, and using these new procedures, to develop specific and sensitive analytical methods for the determination of substrates, activators, and inhibitors of such systems. Preliminary results indicated that HVA could be used for the determination of the oxidative enzymes peroxidase, glucose oxidase, and xanthine oxidase, and for the substrates of these enzymes systems peroxide, glucose, hypoxanthine, and xanthine in biological samples, such as urine and blood. Finally, analytical methods were developed for various inorganic and organic substances which inhibit these enzyme systems : cyanide, sulfide, dichromate, sulfite, Cu(II), Fe(I1 or 111), Mn(II), Pb(II), Co(II), Cd(II), hydroxylamine, and cystine.
+ H202 Hypoxanthine + H 2 0 + ~xidasexanthine + H202 gluconic ccid
(1)
Xanthine
0 2
Xanthine H202
+ HzO +
+ HVA
Xanthine
0 2
Peroxidase
uric acid
+ H~OZ
fluorescent compound I1
(2) (3)
(4)
In general, all substrates, enzymes, and inhibitors were determined with a relative standard deviation of =t3Z and an accuracy of less than 2%. EXPERIMENTAL
Solutions. All solutions were prepared using triply distilled water, which is obtained by passing once distilled water through a charcoal column, followed by a double distillation from permanganate. Enzymes. PEROXIDASE. A stock 0.75-mg/ml solution of horse radish peroxidase (Calbiochem. Co., Los Angeles, Calif.) was prepared by dissolving 7.5 mg of enzyme in triply distilled water. The enzyme was assayed (15) and found to have an activity of 125 units per mg. GLUCOSEOXIDASE.Fungal glucose oxidase (Calbiochem. Co., specific activity 20 enzyme units per mg) was dissolved in triply distilled water. The enzyme was assayed colorimetrically using o-dianisidine (16). XANTHINEOXIDASE.All standard solutions were prepared by diluting xanthine oxidase, obtained from milk (14) G. G. Guilbault, D. N. Kramer, and E. Hackley, ANAL. CHEM., 39, 271 (1967). (15) A. C. Maehly and B. Chance, “Methods of Biochemical Analysis,” D. Glick, Ed., Vol. 1 , Interscience, New York, 1954, p. 337. (16) A. St. G. Huggett and D. A. Nixon, Bioclwn. J . , 66, 12P (1957).
(Worthington Chem. Co., Freehold, N. J.) in water. The enzyme was assayed using the colorimetric procedure of Kalckar (17), and was found to contain 9.5 units per ml of preparation. Substrates. PEROXIDE.A 3% solution of hydrogen peroxide was prepared by diluting a 3 0 z stock solution (Merck and Co.), which had passed through an ion exchange column to remove metal ion impurities, with triply distilled water. The peroxide concentration was assayed titrimetrically with standard ceric sulfate. HYPOXANTHINE AND XANTHINE.Stock 10 mg/ml solutions were prepared by dissolving the compounds (Sigma Chemical Co., St. Louis) in tris(hydroxymethy1)aminomethane buffer, pH 8.00. GLUCOSE.A standard solution (0.1M) was prepared by dissolving 1A072 grams of P-D-glucose (Sigma Chemical Co., St. Louis) in 100 ml of tris buffer, pH 8.00. HOMOVANILLIC ACID. Solutions were prepared by dissolving the CP compound (Calbiochem. co.) in triply distilled water. Apparatus. Fluorescence measurements were made with an Aminco-Bowman spectrophotofluorometer equipped with a thermoelectric cooler to maintain a constant temperature at 30” C, and a Beckman linear recorder for automatic recording of the changes in fluorescence with time. Alternatively, an Aminco filter fluorometer was used for some studies described. It had a CS 7-60 primary filter, a 47B, 2A combination secondary filter, a circulating water bath to control the temperature (at 30” C ) , and a Beckman linear recorder. Calibration. In order to obtain reproducible rates from day to day, the SPF is set to a total of 0.20 fluorescence unit (F.U.) daily with a standard 0.1 pg/ml solution of quinine sulfate in 0.1N sulfuric acid, A,, = 350 mp, A,, = 450 mp. Procedures. DETERMINATION OF PEROXIDASE. To 2.7 ml of 0.1M tris buffer (pH 8.50) is added 0.1 ml of 0.3% peroxide solution, 0.1 ml of a 2.5 mg/ml homovanillic acid solution and 0.1 ml of the peroxidase to be assayed (containing 0.003 to 7 units). The rate of change in the fluorescence of the solution is recorded at a A, of 315 mp and a ,A, of 425 mp. From calibration plots of AFjminute us. the peroxidase concentration, the amount of this enzyme present can be calculated. DETERMINATION OF GLUCOSE OXIDASE.T o 2.7 ml of 0.1M tris buffer (pH 8.00) is added 0.1 ml of the stock 0.1M glucose solution, 0.1 ml of a 2.5 mg/ml solution of homovanillic acid and 0.1 ml of a 0.75 mg/ml peroxidase solution. At zero time, 0.1 ml of a solution of the glucose oxidase to be assayed is added, (containing 0.003 to 0.75 unit) and the rate of change of the fluorescence with time is recorded. The amount of glucose oxidase present is calculated as above. DETERMINATION OF XANTHINE OXIDASE.The procedure as described above for glucose oxidase is followed, except that 0.1 ml of the xanthine oxidase solution to be determined (0.0010 to 0.10 unit) is added. From calibration plots of AFlminute cs. enzyme concentration the amount of xanthine oxidase present is determined. DETERMINATION OF PEROXIDE.To 2.7 ml of 0.1M tris buffer (pH 8.50) is added 0.1 ml of a 2.5 mg/ml solution of homovanillic acid and 0.1 ml of 0.75 mg/ml of peroxidase solution. At zero time, 0.1 ml of the peroxide to be determined (containing 0.01 to 1.5 pg) is added, and the rate of change in the fluorescence with time is recorded. From calibration plots of AFlminute 6s. peroxide concentration, the amount of this substrate present can be determined, DETERMINATION OF GLUCOSE,The following are mixed in a 1.0-cm cuvette: 2.7 ml of tris buffer (pH 8.0), 0.1 ml of 2.5 mg/ml of homovanillic acid, 0.1 ml of the glucose solution to be analyzed (0.5-50 pg) and 0.1 ml of 0.75 mg/ml peroxi(17) H. Kalckar, J. Biol. Chem., 167,429 (1947). VOL. 40, NO. 1, JANUARY 1968
191
Table I. Determination of Peroxidase, Glucose Oxidase, and Xanthine Oxidase Peroxidase, units/ml Glucose oxidase, units/ml Xanthine oxidase, units/ml Added Founda Added Found" Added Founds 0.00466 0.00476 0.00129 0.00128 0.000200 0,000206 0.0233 0.0236 0.00666 0.00676 0.00210 0.00204 0.0548 0.0547 0.0250 0.0250 0.00420 0.00420 0.110 0.110 0.0472 0.0463 0.00800 0.00808 0.466 0.457 0.0666 0.0680 0.0170 0.0167 2.33 2.37 0.250 0.255 0.0330 0,0333 Peroxidase +2.0 $1.3 -0.2
Re1 error, % Glucose oxidase -0.7 +1.5
Xanthine oxidase +3.0
-2.8
0
0 +I .o -1.7 +0.9
-1.9 +2.1 -2.0
0
-1.9 +2.0
Av re1 error h1.6 zk1.9 12.0 a Represents an average of three or more determinations with a relative standard deviation of 1 3 z .
Peroxide, pg/ml Added 0.00340 0.0100 0.0500 0.100 0.200 0.500
Table 11. Determination of Substrates Glucose, pg/ml Added Founda 0.287 0.292 0.596 0.584 1.75 1.75 2.97 2.92 5.72 5.84 17.5 17.8
Founda 0.00330 0.0102 0.0490 0.100
0.202 0.515
Re1 error, % Glucose -1.7 $2.0
Peroxide -3.0 +2.0 -2.0
0
+1.7 -2.1 +1.7
0
$1.0 +3.0
5
Hypoxanthine, pg/ml Added Found0 0.0300 0,0303 0.0750 0.0740 0.100 0.102 0.200 0.194 0.500 0.500 1.00 1.02
Hypoxanthine $1.0
-1.3 +2.0 -3.0 0
+2.0
Av re1 error h2.2 11.8 f1.9 Represents an average of three of more determinations with a relative standard deviation of &3%.
dase. At zero time, 0.1 ml of a solution of glucose oxidase (2 mg/ml) is added, and the analysis is performed as above. DETERMINATION OF HYPOXANTHINE AND XANTHINE.The analysis is performed as in the analysis of glucose except that a xanthine or hypoxanthine solution to be analyzed (0.1 to 3 pg) is added in place of glucose. At zero time, 0.1 of a 0.5 unit/ml solution of xanthine oxidase is added. DETERMINATION OF INHIBITORS. To 4.0 ml of tris buffer (PH 8.50) is added 0.1 ml of HVA (2.5 mgjml), 0.1 ml of peroxide (0.3% solution), and 0.1 ml of the inhibitor to be determined. The fluorescence is adjusted to read zero, and 0.1 ml of a 0.5 mg/ml solution of peroxidase is added. The rate of change in fluorescence with time, AFlminute, is determined, and is compared to a control containing no inhibitor, From calibration plots of per cent inhibitor us. the log of inhibitor concentration (Figure 1) the amount of inhibitor present in solution can be calculated.
% Inhibition
=
[AFjmin (no inhibitor)] - [AF/min (inhibitor)] AF/min (no inhibitor)
192
ANALYTICAL CHEMISTRY
x
100
RESULTS The results of the determination of samples of the enzymes peroxidase, glucose oxidase, and xanthine oxidase are given in Table I. Samples of peroxidase and glucose oxidase ranging from 0.00466 to 2.33 and 0.00129 to 0.250 units per ml were determined with average relative errors of h1.6 and 1.9%. Samples of xanthine oxidase ranging from 0.00020 to 0.400 unit per ml were analyzed with an average relative error of 2%. The substrates peroxide (0.0034 to 0.50 pg per ml), glucose (0.292 to 17.5 pg per ml), and hypoxanthine (0.030 to 1.0 pg per ml) were analyzed with average relative errors of k2.2, 1.8, ad 1.9%) respectively (Table 11). Some typical results obtained in the determination of various inhibitors of peroxidase are listed in Table 111. From 1-100 pg/ml of CN-, 0.3-3.2 pg/ml of S2-, 0.3-12 pg/ml of Mn2+, 2-60 pg/ml of NH20H, Fez+, and Fe3+, and 0.5-20 pg/ ml of Co2+were determined with an average relative error of about =t2%. All results given represent an average of
90
three or more results, with a standard deviation of 5 3 % . Results reported for sulfide, Mn(II), and hydroxylamine are those obtained in the analysis of complex mixtures.
80
-
C
dS;/'
70 -
DISCUSSION /"
Use of Homovanillic Acid as Substrate. The oxidation of homovanillic acid to form a fluorescent compound (11) upon peroxidatic activity is believed to proceed via the following mechanism :
/
(a)
2H202-+4HO*
CH,COOH
FH,COOH
I
I
OH I
FH,COOH
