4-Methoxy-a-Naphthol as a Spectrophotometric Reagent Substrate for Measuring Peroxidatic Activity GEORGE G. GUILBAULT and DAVID N. KRAMER Detection Research Branch, Defensive Research Division, Directorate of Defensive Systems, CRDI, Edgewood Arsenal, Md.
b An improved substrate is described for rapid spectrophotometric assay of the enzymes peroxidase, glucose oxidase, and xanthine oxidase. The compound, 4-methoxy-a-naphthol, is a colorless material which is oxidized to an intensely colored blue compound (E = 1.8 X a t 620 mb) upon peroxidatic action. By this procedure 0.010 to 0.700 units/mI. of peroxidase, 0.00550 to 0.220 and 0.000330 to 0.00666 units/ml. of glucose oxidase and xanthine oxidase were assayed with standard deviations of 1.9, 1.8, and 1.6, respectively. The oxidized material was found to be a stable compound, whose color and production are independent of pH.
lo4
B
of the importance of the enzymes peroxidase, glucose oxidase, and xanthine oxidase in the detection of submicrogram quantities of sulfhydryl-binding compounds-e.g., oiodosobenzoic acid (@-there was a need in these laboratories for an improved spectrophotometric reagent for rapid assay of these enzymes. I n particular. a stable colorless substrat,e was desired, which would give a n intense coloration upon peroxidatic activity a t any pH employed. Present spectrophotometric procedures for peroxidase include determination of dyes formed after aerobic oxidation of guaicol (S), or mesidine (4)with horseradish peroxidase (HRP) and peroxide ( 1 2 ) . Other oxidizable substrat,es include ascorbic acid, uric acid, cytochrome C, and eugenol (a). All of these substrates suffer the disadvantage of instability, of having their color formation dependent, on pH, or of requiring measurement in the ECAUSE
Table
Peroxidase ____--Added Founda 0 0100 0 0400 0 105 0 140 0 400 0 700 a
0 0103 0 0392 0 105 0 137 0 404 0 700
-
I.
EXPERIMENTAL
Reagents. All solutions were prepared from reagent grade chemicals and triply distilled water. MACILVAINE BUFFERS, pH 5.7 and 6.76 were prepared by dissolving the appropriate amounts of ?r'a2HP04,citric acid, and KC1 in 100 ml. of triply distilled water ( 7 ) . PEROXIDASE. -1 stock, 40 units/nil. solution of horse radish peroxidase (California Biochemical Co., Los Angeles, Calif.; specific activity 400 purpurogallin units per mg.) was prepared by dissolving 10 mg. of enzyme in 100 ml. of distilled water. GLUCOSEOXIDASE. Fungal glucose oxidase (California Biochemical Co.; specific activity 34 enzyme units per mg.) was dissolved in distilled water. XANTHINE OXIDASE. -411 standard solutions were prepared by diluting xanthine oxidase, prepared from milk by the method of Ball ( I ) (Worthington Chemical Co., Freehold, N. J.) in tris buffer, pH 7.40. The enzyme was assayed using the colorimetric procedure of Kalckar (IO) and was found to contain 11.2 enzyme units per ml. of preparation. HYDROGCX PEROXIDE. A 2 x 10-3M solution was prepared by diluting a 30y0 stock solution of hydrogen peroxide (Xlerck and Co., Rahway, ?;. J.) with tris buffer, pH 7.40. GLUCOSE. A standard solution (0.05M) was prepared by dissolving D-glucose (Eastman Organics, Rochester, S . Y.) in tris buffer, pH 7.40.
Determination of Peroxidase, Glucose Oxidase, and Xanthine Oxidase
oxidase ~ Glucose - - ~ - _ _ Added Found. 0 0 0 0
00550
0110 0220 0350 0 110 0 220
_
0 00556 0 0110 0 0225 0 06.50 0 107 0 224
Represents an average of three or more determinations.
2494
The resulting solution was allowed to sit overnight before using. HYPOXAKTHIXE. ;1 stock solution, 10-4.Tf, was prepared by dissolving hypoxanthine (Sigma Chemical Co., St. Louis, Mo.) in 1 ml. of lJ1 sodium hydroxide, and diluting to 100 ml. with llacllvaine buffer, pH 6.76. 4-11ETHOXY-a-SAPHrHOL. -1 stock 2 X 10-3X solution was prepared by dissolving 4-niethoxy-a-naphthol (Allied Chemical Co., Kational Aniline Division, Buffalo, S.Y . ) in 15 ml. of methyl cellosolve, and diluting to 100 ml with MacIlvaine buffer, pH 5.70. Apparatus. A11 photometric measurements were made with a Beckman model D B spectrophotometer using 1.0-em. cells and a Beckman linear recorder. Procedure. UETERMIKATIOX OF PEROXIDASE. To 3 ml. of a 2 x 10-3Alf solution of hydrogen peroxide in a 1.0cm. cuvet is added 0.1 cc. of a 2 x 10-351 solution of 4-methosy-a-. naphthol and 0.1 ml. of a solution of the peroxidase to be determined (containing 0.03 to 2.0 units). The rate of change in the absorbance of the solution with time is automatically recorded at 620 m p . From calibration plots of 4A /At us. enzyme concentration, the amount of unknown peroxidase present in solution may be calculated. DETERMINhTION O F GLUCOSE0x1DASL. Three milliliters of a 0.05X glucose solution, 0.1 nil. of a 40 unit5 per ml. solution of H R P and 0.1 nil. of a 2 x l o - 3 x solution of 4-methoxya-naphthol are placed in a 1.0-cnl. cuvet. At zero time, 0.1 ml. of a solution of glucose oxidase, containing 0.007 to 0.80 unit, is added, and the amount of glucose oxidase present is determined as described above. DETERMINdTION OF X A S T H I N E 0x1DASE. The procedure as described above for glucose oxidase is followed, except that 3 ml. of a 1 X 10-4J1 solution of hypoxanthine is used, and at zero
ultraviolet. Preliminary experiments indicated that the compound 4methoxya-naphthol might be an improved substrate for measuring peroxidatic activity. The blue color of the oxidized material is stable, independent of pH, and has a large molar absorptivity ( e = 1.8 X lo4 a t 620 mp).
ANALYTICAL CHEMISTRY
Xanthine oxidase Added Found. 0 0 0 0 0 0
000330 000660 00165 00266 00333 00666
0 0 0 0 0 0
000336 000657 00165 00271 000327 00654
Peroxidase +3 0
Std. dev.
-2 0 0 0 -2 1
+lo
0 0 19
Glucose
Xanthine oxidase
+I 1 0 0 +2 3 0 0 -2 7
+ I S -0 5 0 0
oxidase
+I 8 1 8
+ 1 Y
-1 8 -1 8 1 6
time 0.1 ml. of the xanthine oxidase solution to be determined (0.0005 to 0.040 units) is added. From calibration plots of A-1 'Al vs. enzyme concentration, the amount of xanthine oxidase present is determined. RESULTS
The results of the determination of samples of the enzymes peroxidase, glucose oxidase, and xant'hine oxidase are given in Table I. Samples of peroxidase ranging froin 0.0100 to 0.700 unit per nil. of total solution were determined with a standard deviation of 1.9 i ~(re1: error) * qtandard deviation =
Y
in - 1)
Sainplez of glucose oxidase and xanthine oxidaye, ranging from 0.00550 to 0.220 arid 0.000330 to 0.00660 unit per nil., respectively, R ere analyzed with. itandard deviations of 1.8 and 1.6, re9pectirely. DISCUSSION
Oxidation of 4-Methoxy-a-Naphthol. T h e oxidation of 4-methoxy-
a-naphthol to form a blue colored compound (111) upon peroxidatic activity is believed to proceed via the following mechanism. 2H,O,
-+
'"y
Figure 1 . Absorption spectra of 4-methoxy-a-naphthol (6) and the product of its oxidation (A)
uuni dried. The elemental analysis (Table 11) showed the compound has the probqble structure I11 (C22H1601)) the results agreeing well with those calculated on the basis of 111. The
4HO.
OH
0
0
OCH3
OCHg
OCH3
I r
1
-
1
0 0 CHs CH3 I11
In stel) ( l ) ,free hydroxyl radicals are presumed to he formed by the action of peroxidase on hydrogen peroxide. Thebe radicals then attack bmethoxya-naphthol to yield the blue colored compound (111) via a free radical mechanism. T o establish the identity of compound 111, the blue dye was prepared in quantity and allowed to precipitate out of solution, recrystallized from methyl cellosolve-water, and vac-
molecular weight via5 found to he 345, indicating a dimer, rather than a monomer. The 1,l' dihydroxy 2,2' binaphthyl compound [(11) minus the methoxy groups] is a known compound, formed upon ferric chloride oxidation of a-naphthol (6). Delannay ( 5 ) and Tkacheva (11) observed compound I11 (minus the methoxy groups) upon permanganate oxidation of a-naphthol. Elemental analysis fails to distinguish
between compound I1 or I11 as the final product. However: the infrared spect'rum of the oxidized inaterial indicates no phenol groups, a strong aromatic ketone (1250 em.-l). a conjugated C=C (1620 cni.-l), and an aryl ether (1140 em.-?). Hencp, the final blue colored compound alqears to be 111, but ot,her techniques ( X l I R ) mass spectrometry) would be needed to definitely est,ablish this as t,he blue product. The material posse melting point of 259-260" C. and had a iiiolar adsorpti\-ity of 2.80 X l o 4 a t 620 mp. The spectra of I11 in methyl cellosolve (Figure 1A) showed it to possess maxima a t 280 nip and 620 nip. The visible spectrum of the oxidized 4-met,hoxy-a- naphthol (Figure 1 1 ) is sufficiently different from that of the original compound (Figure 1B) as to periiiit ineasureinent of peroxidase concentration based on the formation of this blue color at 620 nip. Furthermore, the enzymes glucose oxidase and xanthine oxidase, lT-hich act upon t'he substrates glucose and hypoxanthine, respectively, to gil-e peroxide, may be rapidly and easily determined if peroxidase is present in excess.
Table 11. Comparison of C, H, 0 Analysis of Oxidized 4-Methoxy-aNaphthol with Various Possible End Products
I1
I11
(311-
c22-
(322-
HI002
HIS04 76 3 5 2
Hdl, 76 7 4 7 18 6
Product 76 5 5 0 lh 5
VOL. 36, NO. 13, DECEMBER 1964
2495
%
C,, / C H
0,Yc
75 8 5 8 18 4
18 5
Table 111. Effect of Peroxide Concentration
W20~1&' 0 4 X low3 0 4 X 1 2 x 10-3
1 2 x 10-3 2 0 x 10-3 2 0 X 4 0 X 4 0 x 10-3
[ P O I, U/ml 0 0665 0 1330 0 0665 0 1330 0 0665 0 1330 0 0665 0 1330
AA/min 0 0385 0 0780 0 0390 0 0800 0 0500 0 100 0 0501 0 100
Table IV. Effect of Glucose and Peroxidase Concentrations in Determination of Glucose Oxidase
4-Alethouy-cu-naphthol = 6 6 X 10-5Xj glucose oxidase = 0 11 units/ml [Glucose] [Peroxidase] 1 'I U/ml AA/min 1 x 10-1 1 21 0 100 5 x 10-2 1 21 0 140 0 084 1 21 1 x 10-2 0 065 1 21 j x 10-3 1 21 0 040 1 x 10-3 1 21 0 030 5 x 10-1 0 121 0 030 5 x 10-2 0 605 0 080 5 x 10-2 0 140 1 21 5 x 10-2 6 05 0 180
