Anal. Chem. 1002, 6 4 , 517-522
517
Hematin as a Peroxidase Substitute in Hydrogen Peroxide Determinations Zhang Genfa and Purnendu K.Dasgupta* Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061
Hematin can substitute for horseradish peroxidase (HRP) as the catalyst In the determinatlon of hydrogen peroxide uslng phenollc substrates such as p-hydroxyphenylacetate or pcresol. Although the peroxldatlc activity of hematin from bovine blood Is not as great as HRP in terms of unit iron content, the actlvity per unit welght is substantially greater. Hematin is 500 tlmes less expensive than HRP per unit peroxidatlc activity. I n hematln-cataiyzed systems, reaction development and fluorescence measurement can both be conducted optimally in the same ammoniacal buffer. Hydroxyalkyi hydroperoxldes are rapidly hydrolyzed to H202at thls pH and are also determlned. On the other hand, for methyl hydroperoxide, hematin exhlbits only -10% of the sensitivity exhibited by HRP. Hematln is signHlcantiy more stable In solutlon than HRP. The use of hematln as catalyst and p-cresol as the substrate leads to a particularly Inexpensive and sensitive system, permittlng a limit of detection (LOD) of 7 nM H202In a flow-injectlon configuration.
The importance of hydrogen peroxide as an analyte is dominated by clinical chemistry applications. Hydrogen peroxide is produced in stoichiometric amounts during the oxidation of substrates of clinical interest (e.g., glucose) by diasolved oxygen in the presence of the correspondingoxidase enzyme.' Presently, Hz02is also an analyte of environmental interest for its role in the oxidation of SOz dissolved in hydrometeors to H804.293The determination of environmental H202typically requires a submicromolar limit of detection, (LOD) and luminometric methods are generally used; electrochemical or absorption photometric methods are commonly used in clinical analysis. Chemiluminescence (CL) techniques used are based on lumino14 or peroxy~xalate,~~~ and fluorescence techniques are based on the formation of a fluorescent oxidation prod~ct.7~~ In common with the colorimetric clinical methods, the fluorometric procedures use HRP as a selective enzyme catalyst for the oxidation of a chromogenic/fluorogenic substrate by Hz02 Presently fluorometric determinations of Hz02largely rely on 4-hydroxyphenylacetate (HPA) as the nonfluorescent substrate,!+20with best case aqueous-phase LODs of 3-5 nMl1J9and gas-phase LODs of 3 X lo-" atm.16 While the use of HRP endows the above methods with a specificity for HzOzcharacteristic of enzymatic techniques, it has its shortcomings. The instability of the enzyme in solution complicates field measurements of ambient H202. If mixed reagents (e.g., HPA + HRP) are used, fresh reagent needs to be made daily even when refrigerated.19 Although HRP is not exorbitantly expensive, the cost is not insigificant. In common with many other laboratories of modest means engaged in long-term programs of H202measurement, we have been seeking alternatives to HRP. Recently the Fenton reaction (Fe2++ H202 'OH) was used to produce fluorescent hydroxybenzoic acids from nonfluorescent benzoic acid.21 Also, UV irradiation has been used to form the fluorescent dimer product from HPA and H202.22 Unfortunately, neither method can reach LODs of M,
-
0003-2700/92/0364-0517$03.00/0
of particular importance in automated near real-time gasphase H202measurements. An alternative is to seek an understanding of the enzyme behavior and thus provide synthetic substitutes for HRP. HRP is a 40 OOO Da globular protein with a heme center, the search for a substitute has therefore concentrated on metal-porphine complexes. Saito et al.23-26 synthesized Mn(II1)-tetrakis(sulfopheny1)porphine. Immobilized on anion exchange resins through the negatively charged sulfonate groups, the metal complex mimicked the behavior of immobilized HRP. More recently, Ci and Wang%" have studied tetrakis(N-methylpyridinium)porphine complexes of Mn, Co, Fe, Ni, Cu, and Zn and found the first three to exhibit peroxidatic activity. The best, the Mn(II1) complex, provided 84% of the activity of HRP. Encouraged by the above reporta, we examined several Mn-porphine complexes as substitutes for HRP and also studied some naturally occurring Fe-porphine complexes that are inexpensive produds from bovine blood. To our surprise, hc matin ([7,12-diethenyl-3,8,13,17-tetramethy1-21H,23Hporphine-2,18-dipropanoato(4-)-N21,N22,N23,N24] hydroxyferrate(2-) dihydrogen; also called hydroxyhemin or ferriheme hydroxide) exhibited very significant peroxidatic activity under optimum conditions. The activity per unit weight exceeded that of any commercially available HRP preparation. Along with a study of the relative merits of various phenolic substances as substrates, the exploitation of hematin for the fluorometric determination of Hz02is reported in the present paper.
EXPERIMENTAL SECTION Reagents. Mn(II1)-rneso-tetraphenylporphine acetate was obtained from Strem Chemicals (Newburyport,MA). Hematin, hematoporphyrinIX, and three other porphines without a metal center were obtained from Aldrich. Mn complexes of the latter three porphines were synthesized by gently refluxing equimolar quantities of MnC12 and the porphine in aqueous solution for several days according to literature procedures.26vn Hematin was also obtained from a second vendor (ICN Biochemicals, Costa Mesa, CA); hemin, bovine, and equine hemoglobin were obtained from Sigma Chemical. Of the principal substrates, HPA (Kodak) was purified by recrystallizing twice,28and p-cresol (Pract., Kodak) was used both without purification and after purification by distillation under reduced preesure. Phenol (Baker), o-cresol (Fisher), rn-cresol (Pract., Kodak), and all other substrates (Aldrich) were obtained as indicated and used without further purification. Hydrogen peroxide (3%, Mallinckrodt analytical reagent) stock solutions were standardized by titration with secondary standard KMn04. Organic peroxides, including methyl hydroperoxide (MHP), hydroxymethyl hydroperoxide (HMHP), and 1hydroxyethyl hydroperoxide (HE") were a gift from researchers at the National Center for Atmospheric Research. The total peroxide content of these samples was determined by iodometric titration.29 Hematin stock solutions were prepared by dissolving 10 mg of hematin in 100 mL of 0.1 M NaOH. This solution is stable for at least 1 month refrigerated; it should be discarded if a precipitate appears. The working solution is prepared by diluting 6.3 mL of stock to 100 mL with an ammonia buffer (5.35 g NH4C1, 60 mL of concentrated ",OH, to 1 L with deionized water); it is nominally 10 gM in hematin. 0 1992 American Chemical Society
518
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992
Table I. Effectiveness of Different Catalysts Studied concentration used, pM
catalyst Mn(III)-tetrakis(4-methoxyphenyl)-21H,23H-porphine(I) Mn(II1)-tetrakis(l-methylpyridyl)-21H,23H-porphine Mn(III)-tetraphenyl-21H,23H-porphine Mn(II1)-rneso-tetraphenylporphine acetate hematoporphyrin IX
hematin peroxidase "Nonfluorescent. -0.4 pM.
nm
L.ax!
405
~emmmax,
relative response
20 4
320 325
405 398,446
7.8 8.4
10 16
nfl"
nfl 410 425 410 410
nfl
40 10
16 units/mLb
320 322 325 325
6.0
2.9 97.9 100.0
P
F
Flgurr 1. Schematlc for Row-inJectlon analysis system. P, pump; W, water carrier; A, phenolic fluorogenic substrate solution; H, hematin sdutlon; C, MnO, cdumn; V, loop Injector; S, sample solution (Iced at submlcromdar ccncenbbatkns); M1, M2, knotted mixing Cays; T, porous tube debubbler; F, flow-through fluorometer.
Equipment. Absorption and fluorescence spectra were obtained on Hewlett-Packard 8451A and Shimadzu RF540 instruments, respectively. A Gilson Minipuls 2 multichannel peristaltic pump, an electromechanicallyactuated &portrotary valve (HVXL 6-6, Hamilton Co., Reno, NV) equipped with a 100- X 1.0-mm and a Nter fluorometer injection loop (calibrated to deliver 75 (Fluoromonitor 111, 50 rL flow cell, Laboratory Data Control, Riviera Beach, FL; equipped with a Cd-lamp and a 326 nm bandpass excitation filter and a 400 nm (50% cutoff) long pass emission fiiter) were used for flow injection analysis (FIA) experiments. FIA System. The arrangement is schematically shown in Figure 1. Pump P pumps carrier water W through a polytetrafhoroethylene (pTFE) column C (30- x 1-mmbed of granular Mn02 (Mallinckrodt), glass wool plugs at each end, to remove residual H202)through valve V. Valve V injects sample S in an automated periodic manner. Past V, the substrate flow A (3 mM HPA or p-cresol) and the ammonia-buffered hematin reagent H are sequentially merged with knotted mixing coil M1 in between. After a knotted reaction coil M2 and a small hydrophobic porous tube debubbler T (10 X 1.0 mm, Gore-Tex TA001, W. L. Gore and Associitea, Elkton, MD),the stream flows through fluorometer F. All conduits were PTFE. Other Procedures. The effectiveness of different catalysts was determined in the batch mode using 1mL of 100 r M Hz02 as sample, 2 mL of H20, 1mL of 3 mM HPA, and 1mL of catalyst (4-40 pM Mn-porphine or hematin/hematoporphyrin IX, in pH 10.5 ammoniacal solution, see Table I). The maximum fluorescenceobserved is reported. The fluorescence intensitywith different substrates (concentration of 1 mg/mL) was measured similarly with each substrate at a concentration of 1mg/mL using hematin as catalyst. Essentially the same relative order of fluorescence intensities was observed with HRP as catalyst; the results are not therefore separately reported. To study Michaelis-Menten behavior, to 4 mL of a solution containing 250 mg/L p-cresol and varying concentrations (7.5-20 pM) of HzOz, 1 mL of hematin (1pM) or HRP (16 purpurogallin units/mL) was added. The solution was continuously pumped through the fluorometer,and the initial rate of fluorescence development was measured. The reaction medium was the pH 10.2 ammonia buffer for hematin, and a pH 5.8 phosphate buffer for HRP with an in-line membrane reactor before the detector for the introduction of ammonia to raise pH."JB RESULTS AND DISCUSSION Effectiveness of HRP Substitutes. Mn(II1) complexes of tetrakis(4-methoxy phenyl)-21H,23H-porphine(I),tetra-
a)
Reaction Time (min)
Figure 2. Kinetic profile for the rate of flwescence devebpment wlth the we of hematin and HRP (2 pM and 3.2 purpurogallin unltslml, respecthrely) as catalysts wlth p-cresol as the substrate and 1 pM H,O,
as the analyte.
kis( l-methyl-4-pyridyl)-21H,23H-porphine (11), tetraphenyl21H,23H-porphine (1111, rneso-tetraphenylporphineacetate (IV), hematoporphyrin IX, and hematin showed different degrees of peroxidatic activity. The fluorescence characteristics of the oxidized HPA product (L 32Ck325 nm, ELgmplIu 400-425 nm) were very similar to those of the corresponding product obtained with HRP as catalyst. An additional weaker emission band at 446 nm was also observed with 11. The products obtained with hematin and HRP as catalysts were spectrally indistinguishable. The fluorescence intensities observed with the six compounds above and HRP (16 purpurogallin units/mL) are presented in Table I. It is intereating that the data clearly show the effectiveness of hematin while Hematoporphyrin IX, the prosthetic group of HRP,SOshows poor peroxidatic activity. Michaelis-Menten Behavior. The kinetic data obtained with hematin (200 nM in final solution) and HRP (36 mg/L in final solution, -80 nM based on an MW of 40000 and an estimated purity of 10% for this preparation) were fitted to the Michaelis-Menten kinetic model31using a nonlinear least squares fitting routine that uses a Marquardt-Levenberg algorithm (MINSQ,Micromath Scientific Software, Salt Lake City, UT). The K, values obtained were 4.5 X and 1.55 X M for HRP and hematin, respectively, indicating comparable binding of H202to both catalysts. The V, valuea (in arbitrary fluorescence units s-l) were 2.59 and 3.75, respectively, for hematin and HRP. Noting the difference in concentrations of the two catalysts used, k, ratio of HRP to hematin is 1.53:l. If the catalytic effectiveness of hematin (FW 633.5) is expressed in terms of unit weight rather than on a molar basis, it has >40 times the activity of the HRP preparation used. As the concentrations we have typically used these catalysts (hematin 10 rM, HRP 16 purpurogallin units/mL in the reagent; both diluted -5 times in the final mixture), kinetic performance is comparable (Figure 2) but the cost differential represents a factor >500 at current retail prices.32 It is also useful to note that despite the apparent conformity of both sets of data to Michaelis-Menten behavior,
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992 Concentration (mM)
p-Cresol oo i
510
01
1
10
$ 1
.,1
00000 5 LIM H2O2 50
7.0
90
11.0
Final Measurement pH 40
90
140
Hematin Concentration (uM) Figure 3. The dependence of the analytical response on the catalyst concentration at two dlfferent concentrations of H,O, and on the
concentratlon of the fluorogenlc substrate.
Figure 2 clearly shows that the kinetic profiles of the two catalytic systems have distinct differences. The initial rate with HRP is higher while attainment of the final value is faster with hematin. Peroxidatic and Catalatic Activities of Hematin. It is well-known that most iron porphyrin and phthalocyanine compounds exhibit some catalatic and peroxidatic activities; hematin-H2Oz adducts have been reported.3O At high HzOz concentrations, simultaneousoxidation has been reported to occur at two opposite methene bridge carbons, resulting in a dipyrrolic compound.33 Reported catalytic activity of hematin is small; catalase is 10' times more effective than hem a t h H Peroxidatic activity has been reported to vary from one specific hematin-substrate combination to another and is highly dependent on pH. Depending on the specific conditions, the observed activity may vary by as much as a factor of 5500.36 With a phenolic substrate like pyrogallol (HRP activity is frequently specified in terms of the rate of formation of purpurogallin from pyrogallol), HRP was reported to be >6 X 105 times more active in terms of unit iron content than hematin.% The same study found hematin 1order of magnitude poorer in peroxidatic activity than hemoglobin. Not surprisingly,more recent benchmark accounts of the chemistry of porphyrins and metalloporphyrins33and the functional relationship between HRP and other heme proteins3' do not even mention peroxidatic activities of hematin and related compounds. Part of this apparent contradiction can be understood from the importance of nitrogenous ligands like ammonia used in the present work in promoting peroxidatic activity of hematin (vide infra). Anyhow, there is little doubt that bovine hematin (obtained from two independent vendors) can be effectively substituted for HRP under the stated experimental conditions. A search of the Chemical Abstracts database in April 1991 indicated that this has not been previously reported. Hemin, which is expected to be rapidly converted to hematin in alkaline solution, displayed essentially the same peroxidatic activity as hematin. The response elicited by injected H20zin our test system using equine and bovine hemoglobin as catalpta (using the same concentration by weight as hematin) were comparable to each other and were less than a third of that obtained with hematin. It is important to note here the fact that the luminescence from the luminol-H202reaction is catalyzed equally well by HRP,hemoglobin, or hemin- is not an indication that hemin or hematin has peroxidatic activity. This CL reaction is catalyzed by a great variety of metal c o m p l e ~ e s . ~ ~ ~ ~ Optimization of Reaction Conditions. The results of independentlyvarying substrate and catalyst concentrations
Flgure 4. The dependence of the analytical response on the final pH used for fluorescence measurement: the trajectories are shown for
several different reaction pH levels.
at low levels of H20zare summarized in Figure 3. Based on these data, 10 pM hematin and 3 mM substrate concentrations were chosen. The use of HPA showed a substrate concentration dependence very similar to that for p-cresol. The optimum pH for using the hematin-HPA system for H2O2determination differs from that of the HRP-HPA system. The common element in both systems is that the oxidized dimer of HPA is fully ionized by pH and therefore the final measurement pH must be 110 for best results. The optimum conditions for the actual oxidation process in the HRP-HPA system is a pH of 5.8; typically the reaction is carried out around this pH and base is then added to render the product f l u o ~ ~ ~ The &.~ hematin-HPA system behaves differently, as shown in Figure 4. The reaction itaelf occurs best at a pH of 10-10.5; the yield decreases to a small extent at still higher pH. The optimum pH for the product fluorescence is not affected by the reaction pH. Results for the hematin-p-cresol system are similar for the optimum reaction pH but the (minimum) optimum measurement pH is lower by 0.5 pH unit. This is expected in that unlike in HPA, a second acidic proton is not present in p-cresol. Note that the data in Figure 4 were obtained in phosphate buffered media with the mixture being allowed to react for 16 h before the pH was adjusted and fluorescence measured. With a short reaction time (as in a practical continuous flow analysis system),the differences between different reaction pH levels are substantially greater. Stimulation of Hematin Peroxidatic Activity by Ammonia. Peroxidatic activity of hematin is dependent not only on the pH but also on the specific buffer used. Although it is not widely recognized, this behavior is also common to HRP.41 Early work of F r i d ~ v i c hshowed ~~ that nitrogenous ligands can greatly extend the optimum range of pH over which peroxidatic activity for HRP can be maintained. For example, the optimum pH for catalysis by HRP with o-dianisidine as the substrate is 5.8. With a pH 9.8 pyrophosphate buffer, the activity decreases to 150 times enhanced. Similarly, imidazole buffers can extend the optimum pH for HRP activity to past 8. The extension of the optimum pH range depends on the pK of the nitrogen base and its concentration in the free-base form. Fridovich postulated that ligation of the nitrogen base with the heme-iron in the enzyme-substrate intermediate adduct is involved. Accessibility of the lone pair of electrons on the nitrogen does seem important-Guilbault et al? reported only marginal differences between phosphate and tris(hydroxymethy1)aminomethane buffers, both at pH 8.5. The differences in the peroxidatic activity of hematin with ammonia, 3-(cyclohexylamino)-l-propanesulfonicacid (CAPS), and
520
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992 80.00
1
Solid lines: 1 U M Hematin Dashed lines: 10 V M H e m a t i n
-a-
-L-F
O.OO0.00
"
' '1O.bb' ' '
"
-
'
' ' 20.00 ' " ' ' ' ' ' ' 30.00 "
Reaction Time (mi.) Flgure 5. The dependence of reaction kinetics on the buffer system used @H 10.3 f 0.1). Results for two dlfferent catalyst concentrations
are shown. phosphate buffer (all at a pH of 10.2-10.4)are shown in Figure 5 for two different hematin concentrations. The differences are obviously substantial, especially at low catalyst concentrations. Choice of a Substrate. For the determination of H202 using HRP, two important studies are available on the relative merits of different fluorogenic substrates. The early work of Guilbault et al.' established four useful substrates: homovanillic acid, HPA, tyrosine and tyramine; with relative product fluorescence intensities of 0.391.00:0.241.19in the order cited. More recently, &tau and OhkwaG have reported the rate and the extent of fluorescence development for 25 fluorogenic substrates. The top seven performers were 4hydroxyphenethyl alcohol, tyramine, N,N-dimethyltyramine, 3-(4-hydroxyphenyl)-l-propanol,4-n-propylphenol, 4-ethylphenol, and 3-(Chydroxyphenyl)propionic acid (HPP). The maximum fluorescence intensities observed in the above seven cases (with HPA being assigned the reference value of unity) are approximately 1.45:1.44:1.43:1.35:1.24:1.22:1.20as estimated from literature graphical data.43 The authors recommended the use of HPP and have since published a FIA procedure for H202using an HRP-HPP system.44 It is appropriate to investigate the relative merits of different substrates with hematin as catalyst since this may not be identical to that with HRP. Moreover, previous studies7@ used a pH 8.5 Tris-buffer; at this pH it is possible to be unduly influenced by the extent of ionization of the oxidation product, rather than the intrinsic utility of the substrate. The results (using 5 pM H202as the analyte) are shown in Table 11. Sensitivity (calibration slope) is not the only determinant of the LOD;the blank fluorescence is a major factor. Although all substrates in Table I1 have low blank fluorescence in pure form, the commercially available products often contain fluorescent impurities that must be removed to obtain the best performance. The procedure for purification can be involved and thus be a deterrent. Cost of the substrate, listed in Table 11, is also a factor; otherwise, (4-hydroxyphenyl)-l-propanol would be the reagent of choice. Based on the above considerations and the performance data shown, we believe that p-cresol is an optimum choice. Even with practical-grade material, excellent LODs can be attained (vide infra). The cost of purified material (which has significantly lower blank fluorescence) is only marginally higher; it is also readily purified in quantity by distillation under reduced pressure. In neutral solution it is indefinitely stable at room temperature in closed bottles; in our experience this is not the case for HPA unless metal chelating agents are added.
Table 11. Sensitivity with Different Substrates substrate o-cresol (99+ % ) trans-4hydroxyL-proline (99+ % ) m-cresol (99%) phenol (99+ % 1 3-hydroxyphenethyl alcohol 4-ethylphenol (99%) tyrosine 4-hydroxyphenethyl alcohol p-cresol (pract.) tyramine hordenine 4-hydroxyphenylacetic acid (98%) 344hydroxyphenyl)propionic acid (98%) (4-hydroxyphenyl)-1propanol
L , m m
nm
Xem,-,
nm
relative responsea blank
per lOOg,$
costb
320
410
5.5
1.6
10.30
300
410
5.8
0.3
89.40
320
410
8.2
0.4
14.30
318
404
10.5
1.5
9.10
320
397
12.8
0.4
108.8Oe
326
412
86.8
4.8
8.50
324 324
412 412
95. 95.4
12.4 24.7
16.40 93.50"
326
415
95.
77.4
5.90
324 324 325
412 412 410
97.8 99.4 100.0
1.8 4.6 3.7
152.80 65.8OC 60.35
325
412
107.3
7.7
109.80
324
410
119.4
1.2
72.20e
aFor 20 rM Hz02 bBased on Aldrich or Sigma Chemical Catalog, 1991. cPer 10 g. dPer 25 g.
System Performance. The calibration data for 0.1-100 pM H202(13concentrations, 25 point at each level) are well described by the equation signal (arb units) = 85.06 f 0.71(C,pM) + 13.65 f 26.99 (r2 = 0.9996) (1) The slope of a log-log plot of response vs concentration for these data is 1.024 f 0.005, indicating acceptable linearity for most applications. Linearity is excellent within smaller bounds (P = 1.OOO for 0.1-1 p M 0.9999for 1-10 pM, and r2 = 0.9989 for 10-100 pM). Similar response behavior is exhibited by the hematin-HPA system, for 0.1-10 pM Hz02we observed signal (arb units) = 72.17 f 0.70(C,pM) + 10.36 f 3.30 (r2 = 0.9997) (2) Typical system output is shown in Figure 6 with practical-grade p-cresol as the substrate. Based on the SIN values observed for the 100 nM HzO2sample (due both to omnipresent residual Hz02in water and instability in dilute solutions, standards cannot be prepared with high accuracy below this level), the LOD is
