Bally and Gribnau: Aspects of 3,3',5,5'-tetramethylbenzidine in a horseradish peroxidase assay 791

J. Clin. Chem. Clin. Biochem.Vol. 27, 1989, pp. 791-796© 1989 Walter de Gruyter & Co.

Berlin · New York

Some Aspects of the Chromogen 33^5,5'-Tetramethylbenzidineäs Hydrogen Donor in a Horseradish Peroxidase Assay

By R. W. Bally and T. C. J. Gribnau

Scientific Development Group Organon Int. U.V. Oss, The Netherlands

(Received October 28, 1988/June 21, 1989)

Summary: Enzyme immunoassays frequently incorporate the use of horseradish perosidase äs the enzymelabel. This enzyme usually catalyses the oxidation of a ehromogen which can be quantified after terminationof the enzyme reaction. A chromogen widely used for this purpose is S^'^S'-tetramethylbenzidine. The twoelectron oxidation of tetramethylbenzidine yields a component with an absorbance maximum at 450 nm. Ifthe enzyme reaction is terminated by lowering of the pH (< 1.0), an additional increase of the absorbance at450 nm is observed.

It is shown that this additional increase is partly due to a l .4-fold increase in the molar lineic absorbance ofoxidized tetramethylbenzidine, caused by the acidic pH, äs well äs a quantitative shift of the existing equilibriumbetween tetramethylbenzidine, oxidized tetramethylbenzidine and their charge-transfer complex. The totalabsorbance increase upon acidification of the reaction mixture depends therefore on the reaction conditionsäs well äs the reaction coordinate.

Introduction

Horseradish peroxidase1) is frequently used äs a labelin enzyme immunoassays. It catalyses the oxidationof a redox indicator by H2O2. A redox indicatorwidely used in this reaction is 3,3^5,5'-tetramethyl-benzidine, a chromogen that is easily quantified bycolorimetry (1).

The reaction invqlves transfer of two electrons (fig.1). In a first step, a cation radical reaction interme-diate is fonned (B) which is the result of one electrontransfer (2).In a second step, äs a result of a second electron.transfer, the cation radical is further oxidized to formthe yellow coloured diimine derivative (D) of tetra-methylbenzidine. Two of the intermediate cation rad-icals may however combine to form a blue colouredcharge-transfer complex (C), which may also be con-sidered to consist of a molecule tetramethylbenzidine

Enzyme:Horseradish peroxidase, Donor: hydrogen-peroxide oxido-reductase, EC 1.11.1.7

(A) and a molecule of its diimine derivative (D); anequilibrium state, that depends on the reaction con-ditions, exists between these various molecules.

The amount of oxidation product(s) fonned, is gen-erally quantified by spectrophotometry. Prior to pho-tometric detection, the turnover of tetramethylbenzi-dine by horseradish peroxidase/H2O2 is commonlyterminated by lowering the pH of the reaction mix-ture, preferably with a strong acid like H2SO4, to avalue below unity. An additional effect of acidifica-tion, after correction for dilution, is a substantialabsorbance increase at 450 nm, the absorbance max-imum of the diimine. This absorbance increase maybe attributed to a number of possible effects, such äs:— a pH effect on the molar lineic absorbance of the

diimine;— a shift in the equilibrium between the charge-

transfer complex and its constituents;— auto-oxidation of the charge-transfer complex un-

der acidic conditions;— a combination of the above.

J. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No. 10

792 Bally and Gribnau: Aspects of 3,3',5,5'-tetramethylbenzidine in a horseradish peroxidase assay

H3C

NH2

Fig. 1. Two electron oxidation of S^'jS^'-tetramethylbenzidine tetramethylbenzidine;(A) - tetramethylbenzidine ( ̂ = 210 and 285 at pH > 3.0);(B) — cation-radical;(C) - charge-transfer complex ( ^ = 380, 650 at pH > = 1.0);(D) — oxidized tetramethylbenzidine (diimine derivative, Xmax == 450).

The mechanism of this acid-induced absorbance in-crease was investigated in a series of experiments, theresults of which are presented in this paper.

Materials and MethodsAll reagents used were of analytical grade. Water used was ofMillipore Super-Q quality. Tetramethylbenzidine (free base) wasobtained from Aldrich; tetramethylbenzidine · 2HC1 fromFmka; horseradish peroxidase (freeze dried powder, RZ = 3.0)from Boehringer; and H2O2 (30%) from Baker.Stock Solutions of the free base of tetramethylbenzidine wereprepared in dimethylsulphoxide. Tetramethylbenzidine · 2HC1,horseradish peroxidase and peroxide Solutions were preparedin aqueous buffer. Incubation mixtures containing the free baseof tetramethylbenzidine were prepared in acetate/citrate buffer(0.1 mol/1 sodium acetate adjusted with citric acid monohydrateto pH 5.5), whereas the tetramethylbenzidine · 2HCl-containingmixtures were prepared in acetate buffer (0.01 mol/1 acetate

buffer, pH 4.0). All experiments were performed at ambienttemperature. Specific reaction conditions are presented in thelegends to the figures. All reactions were terminated by theaddition of l mol/1 H2SO4 in a volume ratio of l: 1.UV/VIS spectra were recorded using a Philips model PÜ8700spectrophotometer. The optical pathlength used was 0.01 meter,unless stated otherwise. Blanks were recorded using incubationmixtures without tetramethylbenzidine.

Results and Discussion

Effect of pH on the UV/VIS spectrum of areaction ihixtureLöwering the pH of a reaction mixture, in which partof the initial ämount of tetramethylbenzidine has beenoxidized, to a value below uriity, has a dramatic effecton the ÜV/VIS spectrum, äs previously shown by

3. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No. 10

Bally and Gribnau: Aspects of 3,3',5,5/-tetramethylbenzidine in a horseradish peroxidase assay 793

Gallati & Pracht (3). This is illustrated by figure 2 inwhich the pH effect is shown for a reaction mixtureallowed to attain maximum absorbance at 650 nm,prior to acidification. This corresponds to 50% turn-over of the initial amount of tetramethylbenzidine. Itis observed that the absorbance band centred around450 nm is increased, whereas any absorbance attrib-uted to the charge-transfer complex (Xmax = 380 and650 nm) becomes negligible.

Effect of pH on the molar lineic absorbanceof the diimine at 450 nm

Reactions were carried out with an excess of horse-radish peroxidase (l mg/1 reaction mixture containingeither 50 or 12.5 μιηοΐ/ΐ tetramethylbenzidine s wells H2O2), ensuring a rapid and s complete s possible

turnover of tetramethylbenzidine into its diimineform. The increase in absorbance at 450 nm wasmeasured with time until a plateau was observed.Spectra recorded between 200 and 700 nm, just before(pH = 5.5) and after tenninatiqn of the reaction (pH= 1.0) are given in figure 3. The spectrum given forpH =1.0 is corrected for the dilution caused byaddition of H2SO4.From the curve at pH = 5.5 it is concluded that theamount of charge-transfer complex in the reactionmixture is indeed virtually zero, s there is no signif-icant absorbance at 650 nm, an absorbance maximumof the charge-transfer complex. Any changes in thespectrum due to changes in the pH of the solutionare therefore to be attributed to the diimine. Ob-viously, the molar lineic absorbance of the diimine ispH-dependent; under both reaction conditions, it isincreased s much s 1.4-fold in the pH r nge 5.5 to1.0. Since there is hardly any charge-transfer complexleft in these mixt res, indicating quantitative oxida-tion of the tetramethylbeiizidiiie present, it is con-cluded from these experknents that a 1.4-fold molarlineic absorbance increase at 450 nm is caused bylowering the pH from 5.5 to unity. It must be notedthat this conclusion does not rely on any knowledgeof molar lineic absorbances, or even on the concen^tratiori of tetramethylbenzidine and its oxidationproducts. It does rely on the correctness of the reac-tion mechanism given by Josephy et al. (2).

Effect of pH on the UV/VIS spectrum of te-tramethylbenzidineA decreased pH also has a marked effect on theabsorbance band of tetramethylbenzidine at 285 nm(fig. 4). Acidification (pH < 3.0) causes the absorb-ance maximum to shift from 285 to 252 nm. The

Fig. 2. UV-VIS spectrum of a mixture of tetramethylbenzidineand its oxidation products at pH = 5.5 (—) and pH= 1.0 (—, corrected for dilution). Conditions: 50%tetramethylbenzidine turnover, 12.5 μτηοΐ/ΐ tetramethyl-benzidine and H2O2, 10 μ§/1 horseradish peroxidase inacetate/citrate buffer.

200 300 400 500X[nm]

600 700

Fig. 3. Effect of pH on the UV-VIS spectrum of the diiminederivative of tetramethylbenzidine; spectra were re-corded at pH = 5.5 (—) and pH =1.0 (—, correctedfor dilution). Conditions: 12.5 μιηοΐ/ΐ tetramethylben-zidine and H2O2, l mg/1 horseradish peroxidase in ace-tate/citrate buffer.

llo

J

8ja

200 300 400 500Xrnm)

600 700

Fig. 4. Effect of pH on the UV-VIS spectrum of tetramethyl-benzidine at pH = 4.0 (—) and pH = 1.0 (—, correctedfor dilution). Conditions: 400 μιηοΐ/ΐ tetramethylbenzi-dine in acetate buffer, optical pathlength 1.0 mm.

J. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No. 10

794 Bally and Gribnau: Aspects of 3,3',5,5'-tetramethylbenzidine in a horseradish peroxidase assay

molar lineic absorbance at 285 nm becomes approx.10 times smaller, whereas the molar lineic absorbancesof the maxima are approximately equal.

The reaction mechanismFrom the comparison of figures 2 and 3, it is con-cluded that the absorbance increase at 450 nm uponacidification is not only dependent on the increase inthe molar lineic absorbance. Acidification causes themolar lineic absorbance to increase by a factor 1.4(fig. 3), whereas the total increase observed in figure2 amounts to a factor 3.2. The total increase thereforeclearly depends also on the reaction coordinate, beingthe ratio of oxidized tetramethylbenzidine over theinitial tetramethylbenzidine concentration.

Füll explanation of the observed absorbance increaserequires a knowledge of the concentrations of all ofthe components involved prior to acidification, andtherefore their molar lineic absorbances.

Using the reaction mechanism proposed by Josephyet al. (2), it is easily verified that the totalized amountof tetramethylbenzidine, i. e. the sum of tetramethyl-benzidine (TMB) itself and its oxidation products, atany time during the reaction is given by:

[TMBJ = [TMB] + 2 · [CHA] + [Ox] (Eq 1)

and therefore

[TMBJ =B285

+ 2£650 6450

(Eq.2)

in which

[TMBJ: the totalized concentrations of tetramethyl-benzidine and its derivatives participating inthe reaction;

[TMB]: the tetramethylbenzidine concentration attime t;

[CHA]: the concentration of Charge-transfer complexat time t;

[Ox]: the diimine concentration at time t;

A: absorbance measurement at the indicatedwavelength;

: molar lineic absorbance at the indicatedwavelength.

Equation 2 basically contains two unknowns, i. e. themolar lineic absorbances at 450 and 650 nm; the molarlineic absorbance of tetramethylbenzidine at 285 nmmay be calculated from an absorbance measurementat the initial (known) concentration.

For calculation of the molar lineic absorbances, itwould be sufficient to measure the absorbances at twodifferent reaction coordinates and the same totalizedamount of tetramethylbenzidine. For the sake of ac-curacy, howeyer, it was decided to measure the ab-sorbances during tetramethylbenzidine turnover byhorseradish peroxidase/H2O2 of a particular mixture,i. e. äs a fünction of time. The molar lineic absorbanceat 450 and 650 um were subsequently calculated bymeans of a least-squares fit to the model given byequation 2.Such an approach is valid only if the absorbances atthe various wavelengths can be uniquely assigned toone coinponent, äs is the case for the band maximaat 285 (tetramethylbenzidine), 450 (diimine) and 650nm (charge transfer complex) but not for the absorb-ance band maximum at 380 nm (charge transfer com-plex). This latter band overlaps both of the adjacentbänds (c. f. figs. 2, 3 and 4).

Determination of the molar lineic absorbances enablesthe calculation of the various concentrations that arepresent prior to acidification. These data are required,in order to Interpret the inechanism(s) leading to theincrease at 450 nm on acidification.

This procedure was carried out using the acetate-buffered chromogen/sübstrate solution with 5 g(miXi) or 2 g (mix2) horseradish peroxidase per litrereaction mixture. The absorbance measurements werecorrected for time differences originating from asyii-chronous measurement at the various wavelengths.The averaged molar lineic absorbances at pH = 4".0were finally found to be:

8450 = 7200 m2/mol for the diimine;£550 = 4000 m2/mol for the charge-transfer complex;

= 2006 m2/mol for tetramethylbenzidine.

The validity of the model was checked by calculatingthe totalized concentration of tetramethylbenzidineand derivatives, which should be the same for evefymeasurement (see figs. 5a and 5b, Eq. 1). In thesefigures, the lines correspönding to the totalizedamount show a slight downward trend, indicating atime effect on the measurements that is not accountedfor by the model. However, the totalized amount oftetramethylbenzidine is considered to be sufficientlyconstant for our purposes. The molar lineic absorb-atices calculated for tetramethylbenzidine and thecharge-transfer complex (2100 and 3900 m2/möl, re-spectively) are in close agreement with those reportedby Josephy et al. (2). The different valües for thediimine (7200 and 5900) must be attribtited to differ-ences in pH (4.0 and 5.0, respectively).

J. CHn. Chem. Clin. Biochem. / Vol. 27,1989 / Nö. 10

Bally and Gribnau: Aspects of 3,3',5,5'-tetramethylbenzidine in a horseradish peroxidase assay 795

50η

25-

;*=: οΙ °Sε50! *>

500 1000 1500t[e]

25-

500 1000t [s]

1500 2000

Fig. 5. Concentration (calculated) of tetramethylbenzidine (+),charge-transfer complex (·), diimine (4-) and the to-talized amount of tetramethylbenzidine (A) s functionof time; initial tetramethylbenzidine concentration 45μηιοΐ/ΐ in acetate buffer pH = 4.0.5.a: mixture 1; horseradish peroxidase 5 μg/l reaetionmixture.5.b: mixture 2; horseradish peroxidase 2 μg/l reactionmixture.

The absorbances t 450 nm prior to and after acidi-fleation are given in table 1. The cprresponding (cal-culated) concentrations prior to acidification are givenin table 2.The following obserVations can be made from thedata presented in these tables.

— Prior to acidification^ the absorbance ratio of mix-ture l to mixture 2 (1.54) equals the ratio of thecalculated diimine (Ox) concentrations (1.54) inboth mixtures· this is to be expected if the molarlineic absorbances used are correct.

— The absorbance ratio of mixture l prior to andafter acidification (1.33) is different from the cor-responding ratio of mixture 2 (1.78). This is(again) an indication of the fact that the absorb-ance increase is not solely dependent on the pHeffect on the molar lineic absorbance, but also onthe reaction coordinate and therefore the initialamount of tetramethylbenzidine;

Tab. 1. Absorbance (ratios) at 450 nm prior to and after aci-dification for the mixtures containing 5 (mixi) and 2(mix2) μg horseradish peroxidase per litre reaction mix-ture.

Absorbance No acid Acida) Acid/no acid

miximix2mixi/mix2

2.001.301.54

2.662.321.15

1.331.78

a) corrected for dilution.

Tab. 2. Vari us (calculated/totalized) concentrations (μιηοΐ/ΐ)of tetramethylbenzidine and oxidation products in re-action mixtures l and 2 just prior to termination of thereaction.

Concen-tration

mixjmix2mixi/mix2

Tetra-methyl-benzidine

4.35.6

Chargetransfercomplex

6.511.0

Diimine

27.918.11.54

Diimine+ Chargetransfercomplex

34.329.1

1.18

— After acidification, the absorbance ratio of mix-ture l over mixture 2 (1.15) is in close agreementwith the ratio calculated for the totalized amountof diimine plus charge-transfer complex (Ox+ CHA = 1.18).

These fmdings and the fact that the absorbance dueto charge-transfer complex completely disappearsupon acidification, lead to the conclusion that themost probable explanation for the observed absorb-ance increase is the complete dissociation of thecharge-transfer complex, combined with a simulta-neous increase in the molar lineic absorbance of thediimine, caused by the change of pH.The molar lineic absorbance of the diimine after aci-dification (pH < 1.0) was found to be approx. 7900m2/mol which is 1.3 times larger than the value re-ported by Josephy et al. at pH = 5.0. The molar lineicabsorbance increases by about 1.1 when the pH isdecreased from 4.0 to (less than) unity.

Conclusions

Acidification, for the purpose of terminating the hor-seradish peroxidase-catalysed oxidation of tetrame-thylbenzidine, s is frequently used in EIA's, has alarge impact on:— the absorbance maximum of tetramethylbenzi-

dine;

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796 Bally and Gribnau: Aspects of 3,3',5,5'-tetramethylbenzidine in a horseradish peroxidase assay

— the equilibrium between the charge-transfer com-plex and tetramethylbenzidine/diimme, which isquantitatively shifted towards the tetramethylben-zidine/diimine side;

decreased from 5.5, 4.0 ör 5.0 to (less than) unity,fespectively.

Depending on the reaction conditipns äs well äs the— the molar lineic absorbance of the diimine, which reaction coordinate, a combination of the latter two

is very pH-sensitive; it changes by a factor of 1.4 effects causes a substantiäl incfease in the absorbanceor 1.1 (this study) or 1.3 (2) when the pH is at450nm. ; f

References1. Bös, E., Doelen, A. van der, Rooy, A. van & Schuurs, A.

(1981) J. Immunoassay 2, 187-204.2. Josephy, R, Eling, Th. & Mason, R. (1982) J. Bioi. Chem.

257, 3669-3675.

3. Gallati, H. & Pracht, I. (1985) J. Clin. Chem. Clin. Biochem.23,453-460.

Dr. R. W. BallyOrganen Int. B. V. DRL-D/RE2201Postbus 20NL-5340BH OssDr. T. C. J. GribnauOrganon Int. B. V. DRL-D/RE 2140Postbus 20NL-5340BH Oss

J. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No, 10