Free Radical Biology & Medicine, Vol. 15, pp. 447-451, 1993 0891-5849/93 $6.00 + .00 Printed in the USA. All rights reserved. Copyright © 1993 Pergamon Press Ltd.

Review Article

L U M I N O L A N D L U C I G E N I N AS D E T E C T O R S F O R 02"-

KEVIN FAULKNER and IRWIN FRIDOVICH

Department of Biochemistry, Duke University Medical Center, Durham, NC, USA

(Received 6 April 1993; Accepted 3 May 1993)

Abst rac t - -Univa lent oxidation of luminol and univalent reduction of lucigenin must precede reaction with 02"-- if that reaction is to lead to luminescence. The assumption that luminol or lucigenin, per se, reacts with O2"- in a way leading to luminescence is incorrect, and leads to misinterpretation of results. The chemical reactions leading to the 02"--dependent luminescences of luminol and of lucigenin are discussed.

Keywords--Luminol , O2"---dependent luminescence of, Lucigenin, O2"---dependent luminescence of, Luminescence, O2"---dependent, Superoxide--dependent luminescence, Superoxide dismutase--inhibitable luminescence, Free radicals

INTRODUCYION

The luminescences of luminol and of lucigenin have frequently been used for the detection of 02"- in bio- logical systemsJ -~3 It is generally assumed that these compounds react with 02"- in a way that leads to re- lease of photons and, therefore, that this photon re- lease is diagnostic of the presence of 02"-. This is not quite correct.

Thus, it is not luminol, but rather its univalently oxidized form, that reacts with O2"-, and it is not luci- genin, but rather its univalently reduced form, which does so. In both cases, light production depends on the formation of an unstable endoperoxide or dioxe- tane, which decomposes to an electronically excited product, which releases a photon as it falls to the ground state. Since failure to appreciate the underly- ing chemistry can lead to misinterpretation ofexperi-

Address correspondence to: Irwin Fridovich, Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

Keven Michael Faulkner majored in chemistry at the Univer- sity of North Carolina-Wilmington and then earned a Ph.D. in chemistry at Duke University where he studied cooperativity in hemoglobins. He is now a postdoctoral fellow in the Department of Biochemistry at Duke University Medical Center where he is study- ing low molecular weight mimics of superoxide dismutase activity.

Irwin Fridovich majored in chemistry at the City College of New York and then earned a Ph.D. in biochemistry at Duke Univer- sity Medical Center. He is currently a James B. Duke Professor at Duke. His research has focussed upon the biology of oxygen radi- cals.

mental results, a brief review seems both timely and useful.

Luminol

Ultrasonication was seen to elicit the luminescence ofluminol over 50 years ago and some activated form 0fO2 was implicated 14'15 That a free radical ofluminol was an intermediate in the pathway leading to lumi- nescence was deduced soon thereafter. ~6,.7 In alkaline DMSO luminol autoxidizes, with intense lumines- cence, producing N2 and aminophthallate as the sole products. The aminophthallate is produced in an electronically excited state and it is the light-emitting species. ~ s, ~ 9

In buffered aqueous solutions the luminescence of luminol can be elicited by a variety of oxidants, pro- vided that 02 is also available. Thus, oxidation of lu- minol with iodine, 2° or with H202 plus a peroxidase, 21 or at the anode of an electrochemical cell, 22 all re- sulted in an oxygen-dependent luminescence.

Xanthine oxidase, while catalyzing the oxidation of its substrates, releases both O2"- and H 2 0 2 (Ref. 23), and the first report of enzyme-induced chemilu- minescence involved the use ofxanthine oxidase. 24 A role for O2"- in the xanthine oxidase-induced lumines- cence of luminol was revealed by the inhibitory effect of superoxide dismutase (SOD). 25 Indeed, SOD strongly inhibited the chemiluminescence ofluminol, whether that chemiluminescence was elicited by xan-

447

448 K. FAULKNER and I. FRIDOVICH

a)

b)

c)

d)

NH 2 O NH2 OH

Nit N

0 OH

NH2 O

OH OH

NH2 O- ]4 NH 2 O"

~ N oxidation ~ !

OH OH

+ H +

NH 2 O NH 2 O

N ~ t ~ ~ NH +

OH O

e)

f)

g)

! +02- --

OH OH

. ° O

N

OH

COOH

+ N 2

1 + hv

COOH COOH

Fig. 1. Reac t ions invo lved in the un iva l en t ox ida t ion o f l u m i n o l .

thine oxidase plus xanthine 25 or by oxidants such as ferricyanide, H202, persulfate, or hypochlorite. 26,27 The explanation for these results is that univalent oxi- dation of luminol yields a luminol radical that can both reduce 02 to 02"- and can react with O2"-, yield- ing an unstable endoperoxide, whose decomposition leads to the electronically excited aminophthallate. The reactions involved in this process are shown in Fig. 1. In this scheme reaction (a) represents a keto-

enol tautomerism and reaction (b) shows a simple ionization. The luminol monoanion can be univa- lently oxidized by a variety of oxidants and this oxida- tion is shown in reaction (c). The luminol radical gen- erated by reaction (c) can reduce 02 to 02"- as in reac- tion (d), and it can also add 02"- yielding the endoperoxide shown in reaction (e). This endoperox- ide is unstable and splits out N2 while going over to the electronically excited aminophthallate as in reac-

Luminol, lucigenin, and 02"-- 449

tion (f). Luminescence occurs when the aminophthal- late drops to the ground state, as in reaction (g).

Luminol is not a reliable detector of O2"- since, in the presence of any univalent oxidant, it can act as a source of O2"-. In the case of xanthine oxidase plus xanthine, the univalent oxidant is HO" or Fe(II)-O, generated by the iron-catalyzed interaction of 02"- with H20 2. In the case of activated neutrophils, the univalent oxidant is myeloperoxidase plus H20 2 .28 In these cases, either catalase or SOD inhibits the lu- minol luminescence.

The requirement for both O2"- and the luminol radical was also clearly demonstrated by Merenyi and Lind. 29 They further noted that decomposition of the endoperoxide adduct was rate-limiting in the lumines- cence pathway and was strongly pH dependent. Thus, at pH 7.7 the rate constant for this decomposition was seen to be 2 × l 0 3 S - l , while at pH 11.0 it was 2 × 105 s -1. Moreover, the difference between pH 7.7 and 11.0 was greater than this 100-fold difference in rate constants would lead one to expect. This is the case because the alkaline decomposition provides a high quantum yield of luminescence whereas the neutral decomposition does not.

Luminol is, thus, not well suited to the detection of O2"- within living cells for at least two reasons. One of these is its ability to generate 02"-, when its univa- lently oxidized form autoxidizes, and the second is its poor quantum yield in the neutral pH range. At- tempts have, nevertheless, been made to use luminol for this purpose with bacteria anaerobically grown and then exposed to air, 3° and with submitochondrial particles. 31 Derivatized isoluminols have also been applied to whole cells. 32'33

Lucigenin

The chemiluminescence of lucigenin, like that of luminol, involves reaction of 02"- with a radical form of the luminescent compound. However, there is a crucial difference in that the reactive radical form of luminol must be produced by univalent oxidation; while in the case of lucigenin, the radical form is gen- erated by univalent reduction. The need for reduction of lucigenin on the reaction pathway leading to its luminescence was recognized over 50 years ago by Gleu and Petsch. 34 They studied lucigenin lumines- cence elicited by H20 2 and presented evidence that HzO2 acted both as a reductant and as an oxidant in this process.

We studied the luminescence of lucigenin caused by the xanthine oxidase reaction and found that re- duction of lucigenin, by xanthine oxidase, preceded its reaction with 02"-. 35 Electrochemical studies by

Legg and Hercules 36 also showed that 02"- was a neces- sary reactant and that the divalently reduced luci- genin (the bis acridene) was not capable of reacting with 02"-. Since reduction of lucigenin was essen- tial, 35 and the divalently reduced lucigenin was inac- tive, the univalently reduced lucigenin must be on the reaction pathway leading to luminescence. 37 The scheme in Fig. 2 accounts for the behavior of luci- genin.

Reaction (I) is the univalent reduction of the bisa- cridynium to the corresponding radical; which then reacts with O2"- in reaction (II) to yield the dioxetane; which decomposes into two molecules of the acri- done, as in reaction III, one of which is electronically excited and emits a photon as in reaction IV.

The luminescence of lucigenin has been used re- peatedly as a measure of O2"- production in biological systems. That there is some validity to this method can be deduced from the work of Peters et al. 5 who noted that suspensions of E. coli elicited this lumines- cence in a dioxygen-dependent way. Moreover, para- quat exerted an enhancing effect that was seen with live but not with heat-killed cells. This luminescence appeared to reflect intracellular events, since exoge- nous SOD failed to inhibit. It should be noted in this regard that the lucigenin luminescence elicited in free solution by the xanthine oxidase reaction is power- fully inhibited by SOD, but not by catalase. The en- zyme which can cause the univalent reduction ofluci- genin within E. coli remains unidentified. Also un- known is the extent to which the univalent reduction oflucigenin limits its chemiluminescence in these and other cells.

Is 02"- sufficient as well as necessary for lucigenin chemiluminescence?

The chemistry discussed above would dictate a neg- ative answer to this question, unless O2"- can serve as a univalent reductant for lucigenin. Early work indi- cates that it cannot do so. Thus, the intensity of luci- genin luminescence in the xanthine oxidase reaction was examined as a function ofpO235 and an optimum pO2 was observed, above and below which lumines- cence decreased. 02"- production by xanthine oxidase increases monotonically with pO2 in the range of 0-100% 02 in the equilibrating gas phase (23).

We can now understand this optimum pO2 on the basis of the need for two reductions by xanthine oxidase. One of these is the reduction of 02 to O2"-, and the other is the univalent reduction of lucigenin. The partition of electron outflow from xanthine oxi- dase between these two reductions will depend upon the relative concentrations of the two electron accep-

450 K. FAULKNER and I. FRIDOVlCH

CH 3

CH 3

+e-

CH3

CH3

ll)

CH 3

CH 3

+ 0 2-

CH 3

"O

CH 3

CH3 CH 3 I

O III) "O

[ ~H3 1 CH3[

iv) ] .

Fig. 2. Scheme representing the behavior of lucigenin as a result of the xanthine oxidase reaction.

tors. At low PO2 lucigen reduction will predominate but there will be little reduction of 02. Hence, at low POz there will not be enough 02"- to react with the univalently reduced lucigenin and it will dismute to the inactive divalently reduced form. At high pOz, on the other hand, there will be ample O2"- but not much univalently reduced lucigenin for it to react with. It follows that 02"- cannot reduce lucigenin and, there- fore, that O2"- is necessary but is not sufficient for lucigenin luminescence.

Acknowledgements-- This work was supported by grants from the Council for Tobacco Research, USA, Inc.; the Johnson and John- son Focused Giving Program; and Eukarion, Inc.

REFERENCES

1. Minkenberg, I.; Ferber, E. Lucigenin-dependent chemilumines- cence as a new assay for NAD(P)H-oxidase activity in particu- late fractions of human polymorphonuclear leukocytes. J. Im- munoL Methods 71:61-67; 1984.

2. Muller-Peddinghaus, R. In vitro determination of phagocyte

Luminol, lucigenin, and O2"- 451

activity by luminol and lucigenin amplified chemilumines- cence. Int. J. lmmunopharmacol. 6:455-466; 1984.

3. GyUenhammer, H. Lucigenin chemiluminescence in the as- sessment of neutrophil superoxide production. J. Immunol. Methods 97:209-213; 1987

4. Storch, J.; Ferber, E. Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase. Anal. Biochem. 169:262-267; 1988.

5. Peters, T. R.; Tosk, J. M.; Goulbourne, E. A., Jr. Lucigenin chemiluminescence as a probe for measuring reactive oxygen species production in Escherichia coli. Anal. Biochem. 186:316-319; 1990.

6. Okuda, M.; Lee, H.-C.; Chance, B.; Kumar, C. Glutathione and ischemia-reperfusion injury in the perfused liver. Free Ra- dic. Biol. Med. 12:271-279; 1992.

7. Tosi, M. F.; Hamedani, A. A rapid specific assay for superoxide release from phagocytes in small volumes of whole blood. Am. J. Clin. Pathol. 97:566-573; 1992.

8. Johnston, R. B.; Lehmeyer, J. E.; Guthrie, L. A. Generation of superoxide anion and chemiluminescence by human mono- cytes during phagocytosis and on contact with surface-bound immunoglobulin G. J. Exp. Med. 143:1551-1556; 1976.

9. Allred, C. D.; Margetts, J.; Hill, H. R. Luminol-induced neutro- phil chemiluminescence. Biochem. Biophys. Acta 631:380- 385; 1980.

10. Vilim, V.; Wilhelm, J. What do we measure by a luminol-de- pendent chemiluminescence of phagocytes. Free Radio. Biol. Med. 6:623-629; 1989.

11. Wang, J. F.; Komarov, P.; Sies, H.; de Groot, H. Contribution of nitric oxide synthase to luminol-dependent chemilumines- cence generated by phorbol-ester-activated Kupffer cells. Bio- chem..L 279:311-314; 1991.

12. Okuda, M.; Ikai, 1.; Chance, B.; Kumar, C. Superoxide dismu- tase, but not catalase or mannitol, decreased oxygen radical formation in perfused rat heart during ischemia-reperfusion as monitored by luminol-enhanced chemiluminescence. ,L Appl. Cardiol. 6:113-121; 1991.

13. Radi, R.; Cosgrove, T. P.; Beckman, J. S.; Freeman, B. A. Per- oxynitrite-induced luminol luminescence. Biochem. .L 290:52-57; 1993.

14. Flosdorf, E. W.; Chambers, L. A.; Malisoff, W. M. Sonic acti- vation in chemical systems: Oxidations at audible frequencies. J. Am. Chem. Soc. 58:1069-1076; 1936.

15. Harvey, E. N. Sonoluminescence and sonic chemilumines- cence. ,L Am. Chem. Soc. 61:2392-2398; 1939.

16. Stross, F. H.; Branch, G. E. K. The chemiluminescence of 3- aminophthalhydrazide. J. Org. Chem. 3:385-404; 1938.

17. Weiss, J. Oxidation and chemiluminescence. Trans. Faraday Soc. 35:219-224; 1939.

18. White, E. H.; Zafiriou, O. C.; Kagi, H. M.; Hill, J. H. M. Chemi- luminescence ofluminol: The chemical reaction. J. Am. Chem. Soc. 86:940-941 ; 1964.

19. White, E. H.; Bursey, M. M. Chemiluminescence of luminol and related hydrazides: The light emission step. J. Am. Chem. Soc. 86:941-942; 1964.

20. Seitz, W. R.; Hercules, D. M. A quantitative study of chemilu- minescence from the iodine-luminol reaction. J. Am. Chem. Soc. 96:4094-4098; 1974.

21. Pilchard, P. M.; Cormier, M. J. Studies of the mechanism of the horseradish peroxidase catalyzed luminescent peroxidation of luminol. Biochem. Biophys. Res. Commun. 31:131-136; 1968.

22. Epstein, B.; Kuwana, T. Electrochemical generation of solu- tion luminescence-If. Luminol and pthalhydrazide. Photo- chem Photobiol. 4:1157-1173; 1965.

23. Fildovich, I. Quantitative aspects of the production ofsuperox- ide anion radical by milk xanthine oxidase. J. Biol. Chem. 245:4053-4057; 1970.

24. Totter, J. R.; Medina, V. J.; Scoseria, J. L. Luminescence dur- ing the oxidation of hypoxanthine by xanthine oxidase in the presence of dimethylbisacridylium nitrate. J. Biol. Chem. 235:238-241; 1960.

25. Henry, J. P.; Michelson, A. M. Studies in bioluminescence. VIII. Chemically induced luminescence ofPholas dactylus lu- ciferin. Biochimie 55:75-81 ; 1973.

26. Hodgson, E. K.; Fridovich, I. The role of O2 in the chemilumi- nescence of luminol. Photochem. Photobiol. 18:451-455; 1973.

27. Miller, E. K.; Fridovich, I. A demonstration that 02- is a crucial intermediate in the high quantum yield luminescence of lu- minol. J. Free Radic. Biol. Med. 2:107-110; 1986.

28. Allred, C. D.; Margetts, J.; Hill, H. R. Luminol-induced neutro- phil chemiluminescence. Biochim. Biophys. Acta 631:380- 385; 1980.

29. Merenyi, G.; Lind, J. S. Role of a peroxide intermediate in the chemiluminescence of luminol. A mechanistic study. J. Am. Chem. Soc. 102:5830-5835; 1980.

30. Allen, R. C. Chemiluminescence from eukaryotic and prokary- otic cells: Reducing potential and oxygen requirements. Photo- chem. Photobiol. 30:157-163; 1979.

31. Cadenas, E.; Boveris, A.; Chance, B. Low level chemilumines- cence of bovine heart submitochondrial particles. Biochem. J. 186:659-667; 1980.

32. Lippman, R. D. The prolongation of life: A comparison of antioxidants and geroprotectors vs. superoxide in human mito- chondria. J. Gerontol. 36:550-557; 1981.

33. Lippman, R. D.; Agren, A.; Uhlen, M. A new method which investigates lysosomal sensitivity to superoxide with endocy- totic, site-specific, chemiluminescent probes; and Application ofchemiluminescent probes in investigatinglysosomal sensitiv- ity to superoxide vs. suspected radical scavengers. Mech. Aging Dev. 17:275-281 and 283-287; 1981.

34. Gleu, K; Petsch, W. Die chemiluminescenz der dimethyldi- acridyliumsalze. Angew. Chem. 48:57-59; 1935.

35. Greenlee, L.; Fridovich, 1.; Handler, P. Chemiluminescence induced by the operation of iron flavoproteins. Biochemistry 1:779-783; 1962.

36. Legg, K. D.; Hercules, D. M. Electrochemically generated che- miluminescence of lucigenin. J. Am. Chem. Soc. 91:1902- 1907; 1969.

37. Allen, R. C. Phagocytic leukocyte oxygenation activities and chemiluminescence: A kinetic approach to analysis. Methods Enzymol. 133:449-493; 1986.