Biochem. J. (1967) 102, 853

Possible Reactions of 1,2-Naphthaquinone in the Eye

By JANCIS R. REES AND ANTOINETTE PIRIENuffield Laboratory of Ophthalmology, Univer8ity of Oxford

(Received 28 June 1966)

1. Reactions of 1,2-naphthaquinone with amino acids, glutathione and proteinsof the lens have been studied in connexion with investigations of naphthalene-induced cataract. 2. Cysteine reacts probably through its amino group with1,2-naphthaquinone to form either purple or brown compounds with characteristicabsorption spectra. 3. Glutathione reacts with 1,2-naphthaquinone through itsthiol group. 4. Spectroscopic evidence suggests that 1,2-naphthaquinone reactswith the amino group of amino acids. This reaction may take place in the aqueoushumour. 5. The proteins of lens react with 1,2-naphthaquinone to form browncoinpounds. 6. There is loss of protein thiol in this reaction and the products are

less easily digestible by pancreatin than normal lens proteins. 7. The compoundof a-crystallin and 1,2-naphthaquinone is soluble at neutrality, but the compoundsof fl-crystallins and of y-crystallins are largely insoluble. 8. The brown reactionproducts of glutathione or cysteine with 1,2-naphthaquinone catalyse theoxidation of ascorbic acid in the same way as 1,2-naphthaquinone itself. 9. Theseresults are discussed in relation to naphthalene-induced cataract.

When rabbits are dosed with naphthalene(1g./kg.) lens opacities develop and the retinadegenerates (Adams, 1930). van Heyningen &Pirie (1967) suggest that the damage to the eye isdue to enzymic formation of 1,2-dihydroxy-naphthalene in eye tissues and its autoxidation to1,2-naphthaquinone in the aqueous and vitreoushumours and possibly in the retina, lens and otherparts ofthe eye. The avascularity of the lens meansthat it is entirely dependent on the aqueous humourfor nourishment. The reactions of 1,2-naphtha-quinone with constituents of aqueous humour andlens have therefore been studied to see what effectnaphthalene feeding may have on the metabolismof the lens. This paper is concerned with thereaction between 1,2-naphthaquinone and aminoacids, GSH and proteins of the lens. The mostdetailed studies have been made with cysteine andGSH. Reactions of 1,2-naphthaquinone have notbeen widely investigated as it is the 1,4-naphtha-quinone derivatives that occur naturally.

EXPERIMENTAL

Methods

Reaction of cy8teine and OSH with 1,2-naphthaquinone.A solution (2.7-10mm) of 1,2-naphthaquinone in water wasprepared, and added to a solution (5-50mM) of cysteine orGSH in water, at the required pH. The solutions of cysteineand GSH had been adjusted to either pH7 or pH4 with0 1 N-KOH. The tubes were then left either at room tem-

perature or at 3°. Anaerobic incubations were carried outin Thunberg tubes. The aqueous solution of 1,2-naphtha-quinone was put in the tube itself, together with buffer,usually lOO,moles of potassium phosphate, pH7.3, or100/Lmoles of sodium acetate, pH4. The acid solution ofcysteine hydrochloride or GSH was placed in the stopper,and was mixed after evacuation on either a water pumpor an oil pump. The total volume of solution was 3ml.The tubes were left either at room temperature or at 3°.

Separation of cyeteine and GSH reaction products. Bothreaction mixtures were fractionated by paper electro-phoresis in buffer atpH4. The pyridine buffer ofGrassmann,Hannig & Plockl (1955) was used in the initial experiments,and O lM-acetate buffer, pH4, in the later experiments,since total N values were required. The electrophoresispapers were cut into horizontal strips, which were elutedwith water by continuous capillary flow. The GSH reactionmixture was also fractionated by descending paper chroma-tography in butan-l-ol-propan-l-ol-water (2:1:1, by vol.)(Booth, Boyland, Sato & Sims, 1960).Aqueous humour. This was taken with a syringe from

ox eyes within 2hr. of death and stored at 4°.Reaction of 1,2-naphthaquinone with aqueous humour. A

20mM solution of 1,2-naphthaquinone in 60% (v/v)ethanol was prepared and added to aqueous humour togive a final concentration of 2mM. In some experimentsaqueous humour was concentrated fourfold by evaporationat 250 and solid 1,2-naphthaquinone added to a finalconcentration of 20mM.

Preparation of lens extracts. These were prepared by themethod of van Heyningen & Pirie (1967).

ae-Cry8tallins. These were separated from extracts of oxlenses by repeated precipitation at pH5. The protein wasdissolved at neutrality and dialysed for 24hr. at 40 againstdistilled water at neutrality.

853

J. R. REES AND A. PIRIE

pl- and y-Crystallins of ox lens cortex. The soft corticalpart of ox lenses was dissected from the hard nucleus,ground and extracted in the usual way. a-Crystallins wereremoved by precipitation at pH5 and the supernatant wasbrought to neutrality and dialysed at 40 against distilledwater at neutrality.

y- and fl-Crystallins of calf lens nucleu8. When the intactcalf lens is cooled to 4° proteins of the nucleus are precipi-tated and this central part of the lens becomes opaque.Calf lenses were cooled in the refrigerator until a centralopacity was visible. This opaque area was then dissectedout on ice and treated exactly as the ox lens cortex.

Reaction of lens proteins with 1,2-naphthaquinone. A10mM solution of 1,2-naphthaquinone in 50% (v/v)ethanol was added with shaking to dialysed lens extractsat neutrality to give a 1,2-naphthaquinone/protein molarratio 5-10 assuming an average mol.wt. 20000 for lensproteins. The mixture was left at room temperature over-night. Samples were gently shaken with ethyl acetate toextract free 1,2-naphthaquinone before examination of theproteins.Enzymic hydrolysis of proteins. A 10mg. sample of

pancreatin (British Drug Houses Ltd.) was dissolved indistilled water and centrifuged to remove insoluble material.The supernatant was added to 500mg. of proteins in lOml.of water. The mixture was brought to pH8 and, after afew drops of CHCOs had been added, incubated at 300 for7-10 days. The pH was adjusted with dilute Na2CO3 andif digestion was slow a further 10mg. of pancreatin wasadded. Digestion was followed by testing for disappearanceof trichloroacetic acid-precipitable protein.

Acid hydrolysi8 of proteins. A 5mg. sample was hydro.lysed with 6w-HCI in a sealed tube for 16hr. at 1150(Waley, 1965).

Separation of amino acids. This was carried out by paperelectrophoresis in formic acid-acetic acid-water [125ml. of90% (v/v) formic acid and 375ml. of acetic acid in 2.51.]followed by chromatography in butan-l-ol-acetic acid-water (40:9:20, by vol.) (Waley, 1965).

Examination of chromatograms and electrophoresis papers.Amino acids were detected by the ninhydrin reaction(Wiggins & Williams, 1952; Bode, 1955). Phenols weredetected by spraying with diazotized sulphanilic acid(Smith, 1960). Quinones were detected by dipping thepapers in leucomethylene blue; a blue spot developingwithin the first 2min. was considered positive (Linn et al.1959). Bivalent sulphur was detected by dipping thepaper in acid K2Cr2O7, followed by an AgNOs spray(Knight & Young, 1958). Fluorescent and absorbing spotswere located with Hanovia ultraviolet lamps of emission254m,u and 360mtu.

Manometric estimations. The 02 uptake was measuredat 250 in conventional Warburg manometers, with KOH inthe centre wells. The total fluid volume was 3-Oml.

Estimations. Total N was determined as described byMarkham (1942). Amino groups in solution were deter-mined by the method of Waley & van Heyningen (1962).Thiol groups in protein, GSH and cysteine were estimatedby the method of Ellman (1959); GSH and cysteine werealso estimated by a modification of the method of Grunert& Phillips (1951), in which no acid was added to the samplebefore estimation.

Absorption spectra. These were recorded on an OpticaCF4R spectrophotometer.

MaterialsPancreatin was obtained from British Drug Houses Ltd.,

Poole, Dorset.Catalase was obtained from Boehringer Corporation

(London) Ltd., London, W. 5.1,2-Naphthaquinone was prepared by the method of

Fieser (1943).L-Cysteine hydrochloride (laboratory reagent) was

obtained from British Drug Houses Ltd.; solutions wereneutralized just before use.GSH was a gift from the Distillers Co., Speke, Liverpool.

RESULTS

Reaction of 1,2-naphthaquinone with cysteine

The reaction, as described in the Methodssection, was followed by colour change, by spectro-photometry and by manometric determination ofoxygen uptake. The reaction products wereexamined before and after electrophoresis.

Colour. The colour obtained by mixing 1,2-naphthaquinone and cysteine varied with theconditions. A purple colour was formed at pH4and a brown at pH 7. Aerobically, greatest depthof colour was produced with 1,2-naphthaquinoneand cysteine in the molar ratio 1: 4. Anaerobically,at pH4, a pinkish-brown blue-fluorescent inter-mediate was formed. This changed in an hour toa light-purple blue-fluorescent compound, whichwas stable until air was admitted, when it becamedeep purple in a few hours. Anaerobically at pH 7,a pinkish-brown colour developed that showedgreen fluorescence; this changed in 17 hr. to acolourless green-fluorescent solution. On openingthe tube, the solution became first light purple,then dark brown and lost its fluorescence.

It seemed possible that admission of air led tothe autoxidative formation of a quinone withrelease ofhydrogen peroxide. The effect of catalaseon the aerobic reaction was therefore investigated.We found that catalase intensified the purple colourat pH4 and prevented development of the browncolour at pH 7, the reaction mixture being finallydeep purple.

Separation of the brown and the purple products.Both the brown and the purple products could beseparated from either the brown (pH 7) or thepurple (pH4) reaction mixtures by electrophoresison paper at pH4, as described in the Methodssection. The same colour pattern was produced inboth cases, with a brown band near the base lineand a purple band nearer the anode, but electro-phoresis of the pH7 reaction mixture gave more-intense brown and less-intense purple bands. Thisconfirmed our supposition that two products areformed from cysteine and 1,2-naphthaquinone ateither pH, but that more of the purple product is

854

REACTIONS OF 1,2-NAPHTHAQUINONE IN THE EYE

formed at pH4 and more of the brown at pH7.Properties of the purple product. Spectroscopic

examination of the dark-purple reaction mixtureshowed a spectrum with absorption peaks at 500,299 and 240m,u, the relative heights of which werevariable, presumably depending on the contribu-tion of unchanged 1,2-naphthaquinone and itsproducts to the 240m,u peak. The eluate of thepurple band after electrophoresis had an absorptionspectrum showing peaks at 500, 299 and 240m,u,the relative heights of the two last being the same(1.0:0-8) in three different preparations (Fig. 1,curve 1). A second electrophoresis of the eluatedid not alter the spectrum. Reduction with sodiumborohydride bleached the purple colour and in6min. changed the spectrum to a single peak at272m,u (Fig. 1, curve 2). The purple eluate showedblue fluorescence under ultraviolet light and thisfluorescence increased several-fold on reductionwith sodium borohydride.On electrophoresis at pH4 the purple product

travelled further towards the anode than eithercysteine or 1,2-naphthaquinone, which suggeststhat the amino group has become bound. When itwas tested on paper after electrophoresis, it gavea positive reaction for bivalent sulphur, an orange-brown colour with diazotized sulphanilic acid(denoting phenolic groups) and a faint reaction forquinones. The ninhydrin reaction for aminogroups was negative with the neutral reagent ofWiggins & Williams (1952), but was positive withthe acid ninhydrin reagent of Bode (1955). Theninhydrin reaction seems unreliable since the resultdepends on the method used.

Properties of the brown product. Spectroscopicexamination of the brown reaction mixture showedpeaks at 237, 263 and 301m,u (Fig. 1, curve 3).The eluate of the major brown band resulting fromelectrophoresis of a purple reaction mixture had awell-defined absorption peak at 235m,u and less-well-defined ones at about 260 and 297m,u. Reduc-tion of this eluate with sodium borohydridebleached the colour almost totally. The spectrumof the reduced product showed absorption peaks at245 and 344mu (Fig. 1, curve 4).When tested on paper this brown band appeared

to give no reaction with diazotized sulphanilic acidfor phenolic groups. Positive reactions for bivalentsulphur and with ninhydrin were of no significancesince the band travels to the same position ascysteine. This shows that it is a less acidic com-pound than the purple reaction product.A brown reaction product with an absorption

band at 238mu was formed when equimolaramounts of 1,2-naphthaquinone and cystine weremixed at pH7. The absorption spectrum closelyresembled that of the products of reaction of otheramino acids such as glycine. There was no simi-

d 06

Wavelength (m,u)

Fig. 1. Absorption spectra of products of reaction ofcysteine and 1,2-naphthaquinone. 1 (-), Spectrum ofpurple product of reaction between 27mM-cysteine and21 mM-1,2-naphthaquinone at pH4 and 200. The reactionmixture was subjected to electrophoresis as described inthe Methods section; the purple band was eluted in waterand again subjected to electrophoresis and elution in waterbefore spectrophotometry. 2 (----), Spectrum of eluentof purple band (1) after reduction with NaBH4. 3 (. ),Spectrum of brown product of reaction between 23mM-cysteine and 23mM-1,2-naphthaquinone at pH4 and 200.The reaction mixture was subjected to electrophoresis asdescribed in the Methods section and the brown bandeluted in water. 4 (-.-.-), Spectrum of eluent of brownband (3) after reduction with NaBH4.

larity with the spectrum of either the brown or thepurple reaction products of cysteine.

Spectrophotometry of intermediate products formedanaerobically. Cysteine reduced 1,2-naphtha-quinone anaerobically at either pH4 or pH 7 togive a small amount of 1,2-dihydroxynaphthalene.This could be detected spectrophotometrically byextracting the solutions into chloroform, whichwas added through the side arms before admittingair, and then extracting the chloroform with 0 1 N-hydrochloric acid for measurement ofthe spectrum.This coincided with that of 1,2-dihydroxynaphtha-lene in 0.1 N-hydrochloric acid, giving peaks at 238,290 and 333m,u. Comparison of spectral intensitywith solutions of 1,2-dihydroxynaphthalene treatedin the same way showed that not more than one-thirtieth of the 1,2-naphthaquinone present canhave been reduced.The main product of anaerobic reaction was

colourless or pale purple at pH 7 and pH4 respec-tively. Both could be stabilized in 01 N-hydro-chloric teid and then gave a spectrum with peaksat 233, 299 and 333m,u. It thus seems that themain product of anaerobic reaction is colourless or

855;Vol. 102

J. R. REES AND A. PIRIE

pale purple, depending on pH. Reduction withborohydride did not change this spectrum or thatof 1,2-dihydroxynaphthalene. When air wasadmitted at pH4 the fluid became deep purple inthe next few hours and the spectrum changed tothat of the aerobic purple product. Similarly, thebrown product of aerobic reaction at pH 7 wasformed when air was admitted to thepH 7 anaerobictube, even in the presence of catalase.To see whether the initial product with spectral

peaks at 233, 299 and 333mu was convertible intoeither deep-purple or brown products in air depend-ing on pH, buffer of pH 7-3 was added by the sidearm to an anaerobic tube at pH 4 on admissionof air. A brown colour developed immediately.

When buffer ofpH 4 was added in the same way toan anaerobic tube at pH 7 on admission of air thefinal colour was purple-brown. In contrast, thefinal dark-purple and the brown products of theaerobic reactions do not seem to be interconvertible.

Table 1 summarizes the colour and spectralchanges that develop under different conditions.Change in thiol during reaction at pH 4-5 and at

pH 7-3. Change in thiol groups was measured(Table 2) by a modification of the method ofGrunert & Phillips (1951), as described in theMethods section. The method of Ellman (1959),with 5,5'-dithiobis-(2-nitrobenzoic acid), could notbe used in the presence of 1,2-naphthaquinone asthis reoxidized the thiol formed. We found that at

Table 1. Colour and 8pectra of product8 of reaction of cy8teine and 1,2-naphthaquinonemixed in the molar ratio 2: 1

pH Time4 3hr.4 2min.4 lhr.7 3hr.7 3hr.7 Immediate7 17hr.

ColourPurplePink-brownLight purpleBrownPurplePink-brownColourless

SpectrumAm... (mP)

240,299, 500

233, 299, 333*237, 263, 301240, 299, 500

ReducedspectrumAma. (m,L)

272

245, 344

233, 299, 333* Unchanged* In 0 1N-HCl.

Table 2. Change in thiol during reaction of cy8teine with 1,2-naphthaquinone

Tubes were set up containing cysteine and 1,2-naphthaquinone at 30 and the given pH in the molar ratio 2: 1except where indicated. Buffer (33,umoles of phosphate/ml. at pH7-3 or 17,moles of phosphate/ml. at pH6-5)was added to the appropriate tubes. 1,2-Naphthaquinone was added last to the tubes containing catalase. Theanaerobic incubations were carried out in Thunberg tubes, the contents being mixed after evacuation. Thiol wasestimated by the modified method of Grunert & Phillips (1951). Results are expressed as decrease in ,umoles ofthiol/jumole of 1,2-naphthaquinone present, after allowance has been made for the decrease in the control tubes,containing cysteine only.

1 Aerobic

2 Aerobic

3 Aerobic

4 Anaerobic5 Anaerobic6 Aerobic

Aerobic+ catalase

TimepH (min.)4-5 160

2777-3 12

55133

7-3 165165

7-3 1307-3 1706-5 165

3306-5 165

330

Decrease in thiol(,umoles/,umole of

1,2-naphthaquinone)0-070-140-090-460-760-461-320-290-180-690*850*090-09

Concn. of 1,2-naphthaquinone

(mM)0-56

1-67

1-670-83*1-671-671-67

1-67

Colour ofmain productPurple

Brown

Brown

Pale brownPale brownBrown

Purple

* Cysteine/1,2-naphthaquinone ratio 4: 1.

ConditionsAirAnaerobicAnaerobicAirAir+ catalaseAnaerobicAnaerobic

FluorescenceBlueBlueBlueNoneBlueGreenGreen

Expt.no. Conditions

856 1967

REACTIONS OF 1,2-NAPHTHAQUINONE IN THE EYETable 3. Oxygen uptake of 1,2-naphthaquinone in the pre8ence of cysteine

Each manometer flask contained lOO1moles of potassium phosphate buffer, pH7.3, or 300umoles of acetatebuffer, pH4*0. Cysteine (10mM) to give the desired concentration was placed in the main flask and 0-5ml. of5mM-1,2-naphthaquinone was placed in the side arm and tipped after equilibration. The total volume was3*Oml. Incubation was at 250. Catalase (IO,ul.) was added to the main flask as indicated. The 02 uptake ofcysteine controls has been deducted.

pH7.37.37-34*04-0

Cysteine/1,2-naphthaquinone

molar ratio2:14:18:14:14:1

pH4-5 aerobically at 30 or 200 there was a smalldecrease in thiol, equivalent to 0'2-0.3,umole/,tmole of 1,2-naphthaquinone present. The decreasein thiol at pH7*3 aerobically during formation ofthe brown product was considerably greater thanat pH4-5 (Table 2, Expts. 1, 2 and 3), both at 30and at 20°. The reaction was easier to follow atthe lower temperature because of the rapid autoxi-dation of cysteine alone at 200.The loss of thiol that took place in air at neutral

pH did not take place under anaerobic conditionsand was also strongly inhibited by the addition ofcatalase (Table 2, Expts. 4, 5 and 6). It seemsclear that loss of thiol is dependent on the presenceof hydrogen peroxide, formed during autoxidationof the primary product of the reaction.

Oxygen uptake of cy8teine and 1,2-naphthaquinone.Cysteine and 1,2-naphthaquinone showed a smalland rapid uptake of oxygen at pH 7. The amountof oxygen absorbed was only slightly greater thanthat equivalent to the quinone present even whenthe cysteine/quinone ratio was 8: 1 (Table 3).Cysteine is not oxidized to completion. Catalasediminished the oxygen uptake, confirming thathydrogen peroxide is formed in the reaction. AtpH 4 the uptake of oxygen was much slower thanat neutral pH, but in 2 5hr. a total of 1 ,0u.mole ofoxygen was absorbed/,umole of 1,2-naphthaquinonewhen the cysteine/quinone ratio was 4: 1. Catalaseagain halved the oxygen uptake, showing that atpH4, as at pH 7, hydrogen peroxide is beingformed. The pH 4 reaction mixture at the end waspurple in the presence of catalase and purple-brown in its absence.

Reaction of 1,2-naphthaquinone with GSH. Thereaction, as described in the Methods section, wasfollowed by colour change, by spectroscopy andby manometry. The reaction products wereexamined before and after electrophoresis andchromatography.

Colour. Mixing GSH and 1,2-naphthaquinone in

02 uptake (moles/mole of1,2-naphthaquinone)

No catalase Catalase added0*61*2

1.0, 1-70*41-1

0 6

0 3-

0-30.9

0-20-4

230 260 290 320 350 380

Wavelength (m,t)

Fig. 2. Absorption spectra of product of reaction of GSHand 1,2-naphthaquinone. 1 (- ), Spectrum of reactionmixture of 0-16mM-GSH and 0 04mM-1,2-naphthaquinoneat pH7 and 200. 2 (----), Spectrum of reaction mixture(1) after reduction with NaBH4.

air produced a brown colour when the initial pHwas between 6 and 8; under more acid or more

alkaline conditions there was no obvious colourchange. The deepest brown colour was obtainedat an initial pH of 7. Addition of alkali to thebrown solution turned it green, a change that couldbe reversed with acid.

Spectro8copy. The brown colour was accom-

panied by a major absorption peak at 254m,u anda minor one at 327m,u (Fig. 2). The appearance ofthe 327m,u peak was used to follow the progress ofthe reaction. Maximum height of this peak was

obtained when GSH and 1,2-naphthaquinone were

mixed in equimolar amounts.If the reaction was carried out at 30 in air the

solution became at first colourless, showed bluefluorescence under ultraviolet light and hadabsorption peaks at 236, 305 and 339m,u, which issimilar to, but not identical with, the spectra of

Time(min.)

10153060150

t,2

ss\

Vol. 102 857

J. R. REES AND A. PIRIE1,2-dihydroxynaphthalene and the products formedanaerobically from cysteine and 1,2-naphtha-quinone (see Table 1). After 18hr. at 30, thesolution was yellow, not fluorescent, and hadabsorption peaks at 254 and 327m,u. If the colour-less blue-fluorescent solution was warmed to roomtemperature, this transition took place in a fewhours and was accompanied by darkening of thesolution to brown.When the reaction was carried out anaerobically

in a Thunberg tube, the solution again becamecolourless and blue-fluorescent, and had peaks at236, 305 and 339m,u, corresponding to the firstreaction product in air at 3°. This condition couldbe stabilized by adding acid through the side arm,but otherwise, after opening the tube, the solutionlost fluorescence and became brown, and theabsorption peaks changed to 254 and 327m,.Reduction of the final brown solution with

sodium borohydride bleached it. The reducedproduct had absorption peaks at 244, 314 and360m,u (Fig. 2). Ascorbic acid reduced the brownsolution to a pale-pink colour, removed by boro-hydride.

Eletrophores8i and chromatography. The brownreaction mixture could be kept for several days atroom temperature, or at least 1 month at - 200,with very little change in the absorption spectrum,but the spectrum was not wholly stable to electro-phoresis or chromatography.During paper electrophoresis (see the Methods

section) the brown compound travelled betweenthe 1,2-naphthaquinone, which was nearest thebase line, and GSH and GSSG, which were nearestthe anode, supporting the theory that the aminogroup of GSH remains free during the reaction.The eluate of the brown band had an indistinctabsorption spectrum. Somewhat better resultswere obtained with chromatography in butan-l-ol-propan-l-ol-water (2: 1: 1, by vol.) and elution withthe same solvent without drying the paper, but theabsorption spectrum was still not so well-definedas that of the original reaction mixture.Examination of electrophores8i papers. The brown

band gave a positive reaction with the potassiumdichromate-silver nitrate stains for bivalent sul-phur, and stained lightly with leucomethyleneblue, indicating a quinone. Ninhydrin reactionswere again unreliable, the results depending on themethod used. The diazotization reaction wasnegative.The eluate of the brown band contained all the

nitrogen recovered from the paper, except for thatin the ninhydrin-staining streaks of GSH andGSSG. Recovery from the brown band was 54%of the nitrogen applied when a reaction mixturecontaining GSH and 1,2-naphthaquinone in equalproportions was used, and 24% when the ratiowas 3:1.

Thiol estimation. Thiol groups were estimatedby a modification of the method of Grunert &Phillips (1951) at different times after mixing GSH

Table 4. Change in thiol during reaction of GSH with 1,2-naphthaquinone

Tubes were set up containing GSH and 1,2-naphthaquinone under the given conditions. Phosphate buffer,pH7-3 (33,umoles/ml.), was added in Expts. 2, 3, 4, 6 and 7, and phosphate buffer, pH6*5 (17,umoles/ml.), inExpts. 1 and 5. 1,2-Naphthaquinone was added last to the tubes containing catalase. The anaerobic incubationswere carried out in Thunberg tubes, the contents being mixed after evacuation on either a water or an oil pump.Thiolwasestimatedbythemodifiedmethodof Grunert&Phillips (1951), described in the Methods section. Resultsare expressed as decrease in thiol in ,umoles/,umole of 1,2-naphthaquinone present, after allowanoe has beenmade for the fall in the control tubes, containing GSH only.

Expt.no. Conditions

1 Air, 200Air, 200Air, 200Air, 200

2 Anaerobic, 2003 Air, 200

Air, 2004 Anaerobic, 2005 Air, 30

Air, 30Air, 30Air, 30

6 Air, 2007 Anaerobic, 200

Catalase

+

Time(min.)

1818

120120120518

1202020180180

1.5120

Decrease in thiol(,umoles/,umole of

1,2-naphthaquinone)0-761-112-01-71.5080-81-20-360-450-560*460*40.9

Conen. of 1,2-naphthaquinone

(mm)0-2250-2250-2250-2250-2250-2250-450-450-450-450-450-450-2250.9

GSH/1,2.naphthaquinone

molar ratio4:14:14:14:14:12:12:12:12:12:12:12:11:11:1

858 1967

REACTIONS OF 1,2-NAPHTHAQUINONE IN THE EYETable 5. Oxygen uptake of 1,2-naphthaquinone,

1,2-dihydroxynaphthalene and GSH

Each manometer flask contained lOO1,umoles ofpotassiumphosphate buffer, pH 7.3, and 10,umoles of GSH. Anaqueous solution of 1,2-naphthaquinone (5mM) was put inthe side arm to give the required final concentration ratioto GSH and tipped after equilibration. The total volumewas 3*Oml. The gas phase was air. Incubation was at 250.The 02 uptake by GSH controls has been deducted.

GSH/1,2-naphtha-quinone molar

ratio1:12:14:14:1

GSH/1,2-dihydroxy.naphthalenemolar ratio

5:15:15:1

02 uptake (moles/mole of 1,2-

naphthaquinone)0O5

0.6,0-750-80-8

02 uptake (moles/mole of 1,2-dihydroxy-

naphthalene)1.50.91.6

and 1,2-naphthaquinone (Table 4). There was a

rapid fall in the first 20min., then the rate of falldecreased and became the same as the rate in thecontrol tube of GSH alone after 3hr. The initialfast reaction involved loss of thiol in amountsapproaching that of 1,2-naphthaquinone present,took place at 30 and at 200 and was increased byabout 30% in the presence of catalase. Catalaseslightly decreased the total decrease in thiol in3hr. Anaerobiosis did not decrease the loss ofthiol, and it is difficult to account for the largelosses of thiol, equivalent to almost twice theamount of naphthaquinone present, that occurredin 2hr. even under anaerobic conditions.

Oxygen uptake of GSH and 1,2-naphthaquinone.GSH takes up oxygen in the presence of 1,2-naphthaquinone but is not oxidized completely toGSSG. The uptake is most rapid in the first 10-20min., corresponding to the time of most rapidloss of thiol. Table 5 shows that more oxygen istaken up as the amount of GSH relative to 1,2-naphthaquinone is increased, but the oxygenabsorbed is less than 1Omole/mole of 1,2-naphtha-quinone, even when the GSH/1,2-naphthaquinoneratio is 4: 1. If the reduced form, 1,2-dihydroxy-naphthalene, is mixed with GSH, an extra 0 9-

1-6moles of oxygen are absorbed/mole of 1,2-naphthaquinone at a GSH/1,2-dihydroxynaphtha-lene molar ratio 5: 1. GSH is not therefore beingoxidized to completion in either case. Catalaseabout halved the oxygen uptake, showing thathydrogen peroxide was formed in the reaction.

Reaction of 1,2-naphthaquinone with amino acidsMason & Peterson (1965) have described the

spectroscopic changes that take place whenenzymically generated o-benzoquinone reacts withamino acids. From the uniformity of the spectralchanges they conclude that amino acids reactonly through their amino groups or the iminogroup of proline and hydroxyproline.We have investigated spectroscopic changes

when 1,2-naphthaquinone is incubated with atenfold excess of different amino acids for 20hr. atpH7-3 and room temperature. A solution of 1,2-naphthaquinone was kept under the same condi-tions. All amino acid-quinone mixtures showed adecrease in the 250m,u peak of 1,2-naphthaquinonethat was greater than the decrease in the control.A peak at 234-238mpu developed with glycine,lysine, arginine, cystine and aspartate togetherwith a shoulder at 300m,u, and alanine and serineshowed a clearer peak at 300mu and little changeat 230-240m,u. The glycine mixture showed abroad absorption band at 475mp,, lysine at 540muand tryptophan at 500m,u.

Since the spectral changes of the different aminoacids were similar it seemed probable that they allreacted through their amino groups, as suggestedby Mason & Peterson (1965). The amino acidstested (apart from cystine and tryptophan) arethose that are relatively abundant in the aqueoushumour (Kinsey, 1965). The reaction between1,2-naphthaquinone and the ascorbic acid inaqueous humour (van Heyningen & Pirie, 1967)made it difficult to determine whether a reactionalso took place with the amino acids present.Mixtures of aqueous humour and 1,2-naphtha-quinone developed a broad spectral band at 480m,uand one at 238mp with a shoulder at 304m,u. Thisis similar to the spectra developed by mixtures ofamino acids and 1,2-naphthaquinone and is someevidence that the amino acids in aqueous humourreact in the same way.Attempts were made to estimate change in

amino groups after reaction with 1,2-naphtha-quinone, but the compounds appeared to be toounstable for this to be possible and the results wereerratic. Mason & Peterson (1965) found reactionproducts of o-benzoquinone and amnino acids to betoo unstable for isolation. Spectral change appearsthe only criterion of reaction at present.

Reaction between 1,2-naphthaquinone and lensproteins

Some properties of the compounds of 1,2-naphthaquinone and lens proteins have beendescribed by van Heyningen & Pirie (1967).Further experiments were directed to finding what

Time(min.)30301530

101560

859Vol. 102

J. R. REES AND A. PIRIEgroups in the proteins were reactive and whetherquinone compounds of a-, ,B- and y-crystallinscould be differentiated. When a dialysed extract ofox or rabbit lens is mixed with 1,2-naphthaquinone(see the Methods section) a brown colour develops.This cannot be separated from the protein bydialysis or by precipitation with ammnoniumsulphate and appears to be the colour of 1,2-naphthaquinone-proteins themselves. The brownsolution showed broad absorption maxima at320-330 and 450m,u. No such bands appear in asolution of 1,2-naphthaquinone kept under thesame conditions. Addition of borohydride bleachedthe brown 1,2-naphthaquinone-protein and re-moved both absorption bands (Figs. 3 and 4). Thereduced solution regained its brown colour onstanding in air. Similar absorption bands appearwhen lens proteins are mixed with 1,2-dihydroxy-naphthalene. There is a small immediate uptakeof oxygen when 1,2-naphthaquinone or 1,2-dihydroxynaphthalene is mixed with lens proteinsand, as in the reaction with GSH or cysteine, thisuptake is diminished in the presence of catalase.Under anaerobic conditions the yellow colour of1,2-naphthaquinone is immediately bleached byaddition of lens proteins and the blue fluorescence

of the solution is greatly intensified. When air isintroduced the solution at once becomes yellow andthen turns brown. The colour of the protein com-pounds changed to a greenish-brown in alkali andto a red at pH 1-2, with absorption maximum near500m,u.

It seemed most probable that the thiol groupsof the proteins would react with 1,2-naphtha-quinone, becoming oxidized or combining directlywith it. Tables 6 and 7 show that addition of1,2-naphthaquinone to lens proteins removes thethiol groups, the loss being related to the con-centration of 1,2-naphthaquinone used.Brown naphthaquinone-proteins were enzymic-

ally hydrolysed in an attempt to detect whichgroups in the protein were linked. Treatment oflens proteins with 1,2-naphthaquinone made themless readily hydrolysable by pancreatin (see the

0-41

0 21

I 0

0-8

0-6

0 4

0-2

Wavelength (m,)

Fig. 4. Absorption spectra of lens protein-1,2-naphtha-quinone compound, before and after reduction withNaBH4, over the wavelength range 550A400m,u. 1 ( ),Lens protein-1,2-naphthaquinone. 2 (----), Lens protein-1,2-naphthaquinone after reduction. 3 (. ), Differencespectrum. The solution of the reduced compound was putin the reference cuvette and the spectrum of the lensprotein-1,2-naphthaquinone was read against it.

320 340 360Wavelength (m,u)

Fig. 3. Absorption spectra of lens protein-1,2-r- r'. V- .1 rr--Jr---_- -J --_ Jr--r_

quinone compound, before and after reduction with NaBH4,over the wavelength range 400-300m,u. 1 ( ), Lensprotein-1,2-naphthaquinone. 2 (----), Lens protein-1,2-naphthaquinone after reduction. 3 (. ), Differencespectrum. The solution of the reduced compound was putin the reference cuvette and the spectrum of the lensprotein-1,2-naphthaquinone was read against it.

Table 6. Effect of exce88 of 1,2-nacphthaquinone onthiol of lens proteins

1,2-Naphthaquinone was added to dialysed extract oflens until the brown colour developed. The mixture was

I mmmmmmmleft at room temperature overnight and then dialysed at400o 4m' for several days to remove free 1,2-naphthaquinone.

Thiol was estimated by the method of Ellman (1959).

naahtha- Thiol content (/imoles/g. of protein)

LensCalfOx

Ox

Rabbit

Untreated50, 53575752

Treated with1,2-naphthaquinone

Nil13513

860 1967

0 d

REACTIONS OF 1,2-NAPHTHAQUINONE IN THE EYE

Table 7. Effect of increa8ing amounts of 1,2-naphtha-quinone on thiol of len8 protein8

Dialysed ox-lens extract (diluted 1:4) was treated with1,2-naphthaquinone as in Table 6. The mixtures wereextracted with ethyl acetate to remove free 1,2-naphtha-quinone before estimation of thiol.

Conen. of 1,2.naphthaquinone

(mm)0

1-44-25-67-0

Thiol content(,umoles/g. of

protein)7538226*6

Loss of thiol(%)

Nil50709292

Methods section), judged by the fact that no

precipitate of tyrosine and cystine appeared in thehydrolysate, whereas a heavy crystalline precipi-tate appeared in the control hydrolysate of normallens proteins. Less amino nitrogen, estimated bythe method of Waley & van Heyningen (1962), wasliberated by pancreatin from the quinone-treatedprotein than from the control, and hydrolysates ofthe 1,2-naphthaquinone-proteins always retainedsome protein precipitable by trichloroacetic acidthat was absent from the control hydrolysate.After electrophoresis at pH 1-6 followed by paper

chromatography as described in the Methodssection the pancreatin hydrolysates of lens proteinsshowed the usual range of amino acid spots whentreated with ninhydrin (Calam, 1962). Cystine andproline spots were absent from chromatograms ofpancreatin hydrolysates of 1,2-naphthaquinone-treated proteins. Both spots were, however,present if such proteins were hydrolysed by 6N-hydrochloric acid by the method of Waley (1965).This result could be due both to the less completeenzymic digestion of the quinone-treated proteinand to reaction of 1,2-naphthaquinone with thiolgroups. Neither enzymic hydrolysates nor the acidhydrolysates showed any new ninhydrin-positivespots, nor were new diazotizable compounds or

quinones present, as judged by tests on thechromatograms.Experiments were carried out to determine

whether the different lens proteins, a-, ,B- andy-crystallins, react differently with 1,2-naphtha-quinone. A precipitate often appeared in thereaction mixture, particularly if calf lenses were

used. The nuclear part of the calf lens contains a

high proportion of y-crystallin, whereas the corticalpart of the adult ox lens contains rather littley-crystallin and a high proportion of ,-crystallins(Kleifeld, Hockwin & Fuchs, 1956; Testa, Armand& Balazs, 1965). Comparison was therefore madebetween the effect of 1,2-naphthaquinone on

28

ac-crystallin, separated by precipitation at pH 5,and on the proteins of the ox lens cortex and thecalf lens nucleus after a-crystallins had beenremoved. These preparations are described in theMethods section.When 1,2-naphthaquinone was added to a final

concentration of 2mm to 1% solutions of theseparated and dialysed proteins at neutrality androom temperature all solutions turned brown. Aheavy precipitate appeared in the y-crystallin-richextract of the calf nucleus within 1 hr., whereasthe other two extracts remained clear. After 18hr.all protein had precipitated from the extract ofcalf nucleus, about half had precipitated from theextract of the ox lens cortex and the solution ofa-crystallin remained clear. The precipitatedproteins could be brought into solution in dilutealkali but were reprecipitated at neutrality. Thisdifference in precipitability by 1,2-naphthaquinonemay be related to difference in thiol content.cx-Crystallins contain less than either of the othertwo groups of lens proteins (Kinoshita & Merola,1958). Bjork (1961) found a particularly high thiolcontent in y-crystallin of calf lens.

Catalytic oxidation of ascorbic acid bycompounds of 1,2-naphthaquinone

van Heyningen & Pirie (1967) found that 1,2-naphthaquinone, like other o-quinones, wouldcatalytically oxidize large amounts of ascorbicacid, and they concluded that the decrease inascorbic acid in the eye fluids of the naphthalene-fed rabbit was probably due to this reaction.

Tests were made of the catalytic effect of thecompounds of 1,2-naphthaquinone with GSHIE andcysteine on the oxidation of ascorbic acid. Thecatalytic activity of 1,2-naphthaquinone can bedemonstrated at a concentration of 1 pg./ml. Itwas therefore necessary to remove free 1,2-naphtha-quinone from the reaction mixtures by extractionwith chloroform to guard against the possibilitythat any catalysis found was due to remainingtraces of 1,2-naphthaquinone. Control tubes of1,2-naphthaquinone alone were carried through inparallel. Table 8 shows that the neutral reactionmixtures of 1,2-naphthaquinone with GSH orcysteine catalysed oxidation of ascorbic acid afterchloroform extraction. Had all the original 1,2-naphthaquinone still been present it would haveamounted to 1.9-26,ug./ml. in the manometerflasks, depending on the dilution used. This is2-20 times the amount needed for catalysis, butthe fact that the GSH and neutral cysteine reactionmixtures are more effective than the control of1,2-naphthaquinone is against the view thatresidual 1,2-naphthaquinone is the catalyst. Whenthe purple cysteine-1,2-naphthaquinone compound

Bioch. 1967, 102

Vol. 102 861

Table 8. Catalytic oxidation of ascorbic acid by reaction products of 1,2-naphthaquinonewith GASH and cysteine

GSH and 1,2-naphthaquinone: A final concentration of 0*8mm-1,2-naphthaquinone and 1*6mM-GSH in30mM-phosphate buffer, pH7-3, was kept at 200 overnight. Cysteine and 1,2-naphthaquinone: A final con-centration of 1*6mm-1,2-naphthaquinone and 3 3mm- or 16mm-cysteine in 30mM-phosphate buffer, pH7-3,or in unbuffered solution at pH4.5 were kept at 20° overnight. Control of 1,2-naphthaquinone: A control tubeof 1,2-naphthaquinone alone was set up in each experiment. All reaction mixtures were extracted twice with anequal volume of CHCI3 before test and excess of CHCI3 was removed in vacuo. The 02 uptakes were measured inWarburg manometers at 25° (gas phase air) with KOH in the centre wells. The volume was 3*Oml. Each flaskcontained 100,umoles of phosphate buffer, pH7*3, 20,umoles of ascorbic acid and 3,umoles of EDTA; catalase(10,ul.) was added where appropriate. The reaction mixtures were placed in the side arm of the manometerflask and added after equilibration. The preparations took up no 02 in the absence of ascorbic acid.

PreparationGSH+ 1,2-naphthaquinone

Expt. Final diln. ofno. reaction mixture1

2

Cysteine+ 1,2-naphthaquinone (pH7.3)

Cysteine+ 1,2-naphthaquinone (pH475)As above with catalase at all stages

1212

1:101:301:101:601:101:101:101:10

02 uptake in 30min. (Iul.)Experimental

198123175511121316323

Control662326Nil53531911

was both formned and tested in the presence ofcatalase there was negligible catalysis, but if therewas brown compound present oxygen uptake was

stimulated.

DISCUSSION

The results reported in this paper show that1,2-naphthaquinone reacts with cysteine and otheramino acids, with GSH and with proteins of thelens. Development of a brown colour in the lensofthe naphthalene-fed animal has often been notedand van Heyningen & Pirie (1967) have producedevidence that this colour is due to combination of1,2-naphthaquinone with lens proteins. The con-

centration of 1,2-naphthaquinone in the eye of thenaphthalene-fed animal must therefore reach a

concentration sufficient to enable the reactions wehave described in vitro to take place. Formationof 1,2-naphthaquinone within the eye depends on

the rapid autoxidation of the reduced form, 1,2-dihydroxynaphthalene, which can be formedenzymically by eye tissues from metabolites ofnaphthalene in the blood (van Heyningen & Pirie,1967). Hydrogen peroxide is produced in thisprocess.Mason & Peterson (1965) considered that

enzymically generated o-benzoquinone reacted withthe amino group of cysteine and the evidencepresented above suggests that 1,2-naphthaquinonealso reacts in this way. The spectrum of the in

product of reaction under anaerobic conditions issmilar to that of 1,2-dihydroxynaphthalene and

we suggest that this productmaybe a 1,2-dihydroxycompound in which cysteine is linked to thenaphthaquinone nucleus through its amino group.This may then oxidize in air to a Schiff base withformation of hydrogen peroxide. The loss of thiolmay be due to oxidation of cysteine to cystine byhydrogen peroxide and possibly formation of athiazolidine ring compound of naphthaquinone.The reduction of 1,2-naphthaquinone by cysteineto form 1,2-dihydroxynaphthalene seems a minorreaction.GSH and 1,2-naphthaquinone combine in equal

proportions, at physiological pH under aerobicconditions, to form a brown compound, with acharacteristic absorption peak at 327m,u. Thiscompound does not travel as far towards the anodeas either GSH or GSSG on electrophoresis at pH4.Attempts to detect intermediates by carrying outthe reaction anaerobically or at 3° showed that thereaction was complex, but the main intermediatewas a colourless blue-fluorescent compound thatshowed absorption peaks at 236, 305 and 339m,.The similarity of its spectrum and fluorescence tothat of 1,2-dihydroxynaphthalene suggests that itis a dihydroxy compound, but as it is insoluble inchloroform and ethyl acetate it cannot be 1,2-dihydroxynaphthalene itself, and it seems probablethat GSH is combined with the naphthalenenucleus and the large loss in thiol during thereaction suggests that the link is through the thiolgroup of GSH. The loss of thiol is the same at 200and at 30 and also occurs anaerobically, and hencemust take place during the formation of the

862 J. R. REES AND A. PIRIE 1967

Vol. 102 REACTIONS OF 1,2-NAPHTHAQUINONE IN THE EYE 863colourless intermediate. It is difficult to accountfor the fact that loss of thiol in amounts abovethat equivalent to 1,2-naphthaquinone presentoccurs anaerobically, unless one invokes the diffi-culties of working anaerobically with dilute solu-tions of such reactive compounds. The slight effectof catalase on change in thiol is surprising, since itdecreases the oxygen uptake, and further work isnecessary.The thiol group seems also to be the most

reactive group in proteins. However, just as 1,2-naphthaquinone shows a slow reaction, judgedspectroscopically, with amino acids, so also itappears able to react with proteins that have noor few reactive thiol groups. Morgan (1924) foundthat it reacted with gelatin and it forms a browncompound with the collagen-like protein of the lenscapsule. Mason (1955) found that the free aminoand imino groups of proteins reacted more readilywith o-benzoquinone than did the amino groups ofamino acids. There is only one cysteine residue/sub-unit of a-crystallin and even this thiol groupis unreactive in the native protein (Waley, 1965),but a brown compound is formed with 1,2-naphtha-quinone. This is soluble at neutrality, whereas thereaction products of 1,2-naphthaquinone withsome of the ,B-crystallins and y-crystallins, whichcontain a greater number ofthiol groups (Kinoshita& Merola, 1958), become insoluble at this pH. Itis possible that the hardness of the brown lens thatdevelops in the naphthalene-fed animal (Goldmann,1929; Gifford, 1932) is due to precipitation ofnaphthaquinone-proteins.

Reaction of cysteine, GSH and proteins with1,2-naphthaquinone is accompanied by uptake ofoxygen and formation of hydrogen peroxide,suggesting that quinone groups are still present inthe products. The brown reaction products of1,2-naphthaquinone with cysteine and GSH cata-lysed the oxidation of ascorbic acid, which againsuggests the presence of autoxidizable quinonegroups. This action may be important in the eyewhere both the aqueous humour and the vitreoushumour have a concentration of ascorbic acid con-siderably greater than that in the blood.The concentration of GSH in the lens is about

10mM, but there is more protein thiol. This mayexplain why the len,s of the naphthalene-fed rabbitcontains brown proteins when there is still GSHpresent. Glutathione peroxidase (Pirie, 1965) isactive in lens, whereas catalase activity is very low(Zeller, 1953), s0 that formation of hydrogenperoxide in the reactions of 1,2-naphthaquinone

with cell constituents may have as great an effecton GSH metabolism as direct combination with1,2-naphthaquinone. Apart from these reactionswith constituents there is also the probability that1,2-naphthaquinone will have toxic effects byvirtue of its action on oxidation and reduction andother enzyme systems. This aspect has still to beinvestigated.

We have pleasure in thanking Dr S. G. Waley, Dr R.van Heyningen and Dr W. S. Pierpoint for their adviceand interest andMr K. Rixon for skilled technical assistance.

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Biochem. J. 77, 182.Calam, D. H. (1962). Exp. Eye Re8. 1, 436.Ellman, G. L. (1959). Arch. Biochem. Biophy8. 82, 70.Fieser, L. F. (1943). In Organic Synthes8e, collective vol.

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Gifford, H. (1932). A.M.A. Arch. Ophth. 7, 763.Goldmann, H. (1929). Kin. Mbl. Augenheilk. 88, 433.Grassmann, W., Hannig, K. & Pl6ckl, M. (1955). Hoppe.

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170,279.Zeller, E. A. (1953). Amer. J. Ophth. 36, no. 6, part 2,51.