Biochem. J. (1965) 94, 705

A Colorimetric Micro-Method for the Determination of Glutathione

By C. W. I. OWENS AND R. V. BELCHERDepartment of Chemical Pathology, University College Hospital Medical School, London, W.C. 1

(Received 15 June 1964)

1. A rapid colorimetric and apparently specific micromethod for the determina-tion of total glutathione in small amounts of tissue is described. Generally, lessthan 30mg. of tissue is sufficient and this is homogenized in ice-cold 3% metaphos-phoric acid. The product is filtered through sintered glass and neutralized or

diluted before being added to a cuvette containing phosphate buffer, pH7-1,5,5'-dithiobis-(2-nitrobenzoic acid), EDTA and glutathione reductase. Additionof NADPH2 to the system initiates a progressive reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by catalytic amounts of GSH, and this causes a colourincrease at 412m,. The rate of this change, calculated over 5min., is proportionalto the total amount of glutathione present, and consequently unknown concentra-tions may be determined by reference to standards. 2. A preparation (based onthat of Racker, 1955) of a suitable sample of glutathione reductase from yeast isdescribed. 3. A less specific and less sensitive determination of extracted thiolgroups with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH8-0, based on observationsof Ellman (1959) and Jocelyn (1962), is also described. 4. Although the precisenature of the reaction is not known, evidence is put forward to support a process

of cyclo-reduction. GSSG is reduced enzymically to GSH, which reacts with5,5'-dithiobis-(2-nitrobenzoic acid) to produce a coloured ion:

02N SH

C02

(E,,.. 412m,u) and a mixed disulphide. This disulphide reacts with furtherquantities ofGSH to liberate another ion and GSSG, which then re-enters the cycle.

After the isolation ofglutathione (Hopkins, 1921)many attempts were made to devise a satisfactorymethod for the determination of small amounts ofthis tripeptide in tissues. Recently, two newreagents have been introduced, namely bis-(p-nitrophenyl) disulphide (Stevenson, McDonald &Roston, 1960) and the more water-soluble DTNB*(Ellman, 1959; Beutler, Duvon & Kelly, 1963;Jocelyn, 1962) both of which have been used forthiol estimation. The procedure described belowutilizes DTNB, but in contrast with other methodsit measures the increase in extinction at 412mm,uwhen either GSSG or GSH, glutathione reductase,NADPH2 and DTNB are brought together at theappropriate pH. Under suitable conditions therate of change in extinction is proportional to thetotal amount of glutathione present. The mechan-ism of the reaction is not yet clear, but it appears

* Abbreviation: DTNB, 5,5'-dithiobis-(2-nitrobenzoicacid), i.e. di-(5-carboxy-4-nitrophenyl) disulphide.

23

to be specific and can be used for approx. 0-2,ug.of glutathione in a lcm. spectrophotometric cell.By using highly active glutathione reductaseand micro-cells it should be possible to increasethe sensitivity five- to ten-fold.

MATERIALS AND METHODS

DTNB was obtained from the Aldrich Chemical Co. Inc.,Milwaukee, Wis., U.S.A. DTNB (39.6mg.) and NaHCO3(15mg.) were dissolved in 5ml. of 01lM-phosphate buffer,pH 7-0, and the volume was adjusted to 10ml. with buffer.The reagent is stable at - 150 in the dark.NADPH2 was obtained from C. F. Boehringer und

Soehne G.m.b.H., Mannheim, Germany. A solution of4mg./ml. of 0*02N-NaOH, stable at -15°, was kept for amaximum of 2 days.NADH2 was obtained from Sigma Chemical Co., St.

Louis, Mo., U.S.A., and 4mg./ml. was dissolved in 0-02N-NaOH.EDTA was obtained from L. Light and Co. Ltd., Coln-

brook, Bucks. A solution of 73-1 mg. was adjusted to pH7-0Bioch. 1965, 94

705

C. W. I. OWENS AND R. V. BELCHERwith 0 1 N-NaOH and made up to 250 ml. with water. Thisreagent was added to eliminate any variations in responsethat could be caused by extraneous heavy-metal ions.

Trisodium phosphate (British Drug Houses Ltd., Poole,Dorset) was used as an aqueous solution containing 150g.of Na3PO4,12H20/L1

Metaphosphoric acid solution (3 0%,w/v), made bydissolving sticks in deionized or glass-distilled water atroom temperature, was prepared daily. Latterly thisreagent was stirred with 2g. of decolorizing charcoal/100ml.for 20min. at room temperature to remove oily impuritiesthat frequently appeared.GSH was supplied by British Drug Houses Ltd. A

standard solution of 2mg. in 20ml. of 3.0% (w/v) HPO3was prepared daily and kept at 00 to limit hydrolysis andprevent oxidation.GSSG was supplied by C. F. Boehringer und Soehne

G.m.b.H., and standard solutions were made up in water.Glutathione reductase was either purchased from the

Sigma Chemical Co. (glutathione reductase type III) orC. F. Boehringer und Soehne G.m.b.H., or prepared fromyeast as described below. Fresh solutions of the Sigmaproduct were made daily by diluting approx. 1:80, andsimilarly the Boehringer sample was diluted 1: 20. Repeatedfreezing and thawing caused considerable loss of activity,and ox-plasma albumin was not added as a stabilizersince it interfered with the assay reaction.

The enzyme was prepared from yeast by a method basedon Racker (1955) but included a number of modifications.A sample (1 lb.) of baker's yeast (The Distillers Co. Ltd.,Speke, Liverpool) is crumbled into a mortar and 20 ml.toluene added. A paste is made that is allowed to stand for4hr.; then 200ml. of 0 66M-Na2HPO4 is added and themixture stirred continuously at room temperature for 24 hr.The suspension is centrifuged at 1600g long enough for a

clear supernatant to form; it is then decanted and kept.The sediment is resuspended in another 200ml. of 0-66M-Na2HPO4 and stirred at room temperature for a further24-36hr. The centrifugation is repeated, and the twosupernatants are combined, heated at 53-58' in a waterbath for 15 min. while being stirred and then centrifuged at14000g for 5min. in an angled centrifuge. The supernatant,which should be clear, is removed and its volume measuredafter cooling to approx. 0°. One-half its volume of acetoneat -12° is added slowly with stirring and the mixture isallowed to stand for 1 hr. at 40 with occasional stirring.After this, Racker's (1955) technique is followed up to theaddition of protamine sulphate, when, in the present case,2-5 ml. of 2% (w/v) protamine sulphate is added slowly at4°. After a few minutes the suspension is centrifuged for5 min. at -3° and 14000g and the supernatant, in a plasticcentrifuge tube, is adjusted to pH5-5 with about 2-5 ml. of1 N-acetic acid and cooled to near freezing in acetone-solidC02 mixture, and 6-8 ml. of 95% (w/v) ethanol at - 12° isadded with stirring. After standing for 5 min. the precipi-tate is collected by centrifugation at 14 000g for 5 min. andkept partly dissolved at -15° in 20 ml. of water. Thissuspension may or may not be diluted further to givesuitable activity, but it should be divided into 1 ml. portionsand frozen at -15° to avoid daily thawing and re-freezing,which results in loss of activity. Under these conditions theenzyme is stable for at least 9 months. When dilution isnecessary it is best done on each 1 ml. sample.

Ox-plasma albumin was purchased from Armour Pharma-

1965ceutical Co., Eastbourne, Sussex and was used directly as

described.Sulphur-containing compounds (described under specifi-

city and interfering substances) were very kindly given byDr H. Heath, Department of Chemical Pathology, Univer-sity College Hospital Medical School, London.

Extinction measurements were made on either an

Optica recording spectrophotometer or a manually operatedinstrument with glass cells of 1 cm. light-path.

RECOMMENDED METHODAND RESULTS

The addition of certain reducing agents, includingGSH, to DTNB results in a change of spectrum andintensification of yellow colour (Ellman, 1959); thepeak of absorption is at 412m,u. In the proceduredescribed below small quantities of GSSG or GSHbring about this change but to a quantitativelygreater degree, suggesting the operation of a

cyclic process (see the Discussion section).Calibration of enzyme. The method depends on

the rate of an enzymically controlled colour changebeing directly proportional to the amount ofglutathione present. It is therefore necessary tofind the range over which this is true for a givenenzyme preparation.

Initially the reductase is diluted so that it givesa change in extinction at 412m,u of about 0-02/min.with 0-5,ug. of GSH in the cuvette, and to ensure

that the enzyme is never limiting it is calibratedwith either acidic or neutral samples of standardglutathione, depending on whether high or lowconcentrations are to be measured. If the tissuebeing investigated contains, e.g., more than 40mg.of glutathione/lOOg. wet wt., then dilution of theextract with water, before estimation, is permis-sible. Under these circumstances standards are

prepared so as to contain between 1 and 20,tg./ml.after 1:1 or 1:2 dilution. With less than 40mg./100g. wet wt. of tissue or with an enzyme of lowactivity the metaphosphoric acid must be removedby neutralization, and hence standards in 3%metaphosphoric acid are treated with 0-3ml. oftrisodium phosphate/ml. of metaphosphoric acidsolution and finally contain between 1 and 20,ug. ofglutathione/ml.A 0-2ml. sample is added to a cuvette containing

2-5ml. of 0-05M-phosphate buffer, pH7-1, 0-8ml. ofImm-EDTA (sodium salt) and 0-03 ml. of DTNB.The cuvette isshaken and 2min. later 01 inl. ofsuita-blydilutedenzynme andO- 1 ml. ofNADPH2 areadded.The cuvette is shaken again, the time recorded andthe uniform change in extinction at 412mp/min.noted from the first to the fifth minute. The experi-ment is repeated with various amounts of gluta-thione and with a blank, since often a backgroundreaction occurs, presumably owing to glutathionecontamination of the enzyme The amounts at

706

DETERMINATION OF GLUTATHIONEwhich glutathione is limiting are thus determinedand the tissue extract, described below, is treatedaccordingly so that its glutathione content fallswithin these values.A stock mixture of buffer and EDTA (25:8, v/v)

may be made. The reference blank contains 3-7 ml.of this solution with 0-03ml. ofDTNB.

Preparation of ti8sue extract. The tissue isexcised from the animal and, after being weighedquickly on a torsion balance (maximum load 50mg.),is dropped into 10ml. of ice-cold 3% metaphos-phoric acid and homogenized thoroughly. Suitablequantities of some organs are as follows: rat liver,adrenal, retina, kidney, mouse adrenal, 20mg.;blood, 0-lml. in 9-9ml. of acid. With blood, toprevent oxidation of GSH during denaturation,carbon monoxide is passed through the sample fora few minutes to displace oxygen from oxyhaemo-globin (Numata, 1940). The acidic tissue extractis removed by Pasteur pipette from the homogenizerand placed in a glass tube (approx. 15 cm. x 1cm.)that has at one end a sintered-glass ifiter, porositygrade 4 (average pore size 5-10,u). The spaceabove the liquid is flushed with nitrogen andpressure applied at 15-201b./in.2 long enough for2-3ml. to be collected in an ice-cooled test tube. Byusing a manifold, any number of such ultrafiltra-tions can be made at the same time.

Experiments have shown that the extract mustbe kept ice-cold at all times. The glutathionerecovered was not affected by the quantities of

either denatured protein or metaphosphoric acidencountered in the recovery experiments and theefficiency of recovery varied from 90 to 105%(Table 1).Determination of total thiol. A 0-5ml. sample of

the filtered extract containing about 5,ug. of GSHis added to a mixture of 1-5ml. of 0-5M-potassium-sodium phosphate buffer, pH8-0, and 0 03ml. ofDTNB. The blank contains 0-5ml. of 3% meta-phosphoric acid instead ofthe tissue extract and thereaction is carried out in very carefully cleanedtest tubes. The stable colour is read at 412m,u after3min. in a 2cm. light-path micro-cell and it iscompared with that from duplicate standardscontaining 5,ug. of GSH/0.5ml. of 3% metaphos-phoric acid. The extinction at 412m,u is linearlyrelated to thiol present; thus the quantity of thiolin the extract may be calculated.

All glassware concerned with this estimationmust be free from reducing substances. A con-venientway ofensuring this is to treat the apparatuswith a very little ethanol followed by concentratednitric acid. After the violent reaction has subsidedthe glass is washed with deionized water and thenacetone, and dried at 1000. Occasionally, withfresh buffer, prepared by adding 0-5N-potassiumhydroxide to 0 5M-potassium dihydrogenphosphateuntil pH 8-0 is reached, a high blank colour isformed. This may be lessened by boiling the bufferfor about 15-20min.

Determination of total glutathione. If the tissue

Tissue in3% (w/v) HPO3Mouse liver(28-3mg./lOml.)

Rat liver(25-0mg./lOml.)

Rat blood(0.1ml./10ml.)

Rat liver(150mg./lOml.)

Table 1. Glutathione recovery from various tinsue extractsExperimental details are given in the text.

Recovery of thiol from anextract sample

Amount Amountadded(pUg).

found Recovery(pg.) (%)

0 10-610 19-620 30 330 40-60 17218 1910 14018 1590 13018 147

9098-5100

106

106

95

Recovery of total glutathione from anextract sample

Amount Amountadded(pg.)09*0

18*00102030400102030

found(pg.)12-021-530-419-429395158-510-720-531*240

Recovery(%)

105102

969810598

9710298

Vol. 94 707

C. W. I. OWENS AND R. V. BELCHER

glhitathione content is low or the enzyme inactivethen the excess of metaphosphoric acid, whichdepends on the amount of protein precipitated,must be neutralized before the enzymic process isinitiated. Trisodium phosphate is a most effectivebase, but simple titration is, however, undesirablesince according to the initial excess ofmetaphospho-ric acid the final Na+ concentration at neutralitywill vary, and this in turn greatly effects the enzymicactivity. Experience has shown that the additionof 0-3ml. of trisodium phosphate solution/ml. ofextract or standard provides a suitable solution forassay, although dilution when possible doesincrease the sensitivity. In favourable circum-stances, as mentioned above, the extracts andstandards may be diluted directly approx. 1 :1with water. In either case a 0 2 ml. sample is takenand assayed (as described under Calibration ofenzyme) and the glutathione calculated by referenceto standard solutions.

Extracts and standards must be treated identi-cally, the latter being prepared at time of homo-genizing and kept at 0° until neutralized or dilutedor both along with the filtered extracts.

Notes on the method. (a) Total thiol. Ellman(1959) specified that the thiol estimation should becarried out at pH8-0. Since the glutathione isextracted into acid, the effect of final pH on colourdevelopment was studied by using 3-0ml. of 0-5M-phosphate buffer, 0-03ml. of DTNB and 5,tg. ofGSH in 0-5ml. of 1% (w/v) metaphosphoric acid.The result is summarized in Table 2.The molarity of buffer did not effect the final

colour formed, and the optimum amount of DTNBneeded was 0 03ml. A blank was always necessary,since occasionally fresh buffer at pH8-0 gave aconsiderable colour, but it did not appear if thebuffer had stood for a few days at room temperature.Neither hydrogen peroxide treatment nor quickboiling prevented this phenomenon, but saturationwith carbon dioxide decreased it.

(b) Total glutathione. After the discovery of areaction between glutathione, DTNB, glutathionereductase and NADPH2, a sample of the enzymewas prepared as described above and used to

Table 2. Effect of pH on colour development in thereaction between reduced glutathione and DTNB

The system contained 3-Oml. of 0-5M-phosphate buffer,0-03ml. ofDTNB and 5,ug. of GSH in 0-5ml. of 1-0% (w/v)HPO3.

Buffer pH6-07-08-0

Time for colourMax. E412mz development (min.)

0-092 50-168 20.174 0 5

estimate 30,ug. of GSSG umder thc conditions ofRail & Lehninger (1952). The observed rate ofchange in extinction at 340m,u was not noticeablyaltered by the addition of OOlml. of DTNB, butOlml. of DTNB completely stopped the reaction.Since the estimation was not inhibited with lowquantities of DTNB, measurements were possibleat 412m,u when very small quantities of gluta-thione were used and when the optimum quantityofDTNB to be added was 0-03ml.The reaction exhibited a sharp optimum at

pH 7 1 in the presence of 0 011% ox-plasma albuminand 0-05M-phosphate buffer, but since the proteinexerted an inhibitory action (Fig. 1) its use wasfinally dropped. Either glutathione or NADPH2may be added to the cuvette last, but since there isa 2min. period in which GSH reacts with DTNBit is better that NADPH2 is added after this reactionis completed, thus giving a uniform change inextinction from the beginning. The rate of colourchange is proportional to the total glutathioneirrespective of the percentage of each form present(Fig. 2), but the colour before the addition ofNADPH2 varies directly with the GSH present andalso depends on the amount of protein in solution.Experiments showed that 3% (w/v) sulphosali-

cylic acid was unsuitable for tissue extraction,since it produced a 66% inhibition in the cuvettethat could not be overcome by increased ox-plasmaalbumin or DTNB. Metaphosphoric acid, neutral-ized carefully with trisodium phosphate, did notinterfere markedly with the reaction.

0 2 4 6 8Finial collCIi. of ox-plasma albumin

(mg./cuvette)

Fig. 1. Inhibitory action of ox-plasma albumin on thesystem used for total glutathione determination. Thecuvette contained: 2-5ml. of 0 5M-sodium phosphatebuffer, pH7-1; 0-8ml. of lmM-EDTA (sodium salt);0-03ml. of DTNB; 01ml. of enzyme; 5,g. of GSSG;0.1 ml. of NADPH2; ox-plasma albumin as indicated. Theprotein was added in aqueous solution. One activity unit isdefined as 10-4 x AE420.1f/min.

708 1965

DETERMINATION OF GLUTATHIONE

2 3 4Time (min.)

Fig. 2. Relationship between rate of colour development,[GSH]+ [GSSG] and [GSH]/[GSSG] ratio. The cuvettescontained: 2-5 ml. of 0-5M-sodium phosphate buffer, pH 7.1;08ml. of lmM-EDTA (sodium salt); 0-03ml. of DTNB;01ml. of enzyme; 01ml. of NADPH2; glutathione. A,[GSH]+[GSSG], 7/>g./cuvette; [GSH]/[GSSG] ratio 6:1;B, [GSH]+ [GSSG] 7,ug./cuvette; [GSH]/[GSSG] ratio 1:6.

Specificity and interfering 8ub8tance8. No detect-able enzymic reaction occurred with any of thefollowing compounds in solution: 10,ug. of taurine,cysteic acid, cystathione, methionine sulphone,methionine, thiolhistidine or djenkolic acid; 5,ug.ofhomocysteine or lanthionine; or 1mg. of ascorbicacid. The compounds listed in Table 3 gave noenzymic reaction but did give a yellow colour withDTNB, and therefore must be considered tointerfere with the thiol estimation.

It appeared that cysteine was the only naturallyoccurring compound that caused any seriousinterference, but further tests showed that it in noway affected the rate of colour formation. Sincenon-glutathione thiol appeared not to interferewith the change in extinction at 412 m, it washoped that thiol groups could be measured fromthe extinction at 412m,t at to (i.e. just before theaddition of NADPH2) and total glutathione fromthe rate of colour change in the first few minutesafter the addition of NADPH2. Experimentsrevealed difficulties. A calibration curve showedthat, the more thiol groups there were present, thelonger it took for a stable colour to be formed, and,moreover, the amount of enzyme in the cuvette

Table 3. Substance8 found to interfere with the determination of thiol groups withDTNB at pH8-0 in 0-5M-pho8phate buffer

The system contained 30 ml. of 0 5M-phosphate buffer, 0 03 ml. ofDTNB and the sulphur compound in 05 ml.of 1% (w/v) HPO3.

SubstanceErgothioneine (IO,ug.)Methionine (10,g.)flf-Diaminoethyl disulphide (10,ug.)2,3-Dimercaptopropanol (BAL) (10kg.)Mercaptoethylamine (1Ot,g.)Cysteine hydrochloride (3,ug.)

E412miA0-020-0080-020*730-62005

Time for colourdevelopment (min.)

535233

Table 4. Glutathione content of various tis8uesExperimental details are given in the text. The values in parentheses are taken from the literature.

TissueNerveAdrenal (mouse)Retina (rat)

Blood plasma (human)ErythrocytesPituitary gland (rat)Liver (rat)

Wt. ofsample(mg.)

401018-5

1.0 (ml.)0.3 (ml.)8

20

Glutathione content(mg./100g. wet. wt.)

GSSG+ GSH17-2114+ 343-1420-2+ 0-0562+ 12 (60*)81

245+ 5 (243t)* Danielli (1954).t Bhattacharya, 1WobsQn & Stewart (1956).

GSH

1103028

60235+ 10 (241t)

Vol. 94 709

C. W. I. OWENS AND R. V. BELCHERaffected the extinction at to. Finally, the volume ofextract needed to give a satisfactory reading beforethe addition of NADPH2 gave too great a changein extinction at 412mu afterwards. Clearly twoseparate estimations were necessary.

Re8ult8. Some tissues have been briefly examinedfrom laboratory animals and the glutathionecontents found are shown in Table 4. In general,they agree with values stated already in the litera-ture.

DISCUSSION

Precisely how the enzymic reaction proceeds isnot known, but DTNB has been observed toproduce a spectrally identical colour with variousreducing agents such as sodium metabisulphite,2,3-dimercaptopropanol, GSH, ferrous ammoniumsulphate (Ellman, 1959), cysteine, sodium sulphide,50% (w/w) hydrazine hydrate and, to a smallerextent, 'quinol' and 'metol' in sodium hydrogencarbonate solution. With alkali a slightly differentcolour is produced, and with ascorbic acid, glucose,formaldehyde, 2-methyl-1,4-naphthaquinone orsodium chloride there is no reaction. It thereforeseems that there is a progressive reduction ofDTNB, triggered off by catalytic amounts of gluta-thioneinthepresenceofthe reductase andNADPH2.Supporting this is the fact that 0-02ml. of DTNB,at pH8-0, treated with 0 05ml. of aq. 1% (w/v)sodium sulphide solution or with the enzyme (with30jug. of GSSG, to facilitate rapid reaction) yieldscolours of identical spectra and intensity (Fig. 3).Similar results are obtained with 2% (w/v) GSH,

E 0.3

0-2

0.1

520 480 440 400 360 320

Wavelength (mp)Fig. 3. Non-enzymic (-) and enzymic ( ) reduction ofDTNB. Both systems contained: 2-5ml. of 005 M-sodiumphosphate buffer, pH7-1; 0f8ml. of lmm-EDTA (sodiumsalt); 0-02ml. of DTNB; Olml. of enzyme; Olml. ofNADPH2; either 30Mpg. of GSSG or 0-05ml. of sodiumsulphide (approx. 1%, w/v).

but 0 2ml. of 2% (w/w) sodium metabisulphiteproduces only 50% of the colour intensity.The necessity for the complete system of buffer,

glutathione, DTNB, NADPH2 and enzyme suggestsa cyclic reduction, ceasing only when the enzyme,DTNB or NADPH2 is spent. NADH2 is only 50%as efficient as NADPH2, an observation of interestin view of the lack of specificity of the reductase forNADPH2 (Racker, 1955). Both NAD and NADPare ineffective. The role of NADPH2 is critical,since when it is spent the reaction stops abruptlyonly, to re-start on the addition of more nucleotide.If the enzyme is not in excess it appears to beexpendable or unstable, since after a few minutesthe reaction stops. Possibly DTNB reacts with theenzyme to yield an inactive form (Mapson &Isherwood, 1963) or directly with the thiol groupsof the active centres. Further, it is relevant thatexcess of DTNB will inhibit the assay even withsubstantial amounts of enzyme present. Thereaction is completely inhibited by 0X 1 mM-p-chloromercuribenzoate, is effected by heat (Fig. 4)and is completely abolished by 0 71M-sodiumchloride in the presence of neutralized 0-28m-meta-phosphoric acid.

Since the yellow colour produced is always inexcess of that expected from the stoicheiometricreduction of DTNB by GSH, the operation of acyclic process seems virtually proven. Briefly, thepostulated mechanism may be summarized in thefollowing series of equations where DTNB iswritten as ArSSAr:

GSSG+NADPH2 - 2GSH+NADP

GSH+ArSSAr GSSAr+ArSH

ArSH (as the ion) is measured at 412mp.

c5

(1)

(2)

Time (min.)Fig.4. Effectoftemperature. Each cuvette contained: 2-5

ml. of 0-05m-sodium phosphate buffer, pH7-1; 08ml. of1 mm-EDTA (sodium salt); 0 03ml. of DTNB; Ol ml. ofenzyme; 0 1 ml. of NADPH2; 2,ug. of GSSG. The cuvetteswere maintained at: A, 200; B, 380; C, 45-50°; D, 600;B was obtained with boiled enzyme. At 60° neither coolingnor the addition ofNADPH2 had any stimulatory effect.

1965710

Vol. 94 DETERMINATION OF GLUTATHIONE 711

The product GSSAr is cleaved either non-enzymically with GSH or enzymically to yieldArSH and GSSG or ArSH and GSH according toone of the following equations:

ArSSG+GSH = GSSG+ArSH (3)

or ArSSG+NADPH = GSH+ArSH (4)

It is probable that GSH completes the reduction ofArSSG (eqn. 3) and relatively unlikely that ArSSGacts as a substrate for enzymic reduction (eqn. 4).Thus the GSH is regenerated and, with excess ofNADPH2 and constant amount of enzyme, therate of colour production is proportional to thetotal glutathione. Never has the colour producedexceeded the theoretical maximum where all theDTNB has reacted. It does not appear necessarytherefore to postulate the formation of a new andhighly coloured complex.

We thank Professor C. Rimington, F.R.S., for hisinterest, encouragement and help in preparing the manu-

script. C. W. I. 0. is indebted to the Rockefeller ResearchFoundation of this School for a supporting grant.

REFERENCES

Beutler, E., Duvon, 0. & Kelly, M. B. (1963). J. Lab. clin.Med. 61, 882.

Bhattacharya, S. K., Robson, J. S. & Stewart, C. P. (1956).Biochem. J. 62, 12.

Danielli, J. F. (1954). Symp. Soc. exp. Biol. 8,502.Ellman, G. (1959). Arch. Biochem. Biophy8. 82, 70.Hopkins, F. G. (1921). Biochem. J. 15, 286.Jocelyn, P. C. (1962). Biochem. J. 85, 480.Mapson, L. W. & Isherwood, F. A. (1963). Biochem. J. 86,

173.Numata, I. (1940). Biochem. Z. 304, 404.Racker, E. (1955). J. biol. Chem. 217, 855.Rall, T. W. & Lehninger, A. L. (1952). J. biol. Chem. 194,

119.Stevenson, T. D., McDonald, B. L. & Roston, S. (1960).

J. Lab. clin. Med. 56, 157.