296 K. SELBY, C. C. MAITLAND AND K. V. A. THOMPSON 1963Reese, E. T. (1956). Appl. Microbiol. 4, 39.Reese, E. T., Segal, L. & Tripp, V. W. (1957). Text. Res. J.

27, 626.Reese, E. T., Siu, R. G. H. & Levinson, H. S. (1950).

J. Bad. 59, 485.Searle, G. 0. (1929). J. Text. Inst. 20, T162.Selby, K. (1961). Biochem. J. 79, 562.

Selby, K. (1963). In Advance8 in Enzymic Hydroly8is ofCellUlo8e and Related Materials, p. 33. Ed. by Reese, E. T.Oxford: Pergamon Press Ltd.

Van Haga, P. R. (1958). Nature, Lond., 182, 1232.Walseth, C. S. (1952). T.A.P.P.I. 35, 233.Whitaker, D. R. (1953). Arch. Biochem. Biophys. 43,

253.

Biochem. J. (1963) 88, 296

The Catalysis of the Auto-oxidation of 2-Mercaptoethanol andother Thiols by Vitamin B12 DerivativesPOLAROGRAPHIC AND OTHER INVESTIGATIONS

BY J. L. PEELAgricultural Research Council Unit for Microbiology, The Univer8ity, Sheffield 10

(Received 7 January 1963)

During a study of the exchange reaction betweenCO2 and the carboxyl group of pyruvic acid cata-lysed by preparations from Pepto8treptococcu8el8denii (previously referred to as rumen organismLC), it was found that the auto-oxidation of 2-mercaptoethanol (monothioethylene glycol) isgreatly accelerated by catalytic amounts ofvitaminB12 derivatives at pH 7 (Peel, 1962a). Such aneffect does not appear to have been reported pre-viously at physiological pH, except for an inci-dental statement by Dubnoff (1950) that vitaminB12 catalysed the oxidation of certain thiols in anon-enzymic system. In view of the currentinterest in vitamin B12 derivatives, the increasinguse of 2-mercaptoethanol as a reducing agent inincubation mixtures and the possibility that thiscatalytic property might be of use for the detectionand assay of vitamin B12 derivatives, the reactionwas investigated further. The present paper reportsthe results, some of which have appeared in brief(Peel, 1962 b).

MATERIALS AND METHODS

Chemical reagent8The thiols used were as follows: 2-mercaptoethanol,

Eastman grade (from Kodak Ltd., Liverpool); 2,3-dimercaptopropanol (BAL) and L-cysteine hydrochloride(from L. Light and Co. Ltd.); thioglycollic acid, laboratory-reagent grade (from British Drug Houses Ltd.); glutathione[from The Distillers Co. (Biochemicals) Ltd., Liverpool];DL-dihydro-6-thioctic acid, assay 98%, and DL-dihydro-6-thioctic amide, assay 100% (from Farmochimica Cutolo-Calosi S.p.A., Naples; gifts from Dr V. Massey). The2-mercaptoethanol, 2,3-dimercaptopropanol and thio-glycollic acid were distilled under reduced pressure beforeuse and then assayed to be 100, 100 and 95% pure respec-

tively by iodometric determination. The 2-mercapto-ethanol was subsequently used over an extended periodduring which it was stored at room temperature. After6 months the purity had fallen to 92% and the materialwas distilled again. In experiments where the amount ofthiol was critical, solutions were standardized iodometri-cally before use. Where necessary thiol solutions wereneutralized to pH 7-1 with NaOH, immediately before use.Aqueous 50% (v/v) methanol was used to dissolve thedihydrothioctic acid amide. Controls showed that thissolvent had no effect on the polarographic measurements.The vitamin B.2 derivatives were obtained as gifts and,

apart from the exceptions mentioned below, were fromDr E. Lester Smith. Their structural relationships to cyano-cobalamin are indicated in Table 3. The 5,6-dimethyl-benzimidazolylcobamide coenzyme (Barker et al. 1960b)was obtained from Dr H. A. Barker. As the cobamidecoenzymes are very labile to light, experiments with thiscompound were done in near darkness with minimum arti-ficial illumination. Factors A and B (Ford & Porter, 1953)and Factor D (Brown, Cain, Gant, Parker & Smith, 1955)were obtained as solutions in aqueous 70% (v/v) ethanolfrom Dr J. W. G. Porter and Dr H. R. V. Arnstein. Thesesolutions were standardized by measurement of the extinc-tion at 367 m,u in 0-1 M-KCN and assuming E to be 30 4 x10 1. mole-' cm.-" as for dicyanocobalamin (Barker et al.1960a). The term Factor B is usually used to denote themonocyano derivative of cobinamide, derived fromcyanocobalamin by removal of the nucleotide, but as thiscompound readily takes up an additional molecule ofcyanide, the scope ofthe term has sometimes been extendedto include the dicyano form as well. The spectrum of theFactor B sample used in all the experiments reported belowcorresponded to a mixture ofthe mono- and di-cyano forms.On adding excess of cyanide, the spectrum changed to oneclosely resembling that for dicyanocobinamide given byFord & Porter (1953). Factor B has not yet been crystal-lized; as far as the author is aware, reliable extinction coeffi-cients, which would permit an evaluation ofthe proportionsof the two forms in the mixture, are not available. The

VITAMIN B12 DERIVATIVES AND THIOL OXIDATIONSextinctions of the sample at pH 7-1 in 5 mM-potassiumphosphate buffer at 367 and 580 m1L (where dicyanocobin-amide has absorption maxima) were 86 and 73% respec-tively of the values in the presence of 0.1 M-KCN. For con-venience, this preparation is referred to as 'Factor B'throughout. A second sample, which spectroscopicexamination showed to be in the monocyano form, was usedin experimentsalready published (Peel, 1962a). This samplewas exhausted before its catalytic activity could be com-pared quantitatively with the other sample, but no qualita-tive differences in behaviour were noticed.The remaining chemicals were of analytical-reagent

grade except the following: ascorbic acid B.P. (from Hopkinand Williams Ltd.); disodium ethylenediaminetetra-acetate, Na2S204, Na2H2P207, NaN3 and 1,10-phenanthro-line monohydrate (from British Drug Houses Ltd.); andFe2(SO4)3, 'pure' (from Towers and Co.).

Extract of boiled yea8tThis was prepared by mixing baker's yeast with water

(2 ml./g. of yeast), heating at 1000 for 10 min. and thenremoving insoluble material by centrifuging for 20 min. at10 000g. The extract contained about 30 mg. dry wt./ml.and, where indicated, was freeze-dried.

Determination of thiolsAqueous solutions of thiols were standardized iodo-

metrically by the method described by Woodward & Fry(1932) for glutathione, but with the following modifications.The preliminary deproteinization was omitted, the titrationwas done in the presence of 0-1 N-H2SO4 in place of sulpho-salicylic acid, and 1 mM-KIO3 was used. For determina-tions on the products from manometric experiments, themanometer contents plus washings were transferred into10 ml. of 0-2N-H2SO4 and titrated without adding furtheracid.

Spectrophotometric measurement8Extinctions were measured with the Unicam SP. 500 or

SP. 600 instruments. Spectra were scanned with an Opticarecording spectrophotometer [Optica (U.K.) Ltd., Gates-head-on-Tyne].

Manometric measurement of thiol oxidationThis was done in Warburg manometers at 370 and with

air as gas phase by conventional techniques.

Polarographic mea8urement of thiol oxidationApparatus. The oxygen uptake due to thiol oxidation was

also measured by following the diminution in the (partial)pressure of oxygen polarographically. The oxygen electrodeused was of the Clark (1956) type, the advantages of whichhave been pointed out in a review by Connelly (1957). Theessential feature of the design is that the Pt/oxygen elec-trode, together with the Ag/AgCl reference electrode andthe connecting electrolyte, are enclosed by a thin poly-ethylene membrane, readily permeable to gases but not toother solutes. This membrane shields the oxygen electrodefrom substances in the test solution, e.g. protein and pre-cipitating ions, that would otherwise inactivate the elec-trode. Apparatus incorporating such electrodes, suitablefor the measurement of respiration, has been described byBellamy & Bartley (1960) and by Stickland (1960). The

current flowing at a given (partial) pressure of oxygen isincreased by movement of the test solution past the mem-brane and in the procedure used by the above authors theoxygen pressure cannot be measured until the transientdisturbance associated with the addition of the last com-ponent to the reaction mixture has subsided. In the writer'sexperiments this difficulty was overcome by stirring thereaction mixture continuously at a constant rate.The apparatus was designed to present the minimum

liquid surface to the outside air and its construction isshown in Fig. 1. It consisted of a cylindrical glass reactionchamber, RC, surrounded by a water jacket, J, throughwhich water of controlled temperature was circulated bymeans of a Tecam Tempunit [Techne (Cambridge) Ltd.].The contents of the reaction chamber were stirred mag-netically, the stirrer slug. S, being made from a piece ofmild-steel rod (12 mm. long x 3 mm. diam.) sealed in a shortsection of polyethylene tube (external dimensions: 18 mm.long x 5 mm. diam.). The jacketed vessel was centred over amagnet, RM, which rotated at about 250 rev./min. A Per-spex plunger, P, fitted loosely into the cylindrical reaction

,RC

RM- -

Fig. 1. Apparatus for polarographic measurement of thioloxidation. RC, Reaction chamber; J, constant-tempera-ture water jacket; S, magnetic stirrer slug; RM, rotatingmagnet; P, Perspex plunger; SR, supporting rod; PS,polyethylene sleeve; 0, vertical groove; E, combined elec-trode assembly; Ag, silver/silver chloride anode; Pt,platinum cathode; PM, polyethylene membrane; 0, 0ring retaining membrane; C, cable leading to electricalcircuit. For simplicity, much of the interior detail of thecombined electrode assembly has been omitted.

297Vol. 88

J. L. PEELvessel leaving an annular gap of about 05 mm. width. Tothis was attached a stainless-steel supporting rod, SR,which passed through a clamp above the chamber and wasused to raise or lower the plunger. A combined electrodeassembly, E, manufactured by the Yellow Springs Co. Inc.,Yellow Springs, Ohio, U.S.A., projected through theplunger and was held in position by a tight-fitting poly-ethylene sleeve, PS. The polyethylene membrane, PM, wascut from a sheet of mean thickness 0002 in. The lowersurface of the plunger was inclined at an angle of 50 to thehorizontal and from the highest point a V-shaped verticalgroove, G, measuring about 1-5 mm. from apex to circum-ference, led to the upper surface of the plunger. When theplunger was lowered on to the surface of a reaction mixtureany air bubbles escaped through this groove so that theonly liquid surfaces remaining in contact with the air werethose in the groove and the annular gap. An adjustable stopwas fitted to the supporting rod above its clamp and was setso that with8 ml. of liquid in the reaction chamber andthe plunger lowered, the liquid surface came halfway up thevertical groove. After use, the plunger was raised and thereaction chamber emptied by suction from a water pump.The plunger and chamber were then rinsed with water, therinsings were sucked away, and the electrode was blotteddry with paper tissue.The combined electrode was connected by the cable, C, to

a polarographic circuit like that of Stickland (1960), exceptthat the condensers across the batteries were omitted. Thevoltage applied across the combined electrode was 0 6v.The galvanometer was a Scalamp model (W. G. Pye,Cambridge) and a 560Q shunt was placed across it to givea convenient sensitivity. All measurements were thenmade with the sensitivity set to '1', and under these condi-tions a scale deflexion of 94 cm./,uA was obtained.

Calibration. The expected relationship between electrodecurrent and oxygen concentration was a linear one (manu-facturer's manual; cf. Bellamy & Bartley, 1960) and thiswas verified by measuring the current flowing when the testsolution was saturated with a series of air-N2 mixtures ofknown composition. For routine calibration, the current atair saturation and at zero oxygen concentration was mea-sured. The upper calibration point was determined by put-ting into the reaction chamber 7-7 ml. of the appropriatebuffer, raising the plunger to give about 4 ml. of air abovethe liquid, and increasing the rate of stirring so that a widevortex formed. After 3-5 min. the stirrer speed was de-creased to normal and the plunger restored to the fullylowered position. The galvanometer reading was then notedas soon as it became steady. This aeration process wasrepeated until the galvanometer reading was constant. The'residual current', i.e. that flowing at zero oxygen pressure,was determined by adding a few milligrams of sodiumdithionite to the air-saturated buffer and noting the read-ing after 2-3 min. This was found to be identical with thatobserved when the buffer was saturated with N2 (02-free).Saturation with N2 or with air-N2 mixtures was achieved ina similar fashion to air saturation except that a Pasteurpipette was inserted through the groove, G, into theliquid, and gas of the appropriate composition was passed.The term 'oxygen current' is used below to denote the

quantity: (observed current -residual current). The cor-responding galvanometer deflexion was directly propor-tional to oxygen concentration for a given buffer solutionand for many purposes it was sufficient to express results in

terms of this corrected galvanometer reading. Where abso-lute values of oxygen concentration were required, it wasassumed that, during determination of the upper calibra-tion point, the air in equilibrium with the test solution wassaturated with water vapour. The concentration of dis-solved oxygen was then calculated from the relationship:

C=axx1000 (i-) x0-21760

where C is the oxygen concentration in 1l./ml., a is theabsorption coefficient for oxygen, Pa is the atmosphericpressure, and p, is the saturation vapour pressure of waterin mm. Hg; 0-21 is the fraction by volume of oxygenin air.The oxygen currentwith the Clark electrode appears to be

determined by the partial pressure rather than the concen-tration of oxygen in solution since the upper calibrationpoints were identical for water, 01M-phosphate buffer andfor 5% (w/v) NaCl, in which oxygen is considerably lesssoluble than in water (e.g. approx. 25% less at 300). Thepresence of salts thus affects the calibration of the instru-ment in terms of oxygen concentration. In the absence ofsolubility data for the appropriate buffers, their effect onoxygen solubility was ignored in calculating rates of oxygenuptake. Data from standard tables for other solutes sug-gest that the error so introduced is probably less than 10 %with 0 1 m-phosphate buffer.Both residual current and oxygen current increased with

temperature and it was necessary to calibrate the instru-ment separately at each temperature. There was no signifi-cant effect of pH on the calibration within the pH rangeused (5 5-90). Although the response of the electrode wasincreased by stirring, stirring rates greater than normal(250 rev./min.) did not give a higher current. The oxygencurrent was dependent on the polyethylene membrane, andfor air-saturation ranged from 1 to 1-25,UA with differentsamples of the same polyethylene sheet. It was thereforeessential to recalibrate after changing the membrane.When temperature, stirring and membrane remained

unchanged the calibration points still underwent certainvariations. Transient high currents were always observedwhen the electrical circuit was switched on. After 5-15 min.readings became steady in the sense that changes over theperiod required for each measurement of thiol oxidationwere negligible. The following remarks refer to currentsafter this initial 'warming up' period. The oxygen currentremained constant apart from small slow fluctuationsattributable to changes in atmospheric pressure. Theresidual current was subject to long-term changes thatappeared to be related to the state of the electrolyte andthe membrane. When both of these had been newly changedthe residual current was high (as much as 1 p{A) but fell to avalue less than 0-1 A after a few hours' running. After this'running in' period it remained constant but finite for alengthy period (e.g. 100 hr.). Eventually the residual cur-rent began to rise and the upper calibration point movedbeyond the galvanometer range. This effect could be offsetby applying a 'backing off' current to the galvanometer asdescribed by Stickland (1960), but this remedy was onlyused to avoid breaking off during a series of experimentalruns. A low residual current could usually be restored bychanging the KCI electrolyte and 'running in' afresh; whenthis failed, changing of the membrane was effective. Thelong-term rise in residual current was believed to be due toslow contamination of the electrolyte or the membrane.

298 1963

VITAMIN B12 DERIVATIVES AND THIOL OXIDATIONSThere were some indications that prolonged exposure todithionite solution could produce this effect and care wastherefore taken to clean out the apparatus as soon as pos-sible after determination of the lower calibration point.

Operation. For measurements of thiol oxidation the pro-cedure was as follows, except for the modifications indicatedin individual experiments. The apparatus was switched onat least 30 min. before it was required. Meanwhile 0 1M-phosphate buffer, pH 7-1 (KHgP04-KOH mixture), wasequilibratedwith airat 370 by shaking inashallowlayer. Theapparatus was calibrated and 7x7 ml. of buffer was placedin the reaction chamber. With the plunger lowered and thestirrer running, the apparatus was left until the galvan-ometer became steady as thermal equilibrium was attained(3-5 min.). The plunger was then raised and the stirrerstopped temporarily while 0 3 ml. of 0 8M-2-mercapto-ethanol was introduced. When the plunger was loweredagain all air was displaced from beneath. Readings weretaken over the next 5 min., by which time a slow blank rateof oxygen uptake was established. The sample of vitaminB12 derivative, in a volume not exceeding 0 1 ml., was nextintroduced through the vertical groove to start the reaction.Galvanometer readings were taken at suitable intervals(usually every 10 sec.) for the next 3 min. or until theoxygen concentration fell to zero.The vitamin B12 derivative was added from a 0-1 ml.

pipette, the end of which was extended by a short length offlexible nylon tube (1 mm. bore) (catalogue no. 3A; Port-land Plastics Ltd., Hythe, Kent) which was inserted throughthe vertical groove. In preliminary experiments the samplewas added from a micrometer syringe but this method wasabandoned after a period of erratic results that were laterattributed to interference by metal ions derived from newsyringe needles. In calculating the final concentration ofsubstances in the reaction mixture it has been assumed forconvenience that they become distributed uniformlythroughout a reaction volume of 8-0 ml. When substancesother than those mentioned above were present, they wereadded in small volume with the buffer and the volume ofbuffer was decreased accordingly.The changes in oxygen concentration in the experiments

described below were more rapid than those previouslymeasured with the Clark type of electrode (e.g. by Bellamy& Bartley, 1960). With the present apparatus, the maxi-mum rate that could be accurately followed was set by theability of the operator to follow the galvanometer and wasabout 5pl. of 02/ml./min. (12 cm. change in galvanometerreading/min.). The speed of response of the combinedelectrode was not a limiting factor, since the linear depen-dence between the rate of change in galvanometer readingand catalyst concentration held up to the above maximum(Fig. 5). Further, on adding excess of Factor B as catalyst,a rate of approx. 40 cm./min. was obtained and this limitappeared to be set by the inertia of the galvanometer.

RESULTS

Manometric measurement of the oxidation of2-mercaptoethanol

When 2-mercaptoethanol was incubated in air at37° in phosphate buffer, pH 7 0, an uptake ofbetween 30 and 60 ,ul./hr. was observed. When35 ,umm-Factor B was added to the system there was

amuch more rapid uptake ofgas (Fig. 2). The actionof the Factor B is clearly catalytic and in someexperiments a significant stimulation was observedwith concentrations as low as 1 emmx. Cyanocobal-amin and hydroxocobalamin also stimulated thereaction, though much larger concentrations (5 and0-5 ,uM) were required to produce a comparable rate(Fig. 2). The progress curves were not linear andthere were indications that this was due to un-known reactions of the vitamin B12 derivatives withmercaptoethanol which altered their catalyticactivity. Light appeared to be unimportant.The gas uptake was believed to be due to the

oxidation of the mercaptoethanol to the disulphideform as follows:

4HO*CEH2CH2*SH+O2->2HO*CH2*CH2*S*S-CH2*CH2*OH+ 2H20

This was checked by following the reaction with50 mM-mercaptoethanol and 35 ,umM-FactorB untilthe oxygen uptake had almost ceased. The totaloxygen uptake at this point was only 85% of thatrequired for complete oxidation of the thiol initi-ally added, but iodometric measurements showedthat some thiol remained (Table 1). When theresidual thiol was taken into account, the observedoxygen uptake was 96% of that expected from theamount of thiol disappearing.

600

- 400

X 300

aAi

100

0I 10 20 30

Time (min.)

Fig. 2. Effect ofvitamin B12 derivatives on the oxidation of2-mercaptoethanol, measured manometrically. Mano-meters contained, in 2-0 ml.: 0 22M-phosphate buffer,pH 7 0 (KH1PO4-NaOH mixture), 0 2M-mercaptoethanoland vitamin B12 derivative as indicated. The last-namedwas initially placed in the side arm in 0-1 ml. of 0-2m-phosphate buffer and tipped after 20-30 min. of equilibra-tion. The temperature was 37°; the gas phase was air.0, No vitamin B12 derivative; 0, 35jumM-Factor B; A,0 5,um-hydroxocobalamin; A, 5jm-cyanocobalamin.

Vol. 88 299

t

J. L. PEEL

Polarographic mea8urement of the oxidation of thiolsDecrease in oxygen concentration in the presence of

mercaptoethanol and vitamin B12 derivatives. Forfurther study of thiol oxidation, the polarographicmethod proved to be more suitable than man-ometry. The reaction could be started by introduc-ing the vitamin B12 derivative into the reactionvessel, adequate measurements could be obtainedwithin the ensuing 3 min. and lower thiol concen-trations could be used. These factors were expectedto diminish any effects due to modification of thecatalyst. Progress curves with 30 mm-mercapto-ethanol and Factor B, hydroxocobalamin or cyano-cobalamin as catalyst are given in Fig. 3. In eachcase the oxygen concentration fell linearly withtime until it reached about one-fifth of the initialvalue, after which the rate decreased progressivelyas zero oxygen concentration was approached. Thefinal concentration corresponded to an oxygenpressure of less than 2 mm. Hg. A slow but con-sistent consumption of oxygen was observed in theabsence of any vitamin B12 derivative. With a fewexceptions, specifically mentioned, all of the cata-lytic substances investigated gave similar progresscurves. Rates were measured over the linear phaseand duplicate measurements agreed within 5 %.

Effect of pH. The effect of pH on the rate of thereaction was investigated within the pH range 5-9by using citrate-phosphate, phosphate and pyro-phosphate buffers. At pH less than 6 the rate wasvery low, but at pH greater than 6-5 it increasedrapidly, reaching a plateau at pH 8-0 (Fig. 4). ThispH-dependence does not reflect titration of themercaptoethanol as the determined pK for thiscompound was about pH 9-5. The maximum ratemay depend on the nature of the buffer since withglycine-sodium hydroxide buffer at pH 8-7 the ratewas about half of that with pyrophosphate. Theblank rate in the absence of catalyst rose markedlytowards pH 9, making it impossible to investigatethe rate with catalyst at higher pH values. Sub-sequent rate measurements were made at pH 7-1

Table 1. Stoicheiometry of the oxidation of2-mercaptoethanot

Experimental conditions were as for Fig. 2 except thatthe indicated amount of mercaptoethanol was added as afreshly standardized solution immediately before assemblingthe manometers. The catalyst was 35,umM-Factor B. Thereaction time was 200 min.

Mercaptoethanol (initial)Mercaptoethanol (final)Mercaptoethanol consumed (a)Theoretical 02 uptake (a/4)Observed °2 uptake

Amounts (jmoles)

With WithoutFactor B Factor B110-4 110-412-0 85-598-4 24-924-6 6-223-5 5-8

8- 12

"en88

400 0 1 2 3 4 5

Time (min.)

Fig. 3. Effect ofvitamin B12 derivatives on the oxidation of2-mercaptoethanol, measured polarographically. Thereaction mixture initially contained, in 8-0 ml.: 95 mm-phosphate buffer, pH 7-1 (KH2PO4-KOH mixture), and30 mM-mercaptoethanol. The vitamin B12 derivatives, in0-1 ml. of 0 2M-phosphate buffer, were added at zero time.Oxygen concentration is expressed in terms of the cor-responding galvanometer deflexion (corrected for residualcurrent) to which it is directly proportional. 1 cm. _0 106 AA=_ 0 41 ,u. of 02/ml., except with COSO4 where1 cm.- 0106 uA _ 0-48M1u. of 02/ml. Other details aregiven in the Materials and Methods section. 0, No vitaminB12 derivative; 0, 9 MmM-Factor B; A, 2-8pM-hydroxo-cobalamin; A, 30 uM-cyanocobalamin; O, 10 ,M-COSO4.For clarity, the curves for hydroxocobalamin, cyano-cobalamin and COSO4 have been displaced 0 5, 1 and 2 min.to the right respectively, and intermediate experimentalpoints on the curves have been omitted.

1.

m 6't--0o) a

8 4o -4

0

- o

05 6 7

pH8 9

Fig. 4. Effect of pH on the oxidation of 2-mercapto-ethanol. Details were as for Fig. 3 except that 8-0 ml. con-tained 7-7 ml. of buffer at the pH indicated. The catalystwas approx. 5 /LmM-Factor B. The buffers were: 0, 50 mM-citric acid-0 1M-Na2HPO4 mixtures; A, 0-l M-phosphate(KH2PO4-KOH mixtures); [1, 0-1M-pyrophosphate(Na2H2P207-NaOH mixtures); *, A and * indicate thecorresponding blank rates in the absence of Factor B. Therates with Factor B have been corrected by subtraction ofthese blank values. The oxidation rate is expressed as therate of decrease in galvanometer deflexion in cm./min.1 cm. _ 0-481u. of 02/ml.

300 ] 963

VITAMIN B12 DERIVATIVES AND THIOL OXIDATIONSin phosphate buffer. This choice is a compromise:the oxidation rate is only about half the maximum,but the blank rate in the absence of catalyst is smallat this pH.

Effect of temperature. With Factor B as catalystthe rates of oxygen consumption at 250 and 300were 27 and 52 % respectively of that at 37°.

Effect of mercaptoethanol concentration. The rela-tionship between rate and thiol concentration up to150 mm showed a saturation effect similar to thatwith enzyme-catalysed reactions, the concentra-tion of the thiol giving half-maximal velocity being30 mm. This concentration was chosen for routinemeasurements on the same grounds as those used inselecting pH. The blank rate without catalyst wasdirectly proportional to thiol concentration(0.55 cm./min. _ approx. 2,u. of oxygen/min. at150 mM).

Effect of catalyst concentration. With Factor B, therate was directly proportional to the amount ofcatalyst present up to the maximum rate that couldbe accurately followed with the apparatus (Fig. 5).A similar relationship was observed with cyano-cobalamin and hydroxocobalamin. Measurementsfor the other vitamin B12 derivatives were limited,but each derivative was tested at more than oneconcentration and gave results consistent with alinear relationship.

Reaction with other thiol8. A number of otherthiols were compared with mercaptoethanol,Factor B being used as catalyst. These tests were

X .=

m

'IO._

2p X

P-4

2,)

.-40 oL

.- e

Ca4

0 005Factor B solution added (ml.)

Fig. 5. Effect of Factor B concentration on the oxidation of2-mercaptoethanol. The indicated volumes of approx.1 8 M-Factor B in 0-2M-potassium phosphate buffer, pH7-1, were added at zero time. Other details were as forFig. 3. The oxidation rate is expressed as in Fig. 4. 1 cm. -

0-48 pl. of 02/ml.

carried out with 30 mM-thiol, except where thisgave inconvenient rates. The reaction was onlyfollowed to completion with 2,3-dimercapto-propanol and the thioctic acid derivatives, but, withthis proviso, all progress curves were similar in formto those with mercaptoethanol. Factor B caused amarked stimulation in all cases, the increase beingseveral times the blank rate in its absence, exceptwith 2,3-dimercaptopropanol (Table 2). Despite re-distillation, this compound gave a considerableblank, and the effect of Factor B with a thiol con-centration of 30 mm could not be tested. With thio-glycollate, cysteine or glutathione at a concentra-tion of 30 mm, the rate due to Factor B was con-siderably less than with mercaptoethanol. The re-duced forms of 6-thioctic acid and of its amide weremuch more active than mercaptoethanol, concen-trations of 2-5 and 1F25 mm respectively giving thesame rate as 30 mM-mercaptoethanol. One non-thiol, namely ascorbic acid, was tested for com-parison, and no catalysis by Factor B was detect-able, although a rapid oxidation resulted on addingCu2+ ions (cf. Meiklejohn & Stewart, 1941). Mer-captoethanol appeared to be the best thiol forgeneral use, as the thioctic acid derivatives, al-though more active, were only available in limitedamounts and are not readily soluble in water.

Activities of other vitamin B12 derivatiVes. A num-ber of vitamin B12 derivatives were tested withmercaptoethanol. All proved to be active and withone exception gave progress curves of similar formto that with Factor B. The exception was Factor A,with which the rate increased progressively duringthe first half of the reaction. The maximum ratewas used to calculate the activity of this compound.Since, in all cases examined, the rate was directlyproportional to catalyst concentration, activitiesare expressed as ,ul. of oxygen uptake/min./,um-mole of catalyst and are given in Table 3, togetherwith structural details.Of the compounds tested, the least active was the

dimethylbenzimidazolylcobamide coenzyme (no. 2in Table 3) with one-third of the activity of cyano-cobalamin (no. 1). In these two compounds, 5'-deoxyadenosine and cyanide respectively are linkedto the cobalt atom; hydroxocobalamin (no. 3),which lacks these substituents, was ten times asactive as cyanocobalamin. Analogues of cyano-cobalamin with modifications in the short sidechains attached to the corrin nucleus (nos. 4-10) orin the base of the nucleotide moiety (no. 11) wereup to 50 times as active. Factor B (no. 12), whichdiffers from cyanocobalamin in containing nonucleotide, was approx. 4000 times as active (thevalue in Table 3 is a revision of that quoted by Peel,1962 b). Factor D (no. 13) was the only otherderivative tested that had an activity approachingthat of Factor B.

Vol. 88 301

Table 2. Rate8 of oxidation with different thiolsReaction mixtures were as for Fig. 3 except that sufficient thiol to give the concentration indicated was added in

a volume not exceeding 1X5 ml. and the volume of phosphate buffer adjusted to maintain a combined volume of8-0 ml. The Table summarizes the results of four experiments in which different thiols were compared withmercaptoethanol and in which different concentrations of Factor B (between 5 and 9 ,umM) were used. Oxidationrates are expressed as in Fig. 4. 1 cm. _ 0*48 pA. of 02/min. (Expt. 1) or 043 pJ. of O./min. (Expts. 2-4). 'Relativecorrected rate' is the oxidation rate with Factor B, corrected by subtraction of the blank without Factor B, andexpressed as a percentage of the corrected rate for mercaptoethanol in the same experiment.

Expt.no. Thiol1l2 2-Mercaptoethanol41 2,3-Dimercaptopropanol

1 Thioglycollic acid

1 Cysteine2 Glutathione3 Dihydro-6-thioctic acid

4 Dihydro-6-thioctic acid amide

4 Ascorbic acid

Oxidation rate (cm./min.)

Concn. Without(mM) Factor B30 0.1030 0.1030 0.1030 0.1530 2-2010 1-05

150 0 4030 0 0530 0-1530 0 305 0-202-5 0.102-5 0-201-25 0.15

30 0-20

Table 3. Catalytic activity of vitamin B12 derivatives in oxidation of 2-mercaptoethanolAll samples were tested under the conditions of Fig. 3. For acid treatment, samples in 0-6 ml. of 1-9N-HCI were

heated at 650 for 30 min., then added to 3-5 ml. of 70 mm-phosphate buffer, pH 7-1, containing NaOH equivalentto the HCI and cooled, and suitable portions tested. The catalyst concentrations tested were such as to give anoxidation rate of at least 3 0 cm./min. except with the untreated lactone, monobasic acids and dibasic acids wherethe rates were from 0-95 to 2-15 cm./min. The activities after acid treatment are calculated per jum-mole of theoriginal untreated derivative. Oxygen uptakes are uncorrected for the effect of the solutes on the solubility ofoxygen. Fuller details of the structure of the coenzyme form are given by Lenhert & Hodgkin (1961) and of theother derivatives by Smith (1960). Activity (p1. of 02 uptake/

min./jom-mole of catalyst)

DerivativeCyanocobalamin (vitamin B12)DimethylbenzimidazolylcobamidecoenzymeHydroxocobalaminMethylamide

Anilide

Lactam

LactoneMonobasic acids

Dibasic acids

Tribasic acids

Factor A

Factor BFactor D

Structural relationship tocyanocobalamin

CN group replaced by 5'-deoxy-adenosineCN group replaced by -OHOne amide group of side chainsmethylatedOne amide group of side chainsphenylated

Acetamide side chain c convertedinto lactam ring fused to ring B

Lactone corresponding to lactamMixture of derivatives with one NH2group lost from side chains

Mixture of derivatives with twoNH, groups lost from side chains

Mixture of derivatives with threeNH2 groups lost from side chains5,6-Dimethylbenzimidazole replacedby 2-methyladenine

Nucleotide absenttStructure unknown

Untreated0-0810-027

0-793.440

0-62

0-571-100-94

3-0

3.4*

3172001

* Maximum rate (see text) t Sample used also contained dicyanocobinamide (see text).$ Based on an assumed molecular weight of 1000.

After acidtreatment

12960

65107

100

198

5992

34

50

169

93

WithFactor B

3-55l6-60t6-00)5-75J

2-952-551.100*851.05

10-86-209.76-000-20

Relativecorrected

rate

100

52623020121801031701040

Compoundno.(1)(2)

(3)(4)

(5)

(6)

(7)(8)

(9)

(10)

(11)

(12)(13)

302 1963J. L. PEEL

VITAMIN Bn DERIVATIVES AND THIOL OXIDATIONS

Some uncertainty exists about the preciseactivities ofthese last two derivatives. The constitu-tion ofFactor D is unknown and the activity quotedis based on an assumed molecular weight of 1000.The Factor B preparation was a mixture of mono-cyano- and dicyano-cobinamide, and the activitiesof the individual forms were not determined.Previous manometric tests with a preparation con-taining only the monocyano form showed it to behighly active (Peel, 1962a). Dicyanocobinamidemay be inactive (see below), but it is neverthelessevident that removal of the nucleotide moiety fromthe cyanocobalamin molecule gives an enormousincrease in catalytic activity. In consequence, thepossibility cannot be rigidly excluded that theactivities observed with derivatives nos. 1-11 mightbe partly or wholly due to cobinamide impurities, asthe highest activity (no. 5) would only requireapprox. 1% of such impurity. This possibility isregarded as unlikely with cyanocobalamin andhydroxocobalamin; these materials were crystal-line, they were tested more extensively than theother derivatives and fresh solutions showed nosignificant increase in activity over several hours.

Effect of acid treatment. When cyanocobalamin iswarmed briefly with strong acid, the nucleotide islost and Factor B is one of the main products(Smith, 1960). Since Factor B has a much highercatalytic activity than cyanocobalamin, it wasdecided to see whether a simple acid treatmentcould be used to enhance the activity of othervitamin B12 derivatives. When cyanocobalamin washeated with hydrochloric acid at 650, maximumactivity was reached after 20 min. It was only 40%of that expected from an equivalent amount ofFactor B (Table 3), possibly owing to the forma-tion of less active derivatives by side reactions.A standard treatment of 30 min. was adopted andin all cases except for Factor D a large increase inactivity was observed (Table 3). The final activitieswere not identical but the range after acid treat-ment (approx. sixfold) is very much smaller thanwith the untreated derivatives. The activity ofFactor D was halved by acid treatment and this,coupled with its original high activity, suggeststhat, like Factor B, it may contain no nucleotide.

Effect of cyanide. Since cyanocobalamin is muchless active than hydroxocobalamin and sincecyanide has been extensively used in extractionprocedures for vitamin B12, the effect of added cy-anide on mercaptoethanol oxidation was examined.With 9 ,anm-Factor B as catalyst, cyanide hadvery little effect at concentrations less than 1 ,M; ata concentration of 1 ,M there was a marked inhibi-tion and this became complete at a concentration of0 1 mm (Table 4). Complete inhibition at this con-centration was also observed with cyanocobalamin(18 FAM). These inhibitions are most easily explained

as being due to the formation of inactive dicyanoforms. A considerable excess of cyanide may beneeded to stabilize such derivatives in the testsystem since one possible action of the mercapto-ethanol is a rapid removal of cyanide groups fromcobinamides. With cyanocobalamin, no removal ofcyanide was detectable spectrophotometricallywithin several minutes of adding mercaptoethanol,but a slow conversion into the more active hydroxo-cobalamin could account for the increasing rate ofcatalysis with cyanocobalamin in the manometricexperiments (Fig. 2).

Effect of metas8, ethylenediaminetetra-acetate andyeast extract. In view of the ability of heavy-metalions to catalyse the oxidation of thiols (e.g. War-burg, 1949), C02+ ions and a number of other metalions were tested in the present system both forcatalytic activity and for possible interference withthe reaction when Factor B is the catalyst: Mn ,

Cr3+, MoOt2 and Fe3+ ions at a concentration of1 mx were all inert (Table 5); C02+, Ni2+ and Cu2+ions all showed catalytic activity, C02+ ions beingthe most active, the initial rate with 10 jAM cor-responding to an activity of 0 35 pl. of oxygen/min./lamg. ion, which is considerably higher than that ofcyanocobalamin. With this concentration of C02+ion, however, the rate fell off sharply as the reactionprogressed (Fig. 3), and at the same time the reactionmixture turned yellow-brown. This fall in rate wasdue to loss of catalyst rather than to the fall inoxygen concentration. This became evident on re-oxygenating at the end of the reaction by slightlyraising the plunger and increasing the stirring ratefor a briefperiod. The subsequent oxidation rate wasonly about one-tenth of that when the C02+ ion wasfirst added. This rapid fall in activity explains whyno catalysis was detectable with 10 jM-CO2+ ion inmanometric tests (Peel, 1962a). A marked fallingoff of the rate was also observed with 0 1 mM-Cu2+

Table 4. Effect of cyanide on the oxidation of2-mercaptoethanol

Potassium cyanide to give the final concentration indi-cated was added in a volume of 0-08 ml. to the phosphatebuffer. Other details were as for Fig. 3. The oxidation rateis expresed as in Fig. 4 and corrected by subtraction ofblank rate in the absence of added catalyst (0.1 cm./min.).1 cm. - 0-361.d. of 02rml. VntI

CatalystFactor B (9 j.mM)

Cyanocobalamin (18 jAM)

Concn. ofcyanide

(M)

10-810-7

10-6

10-510-4

10-4

%w4rru v&uoxidation

rate(cm./min.)

6-16-56-34-20-6502-90

303Vol. 88

Table 5. Effect of metal ions, ethylenediaminetetra-acetate and yeast extract on the oxidationof 2-mercaptoethanol

The metal ions were added as MnSO4, CrK(SO4)2, (NH4),Mo7024, Fe2(S04)., CUS04, NiSO4 and CoS04 respec-tively. In Expt. 1, metal ions and extract of boiled yeast were introduced in a volume of 0-08 ml. in the samemanner as the vitamin B12 derivatives. EDTA was added with the phosphate buffer; likewise the yeast extractsin Expt. 2. When Factor B and metal ions or yeast extracts were both present, the Factor B (final concn. approx.9 ,umM) was added last in a volume of 0-04 ml. Concentrations (M) of metal ions in the Table and in the text arecalculated as g.ions/l. Other details were as for Fig. 3; oxidation rates are expressed as in Fig. 4. 1 cm. - 0-48(Expt. 1) or 0 45 (Expt. 2) 1l. of 02/ml.

Rate of oxidation (cm./min.)

Expt.no.1

AdditionsNone

Mn2+ ionCrs+ ionMoO4 ionFe3+ ionCu2+ ion

Ni2+ ion

Co2+ ion

Extract ofboiled yeast

2 NoneExtract ofboiled yeast

Difco yeastextract

Conen.(M)

10-310-3i-3

10-3

10-4

10-5

10-610-5

1 mM-EDTA

+

+

+

+

+0 3 mg./ml.

+

6-3 mg./ml.21 mg./ml.

±2-1 mg./ml. -

+

No Factor B With(a)0-10-10-10-10-10-10-50-51.0*0-80-50-054-60-10-557-2*0-10-350-10-1030-551-650-20-1

Factor B(b) (b - a)6-1 6-06-0 5.96-1 6-06-0 5.95-7 5-65-7 5-66-1 5-6

8-0 7-55-8 5-75

6-4 5-85

6-2 5:85

6-5 6-46-1 5-85-5 4.956-0 4-351-2 1.01-2 1.1

* Initial rate; the rate fell progressively with time.

ion, but not with Ni2+ ion although a red-browncolour developed with the latter. At concentrationsgiving low catalytic rates (10 pM for Cu2+ and Ni2+ions; 1 ,zM for C02+ ion), Cu2+ and C02+ ions had verylittle influence on the rate due to Factor B [(b -a)in Table 5], but Ni2+ ion caused a significantenhancement.EDTA (1 mm) completely reversed all the effects

observed with Ni2+ and Co2+ ions but barely in-hibited the catalysis with Cu2+ ions. It did notaffect the reaction with Factor B. The effect ofmetal-ion-binding agents on the blank oxidationwith mercaptoethanol in the absence of addedcatalyst was also investigated, 0-15M-mercapto-ethanol being used so as to give a more easily mea-sured rate. Little if any effect was observed with10 mM-EDTA, 1 mM-sodium azide, 1 mM-1,10-phenanthroline or 0-1 mM-potassium cyanide.To gain some indication whether other biological

materials affect mercaptoethanol oxidation, a boiledextract of baker's yeast was used, as this organism

contains almost no vitamin B12 (Jukes & Williams,1954). When tested in the same way as the metalions, 0-3 mg./ml. gave a slight catalysis that wasabolished by 1 mm-EDTA and did not interferewith the catalysis by Factor B (Table 5: Expt. 1).Higher concentrations were tested by addingfreeze-dried material to the buffer. These gave littlefurther increase in the blank rate (a); the chiefeffect was an inhibition of the rate due to Factor Bwhich became marked at 21 mg./ml. (Table 5:Expt. 2). Difco yeast extract inhibited much more(80% at 2-1 mg./ml.). EDTA caused an unexpectedincrease in the blank rate with the highest concen-tration of boiled extract, but did not relieve theinhibitions. The blank rate may be due, in part, tometalloporphyrin derivatives, since haematincatalyses the oxidation of cysteine (Harrison, 1924;Krebs, 1929). In preliminary experiments with theauthor's system, haematin catalysed mercapto-ethanol oxidation with an activity similar to that ofcyanocobalamin.

304 J. L. PEEL 1963

VITAMIN B12 DERIVATIVES AND THIOL OXIDATIONS

Spectral studiesWhen cyanocobalamin or hydroxocobalamin

solutions are allowed to stand with thioglycollate atalkaline pH, the colour changes from pink toorange-brown, but on subsequent shaking the pinkcolour is instantly restored. This process can berepeated indefinitely, and Smith (1960) pointed outthat under these conditions the cobalamin catalysesthe atmospheric oxidation of the thioglycollate. Theorange-brown derivative is almost certainly vita-min B1fr, a compound first produced by the reduc-tion of cyanocobalamin with hydrogen (Diehl &Murie, 1952). Beaven & Johnson (1955) also statethat a compound with the same spectrum is pro-

duced when cyanocobalamin is treated with cysteineat alkaline pH. The structure of vitamin B12 is un-known but it is rapidly oxidized by air to givehydroxocobalamin and there is evidence that itcontains bivalent cobalt as opposed to the tri-valent cobalt in cyanocobalamin. The alternateformation and oxidation of vitamin B12r could thusaccount for the catalysis of thioglycollate oxidationby cyanocobalamin andhydroxocobalamin. A simi-lar mechanism appeared possible in mercapto-

20

300 400 500Wavelength (m,)

Fig. 6. Effect of 2-mercaptoethanol on the spectrum ofhydroxocobalamin. Spectrophotometer cells of 1 cm. light-path contained initially, in 2-9 ml.: 100 /umoles ofpotassiumphosphate buffer, pH 7-1, or 100l moles of Na2CO3 (giving afinal pH of 11), and 0.1 tmole ofhydroxocobalamin; 0- 1 ml.of 0-2M-mercaptoethanol was added at zero time. Measure-ments were made at room temperature (23-25°). Referencecells contained 33 mM-phosphate buffer or 33 mM-Na2CO3.Curves are continuous traces from the Optica recordingspectrophotometer: , at pH 7-1 before adding mer-

captoethanol; - - - -, at pH 7-1, 20 min. after addingmercaptoethanol;. , at pH 11, 15 min. after addingmercaptoethanol. Times are those after which recording ofthe spectrum began; the time required to record eachspectrum was approximately 6 min.

20

ethanol oxidation and spectral evidence for areduced catalytic intermediate was thereforesought. A marked change in the spectra of Factor Band of hydroxocobalamin after a few minutes withmercaptoethanol at neutral pH had been noted atan early stage in the investigation. Hydroxo-cobalamin was selected for more detailed study as itwas more easily available, the material was crystal-line and what little information is available on thereduction of vitamin B12 derivatives refers tocobalamins. Cyanocobalamin was not used becauseof its low catalytic activity and because it does notgive detectable spectral changes in a short time atneutral pH.The situation at neutrality proved to be more

complicated than was anticipated and a simplepicture only emerged under alkaline conditions,where colour changes similar to those with thio-glycollate were observed. The colour change frompink to orange-brown was accompanied by the dis-appearance of the absorption peak of hydroxo-cobalamin in the 350-360 m,u region. At pH 11 thismaximum lies at 360 m,u and the progress of thereaction was followed by extinction measurementsat this wavelength. The reaction was complete after15 min. and the spectrum of the product (Fig. 6)was almost identical with that of vitamin B12r, thewavelengths ofmaximum absorption being 313, 405and 475 m,, in close agreement with the values of312-5, 405 and 473 given by Diehl & Murie (1952).On shaking with air, the original pink colour wastemporarily restored and these colour changescould be repeated indefinitely. Vitamin B12 thusappears to be a catalytic intermediate in the oxid-ation of mercaptoethanol under these conditions.

It was initially believed that similar processesoccur at neutral pH (Peel, 1962a) on the basis ofpreliminary spectral data with hydroxocobalaminand Factor B. For technical reasons, observationsat that time were limited to the region above340 m,u, and more detailed experiments did notsupport the previous conclusions. First, a markedchange in colour, to violet, was noted immediatelyon adding the mercaptoethanol. This was followedby a slow change to a yellow-brown over a period ofseveral minutes and this colour remained un-changed on shaking with air, even when limitingamounts of thiol were used. In addition, a furtherslow change to yellow-green became apparent afterabout 1 hr. Secondly, when the spectrophotometricexamination was extended to lower wavelengths itbecame clear that the yellow-brown product wasnot vitamin B12,. The progress of the reaction wasfollowed at 353 m,u, this being the maximum for thenear ultraviolet peak of hydroxocobalamin at pH7-1. After 20 min., the initial rapid fall in extinc-tion had ceased but a slow drift was still detectable;scanning of the spectrum was begun at this point.

Bioch. 1963, 88

Vol. 88 305

J. L. PEEL

The large peak at 313 m,u characteristic of vitaminB12r was absent, although there was a smallshoulder at this wavelength. Neither did thespectrum show any resemblance to the reducedcobalamin derivative of Boos, Carr & Conn (1953)which is produced by treating cyanocobalaminwith an excess of chromous derivatives and hasabsorption maxima at 385, 460 and 554 m,.

These experiments show that the action of mer-captoethanol on hydroxocobalamin at neutral pH iscomplex and is not limited to the formation andreoxidation of a reduced derivative such as vitaminB12r. The mechanism of catalysis at neutral pHthus remains obscure.

DISCUSSION

The quantitative findings presented above showthat the catalytic effect of vitamin B12 derivativeson thiol oxidations can be very potent and permitsome assessment of the possible implications andapplications of this activity. The magnitude of thereaction in a given situation is greatly influenced bysuch factors as pH and the nature of the thiol andvitamin B12 derivative, but it is evident that signifi-cant effects may be expected under conditionslikely to be encountered experimentally. Thiscatalysis was observed with all the thiols so fartested; most of these are frequent components ofincubation mixtures and some are of natural occur-rence, including the thioctic acid derivatives whichwere by far the most readily oxidized thiols.Present indications are that the predominantnatural forms of vitamin B12 in living cells are thecoenzymes and other cobamides containing anucleotide (e.g. Volcani, Toohey & Barker, 1961;Toohey & Barker, 1961); in general, these com-pounds are relatively poor catalysts of thiol oxid-ation. The much more active cobinamides are,however, known to occur naturally, e.g. in faecesand in rumen contents (Ford & Porter, 1953), andthey may also arise as metabolic intermediates or asimpurities in cobamide preparations.

Three consequences of non-enzymic interactionsbetween thiols and vitanin B12 derivatives mayaffect the interpretation of experimental observa-tions obtained in vitro. First, catalytic oxidationmay lead to an unsuspected removal of reducedthiol. This might perhaps be of importance wheresmall amounts of thiol are commonly used, e.g. withthioctic acid or with other thiols not so far testedsuch as CoA or proteins. Secondly, such reactionsmay result in a rapid removal of dissolved oxygenfrom solution, with accompanying effects onenzyme systems. Thus the requirement for vitaminB12 derivatives in the C02-pyruvate exchangereaction observed with bacterial preparations inthe presence ofmercaptoethanol (Rabinowitz, 1960;

Rabinowitz & Allen, 1961) can be best explained asthe protection of a labile enzyme by the removal ofdissolved oxygen, rather than as a true coenzymerequirement (Peel, 1962a). Thirdly, the action ofthiols, in catalytic oxidation processes or otherwise,may convert vitamin B12 derivatives into productshaving different biological activities.The physiological significance of thiol oxidations

catalysed by vitamin B12 derivatives is more diffi-cult to assess. These reactions bear no strict parallelwith any of the known enzymic reactions requiringvitamin B12 coenzymes, and the one coenzymetested in the non-enzymic thiol oxidation had verylow activity. Catalytic mechanisms involving theoxidation and reduction of cobamides have beenproposed for the succinyl-CoA-methylmalonyl-CoAinterconversion (Eggerer, Stadtman, Overath &Lynen, 1960), the glycol-aldehyde interconversions(Brownstein & Abeles, 1961) and the methylation ofhomocysteine (Guest, Friedman, Woods & Smith,1962), and it is possible that thiol groups might beinvolved in these oxido-reductions. The formationofdisulphide linkages in proteins is a physiologicallyimportant thiol oxidation in which vitamin B12derivatives might play a part, either enzymically ornon-enzymically. On the other hand, the availableevidence with intact organisms suggests that vita-min B12 promotes the reduction of thiols (Smith,1960). For example, Dubnoff & Bartron (1956)found that the concentration of protein thiolgroups in washed cells of the Escherichia colimutant 113-3 fell after aging but was restored onincubating with vitamin B12, especially in thepresence of glutathione. A possible explanation ofsuch effects is that vitamin B12 derivatives maycatalyse oxidation-reduction reactions betweendifferent thiols or the reduction of thiols by otherhydrogen donors.The susceptibility to oxygen ofthe C02-pyruvate

exchange system from P. el8denii appears similar tothat reported for cytochrome c reductase (Dixon,Maynard & Morrow, 1960) and yeast lactate de-hydrogenase (Armstrong, Coates & Morton, 1960),in that inactivation is rapid and irreversible (Peel,1962a; and unpublished work). The effectiveness ofmercaptoethanol together with vitamin B12 deriva-tives in maintaining the activity of the C02-pyruvate exchange system suggests that this com-bination of reagents may prove of wider use inhandling other enzymes showing this type ofsensitivity to oxygen.A further use of these reagents as 'oxygen

scavengers' is in the culture and handling ofstrictly anaerobic micro-organisms. The addition ofmercaptoethanol (4 mm) and Factor B (1#lM) toculture media a few minutes before inoculation hasalready been successfully used to establish andmaintain anaerobiosis in nutritional studies with

306 1963

Vol. 88 VITAMIN B12 DERIVATIVES AND THIOL OXIDATIONS 307P. el8denii involving growth from small inocula(H. J. Somerville and J. L. Peel, unpublished work).This method ofremoving oxygen has the advantagethat the thiol remains relatively stable until thevitamin B12 derivative is added but that thereafteroxygen is removed very rapidly.The polarographic method permits a convenient

and reasonably precise measurement of auto-oxidation rates, and the kinetics of mercapto-ethanol oxidation are suitable for a quantitativedetermination of vitamin B12 derivatives. A deter-mination based on this principle would be highlysensitive, at least with cobinamides. Thus a rate ofabout 1 cm./min. is given by 10 jpmg. of Factor B,and with a smaller reaction chamber the sensitivitycould be brought close to that of microbiologicalassays where 0.1-05B,umg. is a common range. Thesensitivity with cobalamins and other commoncobamides would be less but could be increasedconsiderably by prior treatment with acid. Theindividual vitamin B12 derivatives have differentactivities in thiol oxidation, even after acid treat-ment, but the same is true of other assays and dif-ferences in activity are in themselves useful instudying interconversions. The mercaptoethanol-oxidation method would have an important advan-tage in speed over microbiological assays and wouldbe applicable to a wider range of vitamin B12derivatives. For instance, it could be used withFactor D, which is devoid of biological activityapart from antagonism in the chick assay (Smith,1960). On present evidence, the most serious inter-fering substances are likely to be cyanide and anunidentified inhibitor present in yeast extracts. Theeffects of acid treatment, cyanide and EDTA areproperties that might be useful in distinguishingcatalysis by vitamin B12 derivatives from spuriousreactions. Where the proportion of interfering sub-stances is low, a method of determination ofvitamin B12 derivatives would thus appear bothfeasible and a useful addition to existing assayprocedures.

SUMMARY

1. At neutral pH, vitamin B12 derivatives cata-lyse the auto-oxidation of 2-mercaptoethanol to thedisulphide form. The effect can be readily demon-stratedmanometricallybutthismethoddoesnotgivereliable rate measurements because of side reactions.

2. A polarographic apparatus suitable for mea-suring changes in the partial pressure of oxygen ofafew minutes' duration is described and has beenused to study mercaptoethanol oxidation.

3. At pH 7-1 and with all vitamin B12 derivativestested except Factor A, the fall in oxygen concen-tration is linear with time for the greater part of thereaction. The final oxygen pressure reached is lessthan 2 mm. Hg.

4. With Factor B as catalyst, the oxidation rateincreases with pH from nearly zero at pH 6x0 to amaximum at pH 8-0, and increases approximatelyfourfold between 250 and 370. The rate shows aMichaelis-type dependence on mercaptoethanolconcentration (half-maximum velocity at about30 mM) and is directly proportional to catalystconcentration.

5. At 370 and pH 7-1 with Factor B as catalyst,activity with different thiols increased in the order:glutathione; cysteine; thioglycollate; 2-mercapto-ethanol; 2,3-dimercaptopropanol; dihydro-6-thi-octic acid; dihydro-6-thioctic acid amide.

6. With mercaptoethanol, the approximatemolar catalytic activities of different vitamin B12derivatives relative to cyanocobalamin (1 0) are:dimethylbenzimidazolylcobamide coenzyme, 0 3;hydroxocobalamin, 10; sundry cobalamin ana-logues, 7-50; Factor B, 4000; Factor D, 2500.Activities of all the cobamide derivatives are greatlyenhanced by brief heating with acid.

7. Co2+, Ni2+ and Cu2+ ions catalyse the oxid-ation of mercaptoethanol; yeast extract has slightcatalytic activity. EDTA (1 mm) completelyabolishes the effect of C02+ or Ni2+ ions, but notthat ofCu2+ ion. Catalysis by FactorB is unaffectedby 1 mm-EDTA, but is completely inhibited by0-1 mM-potassium cyanide. Large concentrationsof yeast extract interfere with the reaction.

8. Spectral studies with hydroxocobalamin andmercaptoethanol indicate that vitamin B12, is acatalytic intermediate in thiol oxidation at pH 11.At neutrality the situation is obscured by sidereactions.

9. The implications ofthese reactions in vitro andin vivo and their possible use for establishinganaerobic conditions and in the determination ofvitamin B12 derivatives are discussed.

I am grateful for the following help: gifts of materialsfrom the donors specified in the text, advice on chemicalmatters (Dr E. Lester Smith and Dr J. W. G. Porter) andon polarography (Dr J. D. Jones), technical assistance(Mrs C. Drabble and Mr R. Bacon), and partial support ofthe work (Rockefeller Foundation).

REFERENCES

Armstrong, J. McD., Coates, J. H. & Morton, R. K. (1960).Nature, Lond., 186, 1033.

Barker, H. A., Smyth, R. D., Weissbaoh, H., Munch-Petersen, A., Toohey, J. I., Ladd, J. N., Volcani, B. E. &Wilson, R. M. (1960a). J. biol. Chem. 235, 181.

Barker, H. A., Smyth, R. D., Weissbach, H., Toohey, J. I.,Ladd, J. N. & Volcani, B. E. (1960b). J. biol. Chem. 235,480.

Beaven, G. H. & Johnson, E. A. (1955). Nature, Lond., 176,1264.

Bellamy, D. & Bartley, W. (1960). BiooIem. J. 76, 78.20-2

308 J. L. PEEL 1963Boos, R. N., Carr, J. E. & Conn, J. B. (1953). Science, 117,

603.Brown, F. B., Cain, J. C., Gant, D. E., Parker, L. F. J. &

Smith, E. L. (1955). Biochem. J. 59, 82.Brownstein, A. M. & Abeles, R. H. (1961). J. biol. Chem.

236, 1199.Clark, L. C. (1956). Tranm. Amer. Soc. artif. intern. Organs, 2,

41.Connelly, C. M. (1957). Fed. Proc. 16, 681.Diehl, H. & Murie, R. (1952). Iowa St. Coll. J. Sci. 26,

555.Dixon, M., Maynard, J. M. & Morrow, P. F. W. (1960).

Nature, Lond., 186, 1032.Dubnoff, J. W. (1950). Arch. Biochem. 27, 466.Dubnoff, J. W. & Bartron, E. (1956). Arch. Biochem.

Biophys. 62, 86.Eggerer, H., Stadtman, E. R., Overath, P. & Lynen, F.

(1960). Biochem. Z. 333, 1.Ford, J. E. & Porter, J. W. G. (1953). Brit. J. Nutr. 7, 326.Guest, J. R., Friedman, S., Woods, D. D. & Smith, E. L.

(1962). Nature, Lond., 195, 340.Harrison, D. C. (1924). Biochem. J. 18, 1009.

Jukes, T. H. & Williams, W. L. (1954).' In The Vitamin8,vol. 1, p. 476. Ed. by Sebrell, W. H. & Harris, R. S.New York: Academic Press Inc.

Krebs, H. A. (1929). Biochem. Z. 204, 322.Lenhert, P. G. & Hodgkin, D. C. (1961). Nature, Lond., 192,

937.Meiklejohn, G. T. & Stewart, C. P. (1941). Biochem. J. 35,

755.Peel, J. L. (1962a). J. biol. Chem. 237, Pc263.Peel, J. L. (1962b). Biochem. J. 85, 17P.Rabinowitz, J. C. (1960). J. biol. Chem. 235, Pc50.Rabinowitz, J. C. & Allen, E. H. (1961). Fed. Proc. 20,962.Smith, E. L. (1960). Vitamin B12. London: Methuen and

Co. Ltd.Stickland, R. G. (1960). Biochem. J. 77, 636.Toohey, J. I. & Barker, H. A. (1961). J. biol. Chem. 236,

560.Volcani, B. E., Toohey, J. I. & Barker, H. A. (1961). Arch.

Biochem. Biophy8. 92, 381.Warburg, 0. (1949). Heavy Metal Pro8thetic Group8 andEnzyme Action, pp. 34-45. Oxford: Clarendon Press.

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Biochem. J. (1963) 88, 308

The Metabolism of Progesterone in ManEXTRACTION AND SEPARATION BY COUNTERCURRENT DISTRIBUTION

OF THE METABOLITES IN URINE

BY R. A. HARKNESS AND K. FOTHERBY*Clinical Endocrinology Research Unit (Medical Research Council), University of Edinburgh

(Received 11 February 1963)

After the administration of [4-14C]progesteroneor [16-3H]progesterone to humans, 15-61 % of thedose was recovered from the urine and 13-28%from the faeces (Gallagher et al. 1954; Pearhman,1957; Davis & Plotz, 1958; Sandberg & Slaunwhite,1958). The known metabolites of progesterone inurine, 5fl-pregnane-30c,20oc-diol (pregnanediol), 3oc-hydroxy-5f-pregnan-20-one (pregnanolone) andtheir isomers, account for less than one-third of theadministered hormone, so that much of the radio-activity present in urine must reside in compoundsother than the above metabolites, if hydrolysis ofthe conjugates of the above steroids in urine hasbeen complete. In an attempt to identify theseunknown metabolites the metabolism of [4-14C]_progesterone in man has been investigated. Thepresent paper describes a study of the hydrolysisand extraction of the metabolites of [4-14C]_progesterone from the urine of ovariectomized

adrenalectomized women who had received thelabelled hormone, and the preliminary separationof these metabolites by countercurrent distribution.

METHODS

SubjectsThe subjects were five women (aged 41-59 years) who

had been adrenalectomized and ovariectomized for mam-mary carcinoma. All subjects were in hospital during theinvestigation and were ambulant; they were receiving 25-37-5 mg. of cortisone acetate daily, by mouth, as replace-ment therapy. There was no evidence of impairment ofhepatic or renal function. All subjects collected complete24 hr. samples of urine for 9-14 days from the day of injec-tion of [4-14C]progesterone.

Admitni8tration of [4-14C]proge8teroneSubject A.W. received 20,uc (03 mg.) of [4-14C]pro-

gesterone (The Radiochemical Centre, Amersham, Bucks.)intravenously over about 15 min. The steroid was admini-stered by dissolving it in 20 ml. of 0-9% sodium chloride in

* Present address: Postgraduate Medical School,Ducane Road, London, W. 12.