236 J. R. QUAYLE, D. B. KEECH AND G. A. TAYLOR 1961Long, A. G., Quayle, J. R. & Stedman, R. J. (1951).

J. chem. Soc. p. 2197.Nason, A. & Little, H. N. (1955). In Method8 ofEnzymology,

vol. 1, p. 536. Ed. by Colowick, S. P. & Kaplan, N. 0.New York: Academic Press Inc.

Ochoa, S., Mehler, A. H. & Kornberg, A. (1948). J. biol.Chem. 174, 979.

Quayle, J. R. & Keech, D. B. (1959a). Nature, Lond., 183,1794.

Quayle, J. R. & Keech, D. B. (1959b). Biochem. J. 72,623.

Quayle, J. R. & Keech, D. B. (1959c). Biochem. J. 72,631.

Quayle, J. R. & Keech, D. B. (1960). Biochem. J. 75,515.

Quayle, J. R., Keech, D. B. & Taylor, G. A. T. (1960).Bi6chem. J. 76, 3P.

Sakami, A. B. (1955 a). Handbook of Isotope TracerMethods, p. 5. Ohio: Department of Biochemistry,Western Reserve University, Cleveland.

Sakami, A. B. (1955 b). Handbook ofIsotope Tracer Methods,p. 8. Ohio: Department of Biochemistry, WesternReserve University, Cleveland.

Sakami, A. B. (1955c). HandbookofIsotope Tracer Methods,p. 55. Ohio: Department of Biochemistry, WesternReserve University, Cleveland.

Shimazono, H. & Hayaishi, 0. (1957). J. biol. Chem. 227,151.

Simon, E. J. & Shemin, D. (1953). J. Amer. chem. Soc. 75,2520.

Stadtman, E. R. (1953). J. biol. Chem. 203, 501.Stadtman, E. R. (1957). In Methods in Enzymology, vol. 3,

p. 938. Ed. by Colowick, S. P. & Kaplan, N. 0. NewYork: Academic Press Inc.

Stadtman, E. R. & Barker, H. A. (1950). J. biol. Chem. 184,769.

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Wolfe, J. B. & Rittenberg, S. C. (1954). J. biol. Chem. 209,885.

Biochem. J. (1961) 78, 236

The Kinetics of Deoxygenation of Human Haemoglobin

BY K. DALZIEL* AND J. R. P. O'BRIENDepartment of BiocheMi8try, Radcliffe Infirmary, Univer8ity of Oxford

(Received 27 May 1960)

Until 1949, kinetic data for the reactions ofhaemoglobin with oxygen and carbon monoxide,obtained almost entirely by Hartridge & Roughton(1923, 1925), Roughton (1934) and Millikan (1933)in experiments with sheep haemoglobin, were con-sidered to conform to simple first-order and second-order rate equations, and to be consistent, there-fore, with the assumption that the four haems in themolecule behave independently. The best equili-brium data, on the other hand, could only be inter-preted by the intermediate-compound theory ofAdair (1925) with the assumption of haem inter-action (Forbes & Roughton, 1931; Courtice &Douglas, 1947). This situation was reviewed byRoughton (1949), who reported that re-inspectionof earlier kinetic data, and new experiments on thedeoxygenation of sheep haemoglobin in thepresence of sodium dithionite (Legge & Roughton,1950), gave evidence of haem interaction, notablya significant upward trend in the value of theoverall first-order velocity coefficient in the earlystages of the deoxygenation reaction. This wasconfirmed with human haemoglobin under variousconditions (cf. Dalziel, 1953, 1954), as will bedescribed here. The further evaluation of the inter-

mediate-compound hypothesis has since beenpursued with remarkable success by Roughton,Gibson and their collaborators. For sheep haemo-globin the four equilibrium constants for theoxygen reaction (Roughton, Otis & Lyster, 1955)and the four combination velocity constants for thecarbon monoxide reaction (Gibson & Roughton,1957 a) have been determined to within narrowlimits. Little further work has been reported forthe deoxygenation reaction, however.For the investigation of the kinetic effects of

haem interaction, the oxygen dissociation appearedto be the reaction of choice, since previous workhad indicated that the reverse reaction could beeliminated by an excess of sodium dithionite, andthat the analysis of consecutive reactions would besimpler than for the reverse, combination reaction.This was the main purpose of the work reportedhere; a secondary objective was to compare thekinetic properties of haemoglobin from severalnormal individuals and from persons suffering fromvarious types of anaemia. The work, which wascarried out during 1948-53, forms part of a Ph.D.thesis approved by the University of London, andbrief accounts have also been published in theliterature (Dalziel, 1954, 1955, 1958). It wasfound that secondary reactions between dithionite-* Sorby Research Fellow, The University, Sheffield 10.

KINETICS OF DEOXYGENATION OF HAEMOGLOBIN

oxidation products and the pigment interferedwith the spectrophotometric analysis (Dalziel &O'Brien, 1951, 1952, 1957; cf. Legge & Roughton,1950), and so far as haem interaction effects areconcerned the full benefits of the precision of theexperimental technique devised for the work(Dalziel, 1953, 1954) could not be realized. Never-theless, reasonably accurate kinetic data wereobtained for the deoxygenation of human haemo-globin under a wide range of conditions of concen-tration, acidity and temperature.

EXPERIMENTAL

Kinetic measurements

The rapid-reaction apparatus consists essentially of aconstant-flow apparatus of the gas-pressure type in con-junction with a Beckman DU photoelectric spectrophoto-meter, and has been described in detail elsewhere (Dalziel,1953, 1954). The mixing chamber and observation tube, of2 mm. bore, was fed by two jets of 1 mm. bore. Totalvolume flow rates of 5-10 ml./sec., or linear flow rates of150-300 cm./sec., were used, and mixing of the two re-actant solutions was 99% complete in 5-8 msec. Tempera-ture was controlled to within 0-10.

Either oxyhaemoglobin (HbO2) reactant or deoxygen-ated haemoglobin (Hb) calibration fluid could be run fromone jet, and either dithionite reactant or HbO2 calibrationfluidfrom the other. Thereactants and calibration fluids werekept in the storage vessels for 20 min. to allow tempera-ture and pressure equilibration and destruction of per-oxide formed by exposure of the dithionite solution to air(Dalziel & O'Brien, 1957). For measurements at the usualwavelength of 430 m,u (Am.. of Hb) the procedure at eachpoint of observation was: (1) a little HbO2 reactant was runthrough jet 1 to displace Hb calibration fluid containingdithionite; (2) with jet 1 closed, HbO2 calibration fluid wasrun from jet 2, whilst the spectrophotometer was adjustedto read E = 0; (3) with jet 2 closed, Hb calibration fluidwas run from jet 1, and AE10 %, the extinction changecorresponding to complete deoxygenation, was recorded;(4) the two reactants were run together, and the extinction,AEt, was recorded; (5) operations (1) and (2) were repeatedto check the stability of the spectrophotometer.The percentage saturation of the pigment with oxygen at

time t after mixing, calculated from the distance of thepoint of observation from the mixing chamber and theflow rate, was calculated as Y = 100 (1- AEt/AElo,(%).

Precision of the measurements. The precision of thespectrophotometric analysis of the reaction mixture(Dalziel, 1953, 1958) depends upon the constancy of therelative delivery rates, and the molar extinction coefficientsof HbO2 and Hb at the wavelength chosen for measure-ments. The most suitable wavelength is 430 mp (Ama.. ofHb), with 415 ml. (Amax of HbO2) as second choice. Thecalculated errors of measurements of the percentagesaturation of the pigment are, at 430 mp, ±0 5% at 95%saturation and ± 1-1% at 5% saturation, and, at 415 mp,+ 1 9 % at 95 %1 saturation and ±0 9% at 5 % saturation.The precision is little affected by variation of AE100%from 0-2 to 1-2. Nominal band widths of 4-6 mye wereused.

MaterialsOxyhaemoglobin. Haemolysates of washed red cells,

diluted with buffer solution, were used without furtherpurification. The HbO2 solutions contained less than 2% ofmethaemoglobin, as shown by spectrophotometric analysisfor cyanmethaemoglobin. The addition of3% ofmethaemo-globin did not affect kinetic data in control experiments.

Clear solutions were obtained by haemolysis withsaponin, which had no effect on the kinetics in controlexperiments. Three experiments with the same sample ofwashed red cells on three successive days gave identicalresults, but experiments were usually made not later thanthe day after the blood was withdrawn. With measurementsat 430 m,u, 15 ml. of normal blood was sufficient for akinetic curve of eighteen points.Sodium dithionite. Commercial samples (British Drug

Houses Ltd.) were used. The solid was stored in a sealedbottle. Dry, freely running samples assayed as 95-98% ofNa2S204,R20 (Vogel, 1939).

Buffers. Phosphate and borate buffers were preparedaccording to Clarke and Lubs (Vogel, 1939).

Preparation of reagentsOxyhaemoglobin reactant. Red cells were separated by

centrifuging from whole blood, to which heparin (0-01 g./100 ml.) had been added, and were washed three times withsodium chloride solution (0 9 g./100 ml.). Saponin solution(3 vol., 0-1 g./100 ml.) was added to 1 vol. of packed redcells, and after 10 min. the haemolysate was centrifuged.The clear supernatant solution was diluted with 1500 ml. ofbuffer solution. The HbO2 concentration, expressed asm-equiv./l. (mEq), that is m-moles of haem or combinedoxygen/l., was calculated from measurements of E576Mand a value for e.Eq of 15-4 (Dalziel & O'Brien, 1954;Lemberg & Legge, 1949).

Dithionite reactant. This solution was prepared withminimal contact with air, and alkali was added to neutral-ize the acid formed. For the usual concentration of 0-4 g./100 ml., 5 g. was dissolved in 1225 ml. of buffer plus 25 ml.of 0-2M-NaOH solution in a separating funnel, and thesolution was immediately covered with a layer of liquidparaffin. After the Hb calibration fluid had been prepared,the remaining dithionite reactant was run into a storagevessel in the kinetic apparatus and covered with 0-5 cm.of liquid paraffin.Haemoglobin calibration fluid. To 250 ml. of HbO2

reactant in a 500 ml. volumetric flask, 250 ml. of dithionitereactant was added from the separating funnel, themixture was quickly poured into the storage vessel withminimal agitation and at once covered with liquidparaffin.

RESULTS

Variation of the initial oxyhaemoglobin concentration

The time required for the deoxygenation of agiven fraction of the oxyhaemoglobin, calculatedfrom spectrophotometric analysis at 430 m,u, isindependent of the initial oxyhaemoglobin concen-tration, and the reaction follows an approximatelyfirst-order course (Fig. 1). The first-order plot isstrictly linear only beyond 60% saturation, how-

237Vol. 78

K. DALZIEL AND J. R. P. O'BRIENever; the velocity coefficient, definedbykY = - dY/dt, increases significantly during the earlier stagesof the reaction (see also Table 2). Values for thefirst-order velocity coefficient calculated over thelast 60% of the reaction, denoted by k6, are givenin Table 1 for a series of experiments at pH 8-4 inwhich the initial oxyhaemoglobin concentrationvaries fivefold. Haemoglobin from a single indi-vidual was used. When corrected to 18.00 bymeans of the temperature coefficient given later,the results of these nine experiments show a varia-tion of + 3.5%, which confirms the precision of thetechnique.

Comparison of spectrokinetic meurementsat several wavelengths

As a test of the validity of the spectrophoto-metric analysis, kinetic curves were obtained bymeasurements at two or more wavelengths with thesame blood sample. In the most detailed experi-ments, with two samples of blood drawn for trans-fusion and containing citrate and glucose, the fivewavelengths of the absorption maxima of oxy-haemoglobin and haemoglobin were used (Fig. 2).Over the whole course of the reaction at pH 8-4 thevalues of the percentage saturation calculatedfrom measurements at 415 mp were significantly

2-0

1-8

1-6

0481

20

0o4

14-

1-2 - \

0 50 100 150 200Time (msec.)

Fig. 1. First-order plots for the rate of deoxygenation ofoxyhaemoglobin at two concentrations. Concn. ofNa2S2O4, 0-2 g./100 ml.; pH 8-4. Y, Percentage saturationof the pigment with oxygen. 0, Expt. no. 11, 17.70,0-017 m-equiv. of HbO2/l; A, Expt. no. 14, 17.80, 0-067 m-equiv. of HbO2/L

100Time (msec.)

Fig. 2. Kinetic curves recorded at three wavelengths inexperiments with the same sample of oxyhaemoglobin:pH 8-4; concn. of Na3S2O4, 0-2 g./lOO ml.; temp. 18-20.0, Expt. 44,430 mi, 0-037 m-equiv. of HbO2/l1; *, Expt.45, 415mI, 0-068 m-equiv. of HbO/l.; A, Expt. 46,578 m,u, 0-33 m-equiv. of HbO,/l.

Table 1. Data of nine experiments with oxyhaemoglobin of a norwal individual

Conditions were: pH 8-4; concn. of Na%,20j, 0-2 g./100 ml.; A 430 m,t. [HbO,], conen. of oxyhaemoglobin.Expt. Flow rate [HbO2] ka ka at 180no. (ml./sec.) (m-equiv./l.) Temp. (sec.-') (sec.-')9 5-1 0-0325 17.60 10-2 10-710 5-1 0-0380 17-6 10-2 10-711 5-1 0-0170 17-7 10-8 11-212 5-1 0-079 17-9 11-3 11-413 5-1 0-0284 17-7 10-7 11-114 5-1 0-067 17-8 10-9 11-216 5-1 0-0268 18-4 11-6 11-017 9-8 0-0327 18-5 11-8 11-019 8-1 0-028 18-5 11-7 10-9

238 1961

Meaai 11-1±z0 4

VKINETICS OF DEOXYGENATION OF HAEMOGLOBINsmaller than those obtained at 430obtained at 578, 560 and 540 mu aanother, but were greater over the

100

80

60

40

20

0 50 100Time (msec.)

Fig. 3. Time course of deoxygenation alat 430 m, (0) and 415 m, (-). Exp0-039 m-equiv. of HbO2/., con¢n. of NagSThe lines are calculated first-order curvesin the range Y 60-20 %.

mnu. The values reaction than those obtained at 430 mru. In eachgreed with one case the first-order plot shows a significant increaselast half of the of slope over the early part of the deoxygenation,

but is linear below 60% saturation. Values for k,calculated from the slopes are 11-8 sec.-' at415 mi, 10-8 sec.-L at 430 m,u and 8-9 see.-' at578 mi/.In all subsequent experiments with fresh blood,

under various conditions of concentration, tem-perature and pH, measurements were made atboth 415 and 430 miA. The results obtained atpH 8-4 (Fig. 3), and at pH 9-5, confirmed thosejust described. Under all conditions the apparenttime course of the reaction was similar at the twowavelengths. The differences between the calcu-lated percentage saturation decreased with de-crease of pH and at pH 6-3 identical values wereobtained at the two wavelengths (Fig. 4).These experiments show that the assumption

that the reaction mixture contains only two ab-sorbing species, characterized by the absorptionspectra of the initial reactant and final product, isnot strictly valid at neutral and alkaline pH.

Variation of dithionite concentration. The results150 200 in Fig. 5 show that deoxygenation is preceded by

an induction period which decreases from about

pH 8-4 recorded 30 msec. with a dithionite concentration ofot. no. 69, 21.10P, 0-05 g./100 ml. to less than 5 nisec. with dithionite320 0.2 g./100 ml concentrations greater than 0-2 g./100 ml. Thesefitted to the data experiments were made at pH 8-4 and 18°, with

measurements at 430 in/. Similar results were

100 r

801-

60 F01-

401-

20 -

I I I I I I I

0 20 40 60 80Time (msec.)

100 120 140

Fig. 4. Time course of deoxygenation at pH 6-3 recordedat 430 mrA (0) and 415 mr (-). Expt. no. 65, 13.80,0-038 m-equiv. of HbO2, conon. of Na2S2O,., 0-2 g./100 ml.The lines are calculated first-order curves fitted to the datain the range Y 60-20 %.

200Time (msec.)

Fig. 5. Kinetic curves at 430 m,u with various dithioniteconcentrations: A, 0-05; *, 0-1; O, 0-2; A, 0-4; *, 0-8 g./100 ml. Expt. nos. 20-24, pH 8-4, temp. 180, 0-031-0-036 m-equiv. of HbO2/l.

Vol. 78 239

K. DALZIEL AND J. R. P. O'BRIEN

obtained with measurements at 415 mpi, and alsoat pH 7 0, where the induction period was shorterthan at pH 8-4. The value of ka increases slightlywith dithionite concentration over the range tried,but the upward trend of k during the first 40% ofthe reaction persists. This is shown in Table 2 byvalues of k calculated from pairs of successivemeasurements in the experiments with dithioniteconcentrations of 0-05 and 0-8 g./100 ml. A 16-foldincrease of dithionite concentration, which practic-ally eliminates the induction period, causes only asmall increase in the rate of haemoglobin forma-tion, and does not accelerate the early stages of thereaction, from Y 90 to 60 %, to a greater extent

Table 2. Variation of velocity coefficient during thedeoxygenation of oxyhaemoglobin in the presence ofdithionite at pH 8-4

Velocity coefficient, k = 2-3 A log Y/At.

Expt. no. 200-05 g. of Na2S204/100 ml.

Mean valueof Y k(%) (sec.-')92 6-283 7.978 6-970 8-757 9.945 10-539 9.934 10-630 10.1

Expt. no. 230-8 g. of Na2S204/100 ml.

Mean valueof Y k(%) (sec.-')90 7-483 10-076 9-868 11-660 12-254 12-648 12-140 12*031 12-823 13-218-5 12-816 12-013 12-2

60 70 80 9-0 100

pH

Fig. 6. Variation of the first-order velocity coefficientwith pH at 180 for the deoxygenation of oxyhaemoglobinsamples from K.D. (0), and three other individuals (0,A). The curve was calculated from pK = 7-0, k. for acidicform = 55 sec.-' and ka for basic form = 10 sec.-'.0-03-004 m-equiv. of HbO2/l; concn. of Na,8204, 0-2 g./100 ml.

than the later stages, and the initial upward trendin k remains. Similar results were obtained withhaemoglobin from another individual, and alsowith a second sample of sodium dithionite.

In all the following experiments, the dithioniteconcentration was 0-2 g./100 ml.

Variation of pH. The reaction was studied atseveral pH values from 5 9 to 9 4, at temperaturesfrom 18 to 210, and with spectrophotometricmeasurements at both 415 and 430 mu. The buffersolutions were Clarke and Lubs borate (pH 8.0-9-4)and phosphate (pH 5.9-7 65), and the ionicstrengths of the reaction mixtures varied from 0.1to 0417. Experiments could not be made in moreacid solutions because of turbidity; even at pH 5 9,the light absorption of the product changedslowly on standing, but at pH 6X3 the product wasstable.

Plots of log Y against time were linear from 60%down to 20% saturation at every pH value. AtpH > 7-0, linearity extended to less than 5%saturation, but in slightly acid solutions there wasevidence of slight deviations towards slower ratesbelow 20% saturation (cf. Fig. 4). The gradualincrease in the first-order velocity coefficient in theearly stages of the reaction was evident over thewhole pH range, but became progressively lessmarked with decrease of pH: at pH 6-3, themaximum value ka was reached at 70% saturation.From studies of the effect of temperature on the

reaction rate, described below, values for ka at 180were calculated, and are plotted against pH inFig. 6.

Variation of temperature. Values of log ka ob-tained from measurements at 430 m,u in experi-ments with oxyhaemoglobin of one individual, atvarious pH values and temperatures from 15 to 300,are plotted against the reciprocal of the absolutetemperature in Fig. 7. The activation energycalculated from the slopes increases slightly withincreasing acidity, and the difference between thevalues of 22 000 cal./mole at pH 6-3 and 26 000 cal./mole at pH 8-4 is outside the random error of themeasurements.To test whether the rate during the first 40% of

deoxygenation, during which k increases, varieswith temperature to the same extent as the laterstages, log Y was plotted against t.ka/2-3 forexperiments at pH 8-4 in the temperature range15-300, over which ka increases ninefold. The slopesof these plots '(Fig. 8) are equal to klka, and theirconstancy shows that this ratio is independent oftemperature. The average value of kIka is about 0 7in the range Y 80-95%. The curves for the severalexperiments are not exactly superimposed becausedifferences of flow rate affect the rate of mixing,and temperature differences alter the duration ofthe induction period.

60

50

- 40C 3IW 30I-,

. ,,320

10

240

KINETICS OF DEOXYGENATION OF HAEMOGLOBINVariation of ionic strength. The effect of ionic evidence of specific ion effects. With increase of

strength on k. was studied over a range of pH by ionic strength, k. increased at pH 8-0-9-5, de-varying the buffer concentration and by the addi- creased at pH 6-3, and varied only slightly attion of sodium chloride. At pH 8-5 and 9-5, the pH 7-0. The results shown in Fig. 9 were obtainedbuffer was borate-potassium chloride-sodium from measurements at 430 m,. The salt effects athydroxide and, at pH 7-0 and 6-3, sodium di- pH 6-3 and 9-5 were confirmed in measurements athydrogen phosphate-sodium hydroxide. AtpH 8-0, 415 mp.both borate and phosphate buffers were used in two Comparative data for oxyhaemoglobin of severalconcentrations, 0-05 and 0-2M. There was no individuals. Values for k. at 18.00 and pH 8-4 for

haemoglobin samples from five normal individuals,calculated from measurements at 430 mjnA, were

20- 11-1, 11-2, 11-5, 11-8 and 11-8 sec.-'. The first ofA\ these is the mean of nine experiments (cf. Table 1),

1-8 _ \and the difference of 6% between this and the lasttwo values is significant. Some of these sampleswere also studied at lower pH values (Fig. 6), but

16 o for these the differences were within the limits ofexperimental error. Samples from patients suffer-ing from polycythaemia, untreated pernicious1-4 \ anaemia and acute haemolytic anaemia (acholuric

o s \ jaundice) gave values of 11-3, 11-4 and 11-3 sec.-'1-2 _ for k. at pH 8-4 and 18.00.

E° Kinetic studies over the pH range 6-5-8-4 were1-0

also made with haemoglobin samples from a14 \ patient suffering from sickle-cell anaemia and an

individual showing sickle-cell trait. These experi-0-8 ments were made at an early stage of the research,

and are less reliable than the later work, but theresults did not differ significantly from those of

3-30 3-34 3-38 3-42 3-46 3-50 control experiments with normal haemoglobin,1031T and showed the same pH effect on the rate and the

usual upward trend in k. The values for k. wereFig. 7. Arrheniusplotsforthe deoxygenation ofoxyhaemo- smaller, by about 15%, than those obtained in theglobin (K.D.) at several pH values: A, pH 6-3; 0, later work. This was probably due to the use of apH 7-35; 0, pH 8-4; *, pH 9*4. 0-03-0-04 m-equiv. of poor sample of dithionite and to neglect of sourcesHbO,Jl.; conen. of Na2S204, 0-2 g./100 ml. of systematic error unsuspected at that time.

Effects of 8alt8 related to dithionite. Possible

20 effects of oxidation products and impurities in thedithionite were sought in experiments at pH 8-5.Sodium sulphate, metabisulphite, thiosulphate and

bO 1I-80

16

1.6 r

1-5 h

01-4

1-3 h

01 02 0-3 0-4 0-5t. kaI2-3

Fig. 8. Plots of log Y against t . ka/2-3 for five experimentsat temperatures from 15 to 300, pH 8-4, 0-03-3-04 m-equiv. of HbO2/L., conen. of Na2S,O4, 0-2 g./l00 ml. ka is thefirst-order velocity coefficient calculated from measure-ments in the range Y 60-20 %, and t is time in sec.

16

1-2 L0-2

0A

I

0.3 0.4 0.5 0-6 0-7 0-8VI

Fig. 9. Variation of the first-order velocity coefficient withionic strength: *, pH9-5; 0, pH8-5; 0, pH 8-0; A,pH 7-0; A, pH 6-3. 0-03-0-04 m-equiv. of HbO211., concn.of Na2S204, 0-2 g./100 ml.

Bioch. 1961, 78

Vol. 78 241

K. DALZIEL AND J. R. P. O'BRIEN

sulphide added to the dithionite reactant (0 4 g./100 ml.) in concentrations of from 0-08 to 0-2 g./100 ml. had no effect on kinetic measurements at415 and 430 m,.

DISCUSSION

The validity of the optical methods which havebeen used to measure the percentage saturation ofhaemoglobin with various gases in kinetic studiesdepends upon the assumption that the reactingmixture contains only two absorbing species,characterized by the absorption curves of the un-reacted and fully reacted mixture. This assumptionhas been tested by the comparison ofmeasurementsat several wavelengths, and the results show thatit is not strictly true for the deoxygenation reactionin the presence of sodium dithionite. The apparentpercentage saturation, after a given time, variedwith the wavelength over a range of about 8% inslightly alkaline solutions. Therefore, one or moreadditional absorbing species must be formed, eitheras intermediates in the reaction or in secondaryreactions. There will in fact be five molecularspecies in a partially saturated mixture, accordingto the intermediate-compound theory, but if eachof the four haems in the haemoglobin moleculecontributes equally and independently to the lightabsorption, regardless of the presence of a ligandon the iron atoms of the other three, there will beeffectively only two absorbing species, oxygenatedand unoxygenated haems, and the light absorptionshould be linearly related to the percentage satura-tion of the pigment. Hartridge & Roughton (1925)found this to be true within the error of theirspectroscopic method of analysis, and it has beenstated that tests with more sensitive spectrophoto-metric methods also support this assumption(Roughton, 1955; Gibson & Roughton, 1957a).The discrepancies between measurements at

different wavelengths can be explained by second-ary reactions between haemoglobin and hydrogenperoxide formned in the auto-oxidation of dithion-ite, in which a small proportion of the haemo-globin is irreversibly degraded (Dalziel & O'Brien,1957). The discrepancies are not large. At everywavelength used, the measurements indicated asimilar time course for the reaction, and belowabout 60% saturation they conformed to the

requirements of a simple first-order dissociation.The velocity coefficient, ka, varied by ± 15%about the value obtained at 430 m,u. It is unlikelythat the results obtained for the rate of reactionand its variation with temperature, pH and ionicstrength are seriously in error.A reproducibility of + 3 5% in k. was realized

consistently in experiments with single bloodsamples and with samples withdrawn from the sameindividual at intervals over a period of manymonths. There were no greater variations betweenthe rates of deoxygenation, at several pH values,of haemoglobin from seven individuals, includingthree suffering from blood disorders, except atpH 8-4, where the highest and lowest values of ka,11 8 and 11 1 sec.-1, differed significantly. It isof special interest that no significant differenceswere detected in the kinetic behaviour, at severalpH values, of haemoglobins from a normal indi-vidual, one with sickle-cell trait, and a patientsuffering from sickle-cell anaemia. Evidently thedifferences between the globins (Itano & Pauling,1949) do not affect the haem-linked acidic groups.In contrast, Gibson, Kreuzer, Meda & Roughton

(1955) reported considerable differences betweenthe rates of deoxygenation of three samples ofhuman haemoglobin; for example, at pH 7-1 rateconstants of 21, 26 and 36 sec.-L were obtained.Unfortunately, no evidence of the reproducibilityof measurements with a single sample of blood wasgiven. Even greater individual variations have,however, been reported for the rates of combinationof human and sheep haemoglobin with oxygen andcarbon monoxide (Hartridge & Roughton, 1925;Gibson et al. 1955; Gibson & Roughton, 1957a).In Table 3, values for ka at 180 obtained in the

present work from measurements at 430 mjt arecompared with specific reaction rates for humanhaemoglobin reported by other workers, correctedwhere necessary to 180 with the temperaturecoefficient reported here. The consistency of thedata is remarkably good in view of the differencesin technique. Millikan (1933) used a constant-flowmethod with colorimetric analysis, and his resultswere consistent with a simple first-order coursethroughout. Dubois (1941) used the deoxygenationof a mixed blood sample as a test of his constant-flow apparatus. Gibson et al. (1955) made measure-ments over the first third of the reaction only, with

Table 3. Specific rates of deoxygenation of human haemoglobin at 180

Specific reaction rate (sec.-")Source pH 6-0 pH 7*1 pH 8-5 pH 9.1-9-8

Present work, ka 51 29 11 11Millikan (1933) 44 24 10 10Dubois (1941) _ - 11Gibson, Kreuzer, Meda & Roughton (1955) - 28 - 11

-24-2

KINETICS OF DEOXYGENATION OF HAEMOGLOBINa stopped-flow method, and the values given in theTable are averages of those reported for three bloodsamples.

Hartridge & Roughton (1923) found the rate ofdeoxygenation of sheep haemoglobin to be inde-pendent of the dithionite concentration above0-05 g./100 ml., and concluded that the role of thedithionite is that of a passive oxygen absorbent,which, after an induction period during whichthe oxygen concentration is reduced sufficiently,permits the dissociation of oxyhaemoglobin andeliminates the reverse reaction. These findingshave been generally confirmed here, although thegreater precision of the method has revealed anincrease of about 10% in the rate of deoxygen-ation, at pH 8-4, for an increase of dithionite con-centration from 0-05 to 0-8 g./100 ml. This smallincrease can be accounted for as a neutral-salteffect, however. The results establish that deoxy-genation by second-order reaction of oxyhaemo-globin with dithionite is very slow, if it occurs atall, but do not of course exclude the possibilitythat dithionite may have some influence on therate of dissociation, for example by reaction withthe globin.The variation of the rate of deoxygenation of

human haemoglobin with pH is very similar tothat of sheep haemoglobin, and can be explained bythe assumption (Henderson, 1920) that oxyhaemo-globin contains an oxy-labile acidic group withpK 7-0 and that the deoxygenation of the un-ionized form is five times as fast as that of the basicform. The corresponding data of Hartridge &Roughton (1923) for sheep haemoglobin werepK 6-7, and a ratio of 7 between the rates of de-oxygenation of acidic and basic forms; the differ-ences are within the experimental error. The pHeffects on the rates of dissociation of the firstcarbon monoxide and nitric oxide molecule re-spectively from the carbon monoxide and nitricoxide compounds of sheep haemoglobin are alsoaccounted for by the assumption of a haem-linkedgroup with pK 6-7 (Gibson & Roughton, 1957b, c).These kinetic effects may all be attributed to theweaker of the two haem-linked groups revealed bypH effects on the dissociation curve (Ferry &Green, 1929) and differential titration of oxyhaemo-globin and haemoglobin (German & Wyman, 1937),for which pK values of 6-7 and 5-8 have beenestimated for horse haemoglobin (Wyman &Ingalls, 1941; Wyman, 1948).

Salt effects on the rate of deoxygenation havenot been recorded hitherto. The increase of ratewith ionic strength at pH 8-4 and 9-4, and thereverse effect at pH 6-3, cannot be explained by asecondary salt effect on the dissociation of ahaem-linked acidic group. A difference of totalcharge between the oxyhaemoglobin molecule and

the activated complex is not consistent with thefact, referred to below, that the entropy of activa-tion appears to be greater in alkaline solution thanin acid solution, and the absence of any evidencethat the isoelectric points of oxyhaemoglobin andhaemoglobin differ significantly. A more likelyexplanation of the salt effects, perhaps, is thatdisorganization of the protein structure accom-panies the formation of the activated complex(cf. Eley, 1943) and that concomitant redistributionof charge and fission of hydrogen bonds is in-fluenced by the relatively high salt concentrationsused here. The situation is complicated by the factthat the processes involved in haem interactionmay also be influenced by salts. It would be ofinterest to investigate salt effects on the dissocia-tion of the first of the four oxygen molecules alone,the rate of which can be determined in the absenceof dithionite (Gibson & Roughton, 1955), and alsoto make similar studies of the deoxygenation ofmyoglobin, in which haem interaction cannotoccur.The activation energies for the overall deoxy-

genation, 22 000 cal. at pH 6-3 and 26 000 cal. atpH 8-4, agree very well with the value of 22 000 cal.obtained by Hartridge & Roughton (1923) forsheep haemoglobin at pH 9-0. The latter workersalso recorded some measurements at pH 5-6 whichindicated a slightly greater activation energy of25 000 cal., but the difference, which is evidentlywithin the experimental error, was not com-mented upon. Nevertheless, Eley (1943) regardedthe difference as significant, and, on the basis of thetransition-state theory, calculated entropies ofactivation of 36 and 24 cal./degree/mole at pH 5-6and pH 9-1 respectively. The pH effect on theactivation energy indicated by the present ex-periments, which give entropies of activation of25 cal./degree/mole at pH 6-3 and 36 cal./degree/mole at pH 8-4, is in the opposite direction, andsuggests that the higher rate of deoxygenation inacid solution is due to a lower activation energyand not to a larger positive entropy of activation,as Eley concluded for sheep haemoglobin.Evidence that the deoxygenation of sheep oxy-

haemoglobin does not follow a simple first-ordercourse throughout, as was concluded by Millikan(1933), was first obtained by Legge & Roughton(1950). Their conclusion that an initial upwardtrend in the velocity coefficient is a genuine featureof the kinetics of the overall reaction is stronglysupported by the present experiments, in whichthe trend is far outside the experimental error andhas been demonstrated under various conditions.Experiments in which the dithionite concentrationwas varied 16-fold, the initial oxyhaemoglobinconcentration sixfold, and the flow rate twofold,together with experimental controls of the tech-

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K. DALZIEL AND J. R. P. O'BRIENnique itself which included kinetic studies of thedehydration of carbonic acid (Dalziel, 1953),established that the effect is not due to insufficientlyrapid removal of dissolved oxygen by the dithion-ite, incomplete mixing, flow anomalies or othertechnical errors. It is especially important, in viewof the occurrence of secondary reactions whichinterfered with the colorimetric analysis by Legge &Roughton (1950), that the deviations from asimple first-order course have been recorded bymeasurements with monochromatic light of fivewavelengths, comprising the absorption maxima ofreactant and product. On the intermediate-compound theory, this feature may be attributedto the fact that kI4, the velocity constant for thedissociation of the first of the four oxygen mole-cules from oxyhaemoglobin, is smaller, relative tothe rate constants for the three succeeding dissoci-ations, than would be expected on a statisticalbasis. This interpretation is consistent with theequilibrium data (Roughton, 1949), and convincingkinetic evidence in its support was obtained byGibson & Roughton (1955).

Direct measurements of the individual constantk4, by a method which does not involve the use ofdithionite, have now been reported for both humanand sheep haemoglobin, and preliminary com-parisons of these data with the overall kinetics inthe presence of dithionite have been made (Gibson& Roughton, 1955; Gibson et al. 1955). The presentfindings will be briefly considered in the light ofthese discussions. First, the overall velocity co-efficient ka is greater at pH 7-1, by a factor of 2-5,than at pH 9- 1. Over this pH range, the overallvelocity coefficient in the very earliest stages of thereaction is estimated, by extrapolation to zerotime, as < 0-6 ka, and should be equal to 0-25 kI4.On the other hand, direct measurements showedthat k4 is independent of pH over this range.Although this is not inconsistent with the markedpH effect on ka, which could be put down to aneffect on one or more of the other three individualrate constants, the initial upward trend of k shouldin this case be considerably more marked at pH 7-1than at pH 9- 1. There was no evidence of this inthe present experiments; indeed there were someindications of the reverse effect.

Secondly, the finding that the initial trend in theoverall velocity coefficient is not significantlyaltered by temperature change, from 150 to 300,indicates that the activation energy for k4 is notgreatly different from that calculated for ka - Inthis case, the overall data are consistent with directdeterminations of k4: for human haemoglobin atpH 7-1, Gibson et al. (1955) obtained an activationenergy of 19 600 cal. for k4, compared with thevalue of 23 000 cal. for ka found here. The differenceis not great enough to produce a significant tem-

perature effect on the time course of overall deoxy-genation.

It is evident that further direct comparisons ofthe overall deoxygenation kinetics and directmeasurements of k4 on the same blood sample areneeded. The value of k4 appears to vary consider-ably from sample to sample (Gibson et al. 1955),but the reproducibility of the data for a singlesample has not been demonstrated. Secondaryreactions accompanying the overall deoxygenationin the presence of dithionite have so far dis-couraged attempts to evaluate all four individualdissociation velocity constants, as has been donefor the carbon monoxide reaction (Gibson &Roughton, 1957 a). These secondary reactions maybe minimized by using high concentrations ofpigment and dithionite (Dalziel & O'Brien, 1957),and controlled by making measurements at two ormore wavelengths corresponding to the absorptionmaxima of haemoglobin and oxyhaemoglobin. Itmay also be possible to obtain evidence of thenature and magnitude of the errors from thissource by spectrokinetic studies of the reduction ofmethaemoglobin by dithionite in the presence andabsence of dissolved oxygen, since the absorptionspectrum of methaemoglobin in the Soret region issimilar to that of oxyhaemoglobin.

It is unlikely, however, that the marked differ-ences between the pH effects on ka and k4 can bedue to secondary reactions of the type demon-strated so far. Dithionite may affect the rate of

. deoxygenation by reaction with the globin (cf.Gibson & Roughton, 1955). It is known that dithion-ite can react with amino groups, and Roughton(1944) has suggested that the weaker of the twooxy-labile groups may be an amino group whichcan bind carbon dioxide. In this connexion studiesof the effect of carbon dioxide on the kinetics ofdeoxygenation would be of interest.

SUMMARY

1. Spectrokinetic studies of the deoxygenationof human haemoglobin in the presence of sodiumdithionite have been made under various conditionswith a constant-flow rapid-reaction apparatus anda photoelectric spectrophotometer. A reproduci-bility of + 3.5 / in estimates of the first-ordervelocity coefficient has been demonstrated.

2. No significant differences were detectedbetween the kinetic behaviour at several pH valuesofoxyhaemoglobin samples fromnormal individualsand from persons suffering from several types ofanaemia, including sickle-cell anaemia. The greatestdifference observed between two haemoglobinsamples was 6% in the specific reaction rate.

3. Significant differences between the results ofanalyses of the reaction mixture, by measurements

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Vol. 78 KINETICS OF DEOXYGENATION OF HAEMOGLOBIN 245

of light absorption at several wavelengths, showthat the deoxygenation is accompanied by second-ary reactions involving the pigment. The errorsthereby introduced are not large, but realization ofthe full benefits of the precision of the techniquein the interpretation of the detailed time course ofthe reaction is restricted.

4. The time course of the overall reactioncannot be accurately described by the simple first-order relation, d[HbO2]/dt = k[HbO2]. The calcu-lated value of k increases from an initial value of< 0-6ka to a constant value of ka when reaction is40% complete. This initial trend in k is not affectedby wide variations of the initial oxyhaemoglobinand dithionite concentrations, and is evident inmeasurements at five wavelengths at pH 8-4 andtwo wavelengths at pH 6 3-9-5, and is thereforeconsidered to be a genuine kinetic feature of thedeoxygenation. This confirms the conclusion ofLegge & Roughton (1950) for the deoxygenation ofsheep haemoglobin, and is in agreement with otherrecent work on the kinetics and equilibria of sheephaemoglobin and with the intermediate-compoundtheory.

5. Variations of the rate of deoxygenation withpH are consistent with the presence of an oxy-labile group with pK 7-0. The specific reactionrate is five times as great at pH 6-0 as at pH 8-5-9*5. There was no evidence of a pH effect on theinitial upward trend in the specific reaction rate.

6. Activation energies of 22 000 cal. at pH 6-3and 26 000 cal. at pH 8-4 have been calculatedfrom the variation of the rate of deoxygenationwith temperature. There was no evidence that theearly and late stages of the overall deoxygenationwere differently affected by temperature change.

7. With increase of ionic strength, the specificreaction rate decreases at pH values below 7 0 andincreases at pH values above 7 0.

8. The results are considered in relation to otherrecent work, and certain anomalies are discussed.

Technical assistance by Mr B. A. Collett during part ofthis work, and financial support from the Nuffield Haema-tology Fund, are gratefully acknowledged.

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