Você está na página 1de 5

CLIN.CHEM.

22/12, 1986-1990 (1976)

A Mechanism for the Conversion of Oxyhemoglobin

to Methemoglobin by Nitrite
F. Lee Rodkey

Each mole of oxyhemoglobin iron converted to methemoglobin causes the oxidation of 1.5 mol of nitrite to nitrate and consumes 1 mol of protons. No oxygen is liberated.

Materials and Methods


Human hemoglobin was prepared from the freshly drawn heparinized or disodium EDTA-treated blood. Erythrocytes were washed three times in 20 volumes of NaC1 solution (9 g/liter). The washed cells were hemolyzed with 10 volumes of de-ionized water at 0 #{176}C. Cell debris was removed by centrifugation (Model PR-2, Internatioal Equipment Co., Needham Heights, Mass. 02194). The clear hemoglobin solution was kept on ice and diluted with phosphate or tris(hydroxymethyl)aminomethane buffers as desired. Methemoglobin was determined by the method of Rodkey and ONeal (4). Total hemoglobin, as cyanmethemoglobin, was measured by the method of Van Kampen and Zijlstra (5). Absorption measurements were obtained with a Beckman DB-GT spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif. 92634) with the cuvette compartment maintained at 28 #{176}C. Nitrite concentration was measured by the method of Schneider and Yeary (6) by use of the neutral ZnSO4-Ba(OH)2 protein-free filtrates as suggested by Wegner (7). I estimated the conversion of O2Hb to metHb by continuous spectrophotometric measurement at 540 nm, recording with a strip-chart recorder. The buffered O2Hb solution, 3.0 ml, was placed in the cuvette and an original absorbance obtained. The reaction was started by adding 0.05 or 0.10 ml of freshly prepared aqueous
NaNO2. The contents of the cuvette were mixed with

The overall reaction has two simultaneously occurring parts. In the beginning the rate-limiting reaction converting O2Hb to metHb is directly proportional to H and N02
concentrations and is independent of metHb. The second

portion accounts in major part for the stoichiometry and rate of the overall reaction. In this portion O2Hb tetramers
and metHbNO2 are the reactants. Essentially no reaction takes place in the presence of CN, which displaces nitrite from the metHbNO2, nor in the presence of 0.5 mol/liter Nat, which converts the O2Hb to af3-dimers. The autocatalytic nature of the overall reaction in the presence of

excess nitrite is the result of metHb, which is formed in both parts of the reaction, associating with nitrite to increase the concentration of one reactant of the cyanidesensitive part. The reaction rates at constant pH in excess nitrite are proportional to the product of the O2Hb concentration and the square of the metHb concentration. The rate increases up to about 66% conversion of O2Hb fol-

lowed by a decrease as the O2Hb becomes limiting. The dissociation constant of metHbNO2 at 25 #{176}C and pH = 6.4 was found to be 1.11 0.11 mmol/liter. The reactions of nitrite ion in the formation of methemoglobin from oxyhemoglobin are not well understood. At low concentrations of nitrite the reaction is very slow, but after a period of time the rate of reaction increases in an autocatalytic fashion (1-3). In contrast to oxidation with ferricyanide, essentially no oxygen gas is released from oxyhemoglobin by nitrite even when conversion to methemoglobin is complete (3). Oxygen is essential for the formation of methemoglobin by nitrite as carboxyhemoglobin is unchanged and the reaction proceeds very slowly, if at all, in nitrogen (2). It was observed by chance that oxyhemoglobin preparations treated with sodium nitrite showed

an induction to the amount aration. This nitrite complex oxyhemoglobin

period that was inversely proportional of methemoglobin in the original prepreport shows that the methemoglobin is required for the rapid conversion of to methemoglobin by nitrite.

a Teflon paddle and spectrophotometric recording was started. When the reaction appeared complete, 2-3 mg of K3Fe(CN)6 was added to ensure that no O2Hb remained and the absorbance, A , was recorded. Finally, all metHb was converted to the cyanide derivative by adding 2-5 mg of KCN. The absorbance of the cyanmethemoglobin so obtained, ACN, was used to estimate the total heme concentration and to calculate the theoretical absorbance of the solution when only O2Hb was present, i.e., 1.3 X ACN. The percentage of the total hemoglobin present as metHb was then calculated from
the equation:

%metHb
The search 20014. Laboratory Institute, of Analytical Biochemistry, Naval Medical National Naval Medical Center, Bethesda, ReMd.

l.3ACN 1.3ACN

A A

100

(1)

The opinions or assertions contained herein are those of the author and are not to be construed as official or as reflecting the views of the
Navy Department or the Naval Service at large. Received Aug. 18, 1976; accepted Sept. 1, 1976. 1986 CLINICAL CHEMISTRY, Vol. 22, No. 12, 1976

where A is the measured absorbance, 1.3 ACN is the absorbance expected when all the heme is present as O2Hb, and A,. is the absorbance observed when all the heme is present as the metHb or metHbNO2 mixture.

Results and Discussion


Effect of pH: Conversion of oxyhemoglobin to methemoglobin was tested as a function of pH in the presence of ninefold excess of sodium nitrite over heme iron. The oxyhemoglobin solution was diluted to approximately 50 mol of heme per liter of 0.2 mol/liter phosphate or tris(hydroxymethyl)aminomethane buffers. Potassium was the counter ion for all phosphate buffers and chloride was the counter ion in all tris(hy8
Tim
-

I 40

droxymethyl)aminomethane buffers used. The formation of methemoglobin was complete in less than 1 mm at pH 6.4 and about 13 mm at pH 7.4. At higher pH the reaction was extremely slow. Only 3% of the O2Hb reacted in 15 mm at pH 8J while less than 2% of the O2Hb was converted to metHb at pH 9.0 in 30 mm. It was clear that the induction period for a given oxyhemoglobin solution was considerably prolonged at higher pH. The freshly prepared hemoglobin solution used in the above experiment contained less than 1% of the pigment as metHb. Air oxidation of the hemolysate at room temperature was permitted until the methemoglobin increased to about 7% of the total pigment. Addition of the same ninefold excess of nitrite to the 93% OHb/7% metHb mixture at pH 8.1 virtually
eliminated the induction period and all O2Hb was

0 mm

14

14

Fig.

1.

Effect of pH on the conversion of oxyhemoglobin to


by nitrite

methemoglobin

Each solution contained the same amount of hemoglobin, 50 tmol of heme per liter, in 90 mmol/liter phosphate buffer at the pH indicated. The reaction was started by addingNaNO2to a concentration of 935 imol/liter

of two reactions occurring together, one of which is insensitive to the presence of CN- and the other so sensitive as to be virtually stopped by cyanide. Absorption curve of methemoglobin derivatives. Methemoglobin was prepared by adding sodium nitrite to oxyhemoglobin in 50-fold excess over heme iron. The mixture was allowed to stand at 25 #{176}C for 2 h, then dialyzed exhaustively with a Bio-Rad hollow fiber (BioRad Laboratories, Richmond, Calif. 94804) against
de-ionized water methemoglobin until no nitrite remained. The dialyzed solution was centrifuged and the sustored at 4 #{176}C until used. The absorption curve

converted to metHb in less than 4 mm. The 93% O2Hb/7% metHb mixture was saturated with 100% CO to remove all O2Hb. Addition of nitrite to the COHb/metHb mixture at pH 8.1 did not cause any detectable formation of metHb in 30 mm, demonstrating that O2Hb is required for the reaction. A few
crystals of KCN were added to the O2Hb/metHb mix-

ture at pH 8.1, to convert the methemoglobin to cyanmethemoglobin. Nitrite was then added to this O2Hb/
cyanmetHb mixture. No measurable conversion of O2Hb to methemoglobin occurred in 2 h. On standing overnight at room temperature less than 5% of the O2Hb was converted to metHb. Cyanide was effective in slowing methemoglobin formation in this mixture even at pH 6.4, where the conversion was complete in less than 30 s in the absence of cyanide. These experiments suggested that free metHb or a metHb-nitrite complex was required as one reactant in the conversion of O2Hb to metHb by nitrite. Both CN and high pH inhibited the oxidation of oxyhemoglobin. I measured the effect of pH on the conversion of O2Hb to metHb by nitrite in a more quantitative fashion in phosphate buffers containing 0.1 mol of phosphate per liter, with use of a nitrite/heme ratio of 17/1. The results in Figure 1 show that the rate of the reaction is slower at higher pH and the induction period is prolonged. Curves similar to those shown in Figure 1 may be obtained at lower pH with less NO2- and at higher pH with greater NO2- concentration. In all cases there is a continually increasing rate up to about 66% conversion and then a decreasing rate as the reaction approached completion. It will be demonstrated later that the overall reaction as shown in Figure 1 is a result

pernate of methemoglobin was measured in 20 mmol/liter potassium phosphate and tris(hydroxymethyl)aminomethane chloride buffers. Curves of identically prepared methemoglobin solutions containing sodium nitrite at 85-fold the molar heme iron concentration were also obtained.
Results shown in Figure 2 indicate that aquamethe-

moglobin (the form present at low pH) has absorption maxima near 630 and 500 nm, with shoulders at 575 and 540 nm. At alkaline pH, the methemoglobin hydroxide has maxima only at 575 and 540 nm. The two isosbestic points of aquamethoglobin and methemoglobin hydroxide at 615 and 522 nm observed by Austin and Drabkin (1) are apparent, with a third isosbestic point near 483 nm. Methemoglobin nitrite, the major form
present at pH 6 in the presence of nitrite, has an ab-

sorption peak near 625 nm and one nearly in common with one for methemoglobin hydroxide at about 538 nm. Methemoglobin nitrite absorbs considerably more strongly at 575 nm than does aquamethemoglobin, but, unlike methemoglobin hydroxide, has no distinct absorption maximum at this wavelength. The similarity
of absorption spectra of methemoglobin nitrite and

methemoglobin hydroxide, together with the competition of nitrite and hydroxide for the methemoglobin, led Van Assendelft and Zijlstra (8) to conclude that the absorption spectra of methemoglobin nitrite was independent of pH. Dissociation constant of methemoglobin nitrite. The dissociation constant of methemoglobin hydroxide at 20 #{176}C is 1.13 X 10 mmol/liter (1, 9). Smith (10) estiCLINICALCHEMISTRY,Vol. 22, No. 12, 1976 1987

0 0

I
4)

4) 0

2..

0-

NoN02
--

-I

K3Fe(CN)6

6.0 pH

70

7.5

Lmm4)

W4)

L094)

Fig. 2. Absorption curves for methemoglobin in the absence (left) and in the presence (right) of NaNO2
Each solution contained heme, 73 zmol/llter as metHb, in 0.1 mel/liter phosphate, pH 7.1, or tris(hyoxymethyI)aminomethane, pH 8.1 and 9.0. SodIum nitrite, where used, was dissolved in the buffers to 6.21 mmol/Iiter, 85-fold the heme concentration

Fig. 3. Proton change when oxyhemoglobin is treated with NaNO2 or


K3Fe(CN)6

at fourfold the molar heme concentration

mated the methemoglobin nitrite dissociation constant spectrophotometrically at 25 #{176}C and pH 7.4. A dissociation constant of 3 mmol/liter was reported, although other data caused him to believe the value to be too large. A number of estimates of the methemoglobin
nitrite dissociation constant were made in dilute tassium phosphate buffers (20 mmol/liter) where methemoglobin is expected to be predominately in tetrameric state. Guidotti (12) has estimated the pothe the tet-

in 20 mmol/liter phosphate buffer at pH 6.4 were then used in which the initial ratio of NO2- to O2Hb varied from 2 to 5. When the reaction was complete, as judged
by the decrease of absorbance at 540 nm, the remaining

nitrite

was determined.

Eight such experiments

were
disapof the

done, which showed that the moles of nitrite pearing correspond to 1.68 0.15 (std. error

mean) times the moles of heme iron oxidized. Clearly, about three nitrite molecules disappear for each two molecules of O2Hb iron converted to metHb. Qualitative data had shown that the conversion of
O2Hb to metHb by nitrite in unbuffered solution resulted in a more alkaline pH. The extent of proton absorption in this reaction was measured with a Radiometer pH Stat (The London Co., Westlake, Ohio 44145) when O2Hb at various pH values was treated with nitrite. Similar measurements were made when K;3Fe(CN)6 was used as oxidant. The results in Figure 3 show that, within experimental error, the reaction with nitrite consumes one proton for each iron atom oxidized at all pH values tested. In contrast, the oxidation with K3Fe(CN)6 actually produces protons if the pH exceeds 7.4 but is quite similar to the nitrite reaction at pH 6.3. It is probable that K3Fe(CN)6 may produce protons by oxidation of thiol groups in addition to iron atoms (10, ii), a reaction that does not occur with nitrite. All data available are consistent with the stoichiometry of the overall reaction according to the following equation: 2O9Fe2 + 3N02 + 2H
=

ramer-dimer

dissociation

constants

for cyanmethem-

oglobin to be similar to those for O2Hb. The present measurements were based on the decrease of absorbance at 630 nm when nitrite was added to aquamethemoglobmn in known amounts. A nitrite concentration 800-fold that of the heme iron was used to establish the

absorbance of methemoglobin nitrite. Essentially no methemoglobin hydroxide is present at this pH. Seven measurements were made in which the methemoglobin nitrite varied from 38 to 84% of the total methemoglobin. The mean value observed was K = 1.11 0.10 (std. error of the mean) mmol/liter. The reason for the difference between this value and the value reported by Smith (10) is not apparent unless it is related to the wavelength and pH at which the measurements were made. At 560 nm as used by Smith (10), the absorbancy of methemoglobin nitrite is nearly identical to that of methemoglobin hydroxide, as shown in Figure 2, and both complexes will be present in significant amounts

at pH 7.4.
Stoichiometry required of the reaction. O2Hb

2Fe

+ 3NO

+ H2O (2)

were made to determine


to convert

A number of attempts the minimum amount of nitrite


to metHb by limiting the

amount of nitrite. The reaction was done in 20 mmol/ liter phosphate buffer at pH 6.4. Although O2Hb was
completely converted to metHb when the molar ratio of N02 to O2Hb (heme iron) was 1.5 or greater, the reaction was so slow that control air oxidation of the

hemoglobin
1988

compromised

the results. Solutions of O2Hb


Vol. 22, No. 12, 1976

where O2Fe2+ and Fe represent the moles of heme iron present as O2Hb and metHb, respectively. Equation 2 also accounts for the failure of nitrite to release molecular oxygen from O2Hb, because all oxygen is consumed in the reaction. Kinetics of the reaction. During these studies I observed that O2Hb is converted to metHb by nitrite much more slowly in the presence of cyanide. Furthermore,

CLINICAL CHEMISTRY,

Table 1. First-Order Velocity Constants for the Cyanide-insensitive Conversion of O2Hb to metHb by Nitrite a
O2Hb oxidized/mm, pH
11,2, mm

o 80 I
0

6.12 6.11 6.30

21.3 27.8 27.8

3.25 2.49 2.49

60

40
4)

6.47 6.68 7.06

10.3 6.0 1.9

6.7 11.6 36.9


1.82 mmol/liter, and [021-lb

t = 28 #{176}C; IKCN] = 1.82 mrnol/liter, (NaNO2I + metHb] 50 Mmol/liter (heme)

5 Time

Solid curve

mm

Dashed curves

Table 2. First-Order Velocity Constants for the


Cyanide-Insensitive Conversion of O2Hb to metHb by Nitrite a
O2Hb oxidized/mm,
mm

Fig. 4. Conversion of oxyhemoglobin to methemoglobin by nitrite at ph = 7.25


Each solution contained phosphate buffer (18 mmol/Ilter) and hemoglobin (50 zmol of home per liter). Solutions corresponding to the dashed curves also contained 0.5 mel of Nal per liter. The reaction was started by adding NaNO2, up to 935 umol/Iiter, or a mixture of NaNO2 + KCN, up to 935 Mmol of each per liter. Note that on the abscissa, units of minutes are used for the solutions with phosphate alone (solid ctrves), but hours for the Nal-containing solutions (broken curves)

pH

N14NO2 KCN mmol/lher

6.03 6.04
6.11

0.47 0.94
1.81

0.47
0.94
1.81

6.12
6.05
a =

1.81

1.81

2.64
28 #{176}C, [O2Hb

2.64
+ metHb]
=

8.2 25.3 27.8 21.3 30.8


(home)

8.45 2.74 2.49


3.25

00

2.25
80
0 0

50 tmol/liter

60

the kinetics of the reaction were strictly first order in O2Hb concentration in the presence of cyanide and followed closely the induction period of the usual reaction represented in Figure 1. I studied this cyanide-insensitive portion of the reaction by evaluating the first-order reaction rate constants as a function of pH at constant nitrite concentration and of nitrite concentration at constant pH. It was found that the reaction rate constant for the cyanide-insensitive reaction was directly proportional to the proton concentration (Table 1) with constant nitrite. When pH was

40

20

0 m,n

12

Time

Fig. 5. Effect of neutral salts on the conversion of oxyhemoglobin

to methemoglobin

by nitrite at pH

7.25

maintained constant, the reaction rate increased with increasing nitrite concentration (Table 2). Variations in the reaction velocity from direct proportionality were
observed but are probably explained by the small differences in pH and in the mixing and starting times for the rapid rates used. It was clear in all these studies that

Each solution contained phosphate buffer (18 mmol/liter) and the indicated concentration of NaCI or Nal. All solutions were 50 zmoI/liter in home iron as hemoglobin, and NaNO2 was added to a concentration of 935 zmol/liter at zero time

no autocatalytic effect occurred in the presence of cyanide at any pH or nitrite concentration used. The exof CN- on the overall reaction is illustrated curves of Figure 4, which shows a typical reaction at pH 7.25 that was complete in mm. In the presence of CN- the autocatawas completely eliminated and the cyanide-insensitive reaction, t112 = 204 mm, accounted for less than 4% conversion in the same time. The rates of the overall reaction, cyanide-insensitive plus cyanide-sensitive, through the major portion of the reaction (20 to 90% metHb), were very closely represented by the product of O2Hb concentration times the treme effect in the solid autocatalytic less than 10 lytic effect

the cyanide-sensitive portion of the reaction might result from the interaction of one unit of O2Hb with two units of methemoglobin nitrite. The previously deter-

mined stoichiometry
must be present

suggested

that the O2Hb, at least,

square of the metHb concentration.

This suggested that

unit. I tested this by measuring the rate of metHb formation of neutral salts known to dissociate O2Hb tetramers into the afl-dimers (12-14). In the presence of 0.5 mol/liter Nal, oxyhemoglobin is about 96% dissociated into dimers (13). The conversion of such O2Hb dimers to metHb by nitrite is shown in Figure 4 and compared with the same reaction in the absence of NaI where about 74% of the O2Hb is present as tetramers. The reaction in the presence of Nal was first order, with t 1,2 = 77 mm, and showed no autocatalytic effect. Addition of cyanide caused the reaction to start somewhat as the tetrameric

hypothesis in solutions

CLINICAL CHEMISTRY. Vol. 22, No. 12, 1976

1989

more slowly, giving t112 = 86 mm for the cyanide-insensitive reaction. Effects of increasing NaC1 concentration are shown in Figure 5. The inverse of the time required to go from 20 to 90% metHb, 1/t20...0, was used to estimate the reaction rate. Tetramer-to-dimer dissociation constants in 0.1, 1.0, and 2.0 mol/liter NaCl reported by Kellett (13) were used to calculate the initial concentrations of O2Hb tetrainer in the solutions. In the absence of added NaC1, with 74% of the total O2Hb present as tetramers, a value of t2oo = 1.4 mm was observed. Increasing the NaCI to 0.97 mol/liter, a concentration at which 52% of O2Hb is present as tetramers, slowed the reaction to t20 = 1.95 mm. Further increase of NaCl to 1.97 mol/liter, a concentration at which 39% of the O2Hb is present as tetramers, caused an additional decrease in rate to t2g0 = 2.9 mm. Thus with NaCl = 0.97 mol/liter the O2Hb tetramer concentration is 70% of that of the control in the absence of NaC1 and the reaction rate is 72% of the control. In 1.97 mol/liter NaCl the O2Hb tetramer is 52% of the control, while the reaction rate was 48% of the control. These data suggest that the autocatalytic cyanide-sensitive portion of the reaction has an obligatory requirement for O2Hb tetramers. The cyanide-insensitive portion occurs with either the dimeric or tetrameric form, although the rates may differ. These suggestions are supported by the data in Figure 4, which show that the reaction in the presence of 0.5 mol/liter Nal (96% a-diniers) proceeds nearly the same in the presence and absence of cyanide. Proposed reaction mechanism. Data presented above show that the overall conversion of O2Hb to metHb in the presence of excess nitrite may be explained as the sum of two reactions. One of these, the rate-limiting step at the start of the reaction, involves the oxidation of O2Hb to metllb and is unaffected by the presence of CN. The rate of this reaction is directly proportional to both W and NO2- concentration but is independent of metHb. The stoichiometry of the cyanide-insensitive reaction has not been determined. The reaction occurs when the O2Hb is present as dimers or as tetramers, although there is some evidence that dimers react more rapidly than tetramers under otherwise comparable conditions. The second reaction dominates both the stoichiometry and rate of the overall reaction. Each mole of O2Hb heme that is converted to metHb heme oxidizes 1.5 mol of nitrite to nitrate and consumes 1 mol of H. The rate at constant pH in the presence of excess nitrite is proportional to the product (O2Hb)(metHb)2. The O2Hb must be in the tetrameric form and metHb must be present as the nitrite derivative, because CN- completely stops the reaction. The preferred tetramerdimer state of the metHbNO2 is not known, as the dissociation constants have not been evaluated for this

compound. Guidotti (12) has shown that the tetramer-dimer dissociation constants for cyanmetHb and O2Hb are similar. This suggests that both O2Hb and metHbNO2 must both be present as tetramers. The autocatalytic nature of this reaction is due to the fact that all metHb present (either endogenous, that formed in the cyanide-insensitive reaction of O2Hb with NO2-, or that formed from the reaction of O2Hb with metHbNO2j will associate with remaining excess NO2and increase the concentration of this reactant. The reaction rate thus continues to increase until the O2Hb becomes limiting at about 66% conversion.
Supported by the Naval Medical Research and Development Command, National Naval Medical Center, Department of the Navy, Research Task No. MRO41.01.01.01039. I wish to acknowledge the assistance of Dennis P. Nelson, LT, MSC, USN, Experimental Medicine Department, Naval Medical Research Institute, in the measurement of the proton utilization during formation of methemoglobin. I also wish to express my deep appreciation to Dr. Louis D. Homer, Clinical and Experimental Immunology Department, Naval Medical Research Institute, for valuable discussions on the interpretation of the kineticdata.

References
1. Austin, J. H., and Drabkin, D. L., Spectrophotometric studies III. Methemoglobin. J. Biol. Chern. 112,67 (1935). 2. Kiese, M., The biochemical production of ferrihemoglobin-forming derivatives from aromatic amines, and mechanisms of ferrihemoglobin formation. Pharmacol. Rev. 18, 1091 (1966). 3. Smith, R. P., Some features of the reaction between cobaltinitrite and hemoglobin. Toxicol. App!. Pharmaco!. 17, 634 (1970). 4. Rodkey, F. L., and ONeal, J. D., Effects of carboxyhemoglobin on
the determination (1974). of methemoglobin in blood. Biochern. Med. 9,261

5. Van Kampen, E. J., and Zijlstra, W. G., Standardization of hemoglobinometry. II. The hemiglobincyanide method. Clin. Chim. Acta 6,538 (1961). 6. Schneider, N. R., and Yeary, R. A., Measurement of nitrite and nitrate in blood. Am. J. Vet. Res. 34, 133 (1973). 7. Wegner, T. N., Simple and sensitive procedure for determining nitrate and nitrite in mixtures in biological fluids. J. Dairy Sci. 55,
642 (1972).

8. Van Assendelft,
hemiglobin using

0. W., and Zijistra,


nitrites. Clin. Chim.

W. G., The formation


Acta 11,571(1965).

of
beActa

9 Brunori,
tween acid

M., Amiconi, G., Antonini, E., et al., The transition and alkaline heme proteins. Biochim. Biophys.

154, 315 (1968). 10. Smith, R. P., The nitrite methemoglobin complex- its significance in methemoglobin analyses and its possible role in methemoglobinemia. Biochem. Pharmacol. 16, 1655 (1967). 11. Smith, R. P., and Gosselin, R. E., On the mechanism of sulfide inactivation by methemoglobin. Toxicol. APP!. Pharmaco!. 8, 159
(1966).

12. Guidotti, 0., Studies on the chemistry of hemoglobin II. The effect
of salts on the dissociation 242, 3685 (1967). of hemoglobin into subunits. J. Biol. Chem.

13. Kellett,
gand-linked (1971).

G. L., Dissociation
dissociation at

of hemoglobin
neutral H. J.

Mo!.

into subunits. LiBiol. 59, 401

14. Tomita, dissociation

S., Maeda, N., Enoki, Y., and Okuda, T., An evidence for of hemoglobin into monomer in concentrated potassium

iodide. J. Mo!. Biol. 69, 303 (1972).

1990

CLINICAL CHEMISTRY,

Vol. 22, No. 12, 1976