AN ELECTROCHEMICAL STUDY OF HEMOGLOBIN.

BY JAMES B. CONANT.

(From the Chemiea2 Laboratory of Harvard University, Cambridge.)

(Received for publication, July 10, 1923.)

In the course of a series of investigations on the physical chemis- try of hemoglobin now being carried on in the Laboratory of Physical Chemistry of the Harvard Medical School, Dr. E. J. Cohn and Dr. R. M. Ferry discovered certain abnormalities in the behavior of a hydrogen electrode immersed in solutions of hemoglobin and oxyhemoglobin. On studying the matter further they found that any inert electrode when dipped into a hemoglobin solution gave a fairly de&rite potential which varied with the degree of oxygenation of the hemoglobin, It was at first thought that this potential might be the oxidation-reduction potential of the system hemoglobin-oxyhemoglobin, and in order to investigate this possibility the present work was undertaken by the author. It was soon found that the titration method of determining oxida- tion potentials, now being applied in this laboratory to a study of quinones (1, 2) and first proposed by Clark (3), was applicable to hemoglobin solutions; the system of oxidized and reduced com- pounds involved, however, was not oxyhemoglobin-hemoglobin, but hemoglobin-methemoglobin. The reactions which are the basis of the method are: hemoglobin + K3Fe(CN)6 + methe- moglobin; and 2 methemoglobin + NazSz04 + 2 hemoglobin.

Procedure.

The experiments were performed in an apparatus identical with the one previously employed in this laboratory in our studies of anthraquinone derivatives (1). A glass cell holding 200 to 300 cc. of solution was fitted with a mechanical stirrer, gas inlet and outlet tubes, and three electrodes, and was connected to a satu- rated calomel electrode by an agar-potassium chloride bridge. The hemoglobin solution was mixed with a suitable buffer solution or

401

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402 Electrochemical Study of Hemoglobin

the requisite salts were dissolved in it so that the total salt concen- tration was about 0.2 M. This solution was placed in the cell which was swept out with nitrogen. Definite increments of reducing agent (sodium hydrosulfite) or oxidizing agent (potassium ferricy- anide) were added from a burette, and the potentials of the elec- trodes were determined after each addition. In this way a titra- tion curve was obtained such as is shown-in Fig. 1. As might be expected, considering the complexity of the hemoglobin molecule, the experimental difficulties were much greater than those exper- ienced with even such substances as the anthraquinones. In order to be sure that the potentials recorded were significant equi- librium potentials it was necessary to be certain that they were constant for at least 15 minutes, and that the potentials of at least two of the three different electrodes were identical. Several hours were required before equilibrium was reached in many cases. The three electrodes employed were platinized platinum, bright plati- num, and gold. Certain electrodes gave erratic potentials and had to be discarded; soaking the electrodes in a solution of methe- moglobin for several days seemed to improve them. Because of these difficulties the actual values of the potentials recorded in this paper must be considered as provisional and possibly to be in error by 30 or 40 millivolts.

A 0.1 N solution of potassium ferricyanide was used as the oxidizing agent; it must be kept away from the light. The sodium hydrosulfite solution which was employed was made up fresh every day and contained a small amount of sodium carbonate which greatly increased its stability. It was protected from oxidation in the burette by a layer of xylene. Each solution was standard- ized by a titration with 0.01 M indigo disulfonate which in turn was standardized against titanous chloride. (The titrations must be carried out in an atmosphere of carbon dioxide.) The hydro- sulfite solution slowly changes in the burette and should be re- standardized at least every 6 hours.

The Hemoglobin-Methemoglobin Potential.

The hemoglobin used in this work was kindly furnished by the Laboratory of Physical Chemistry of the Harvard Medical School; it was prepared from horse blood according to the method of Ferry.l

*Ferry, R. M., J. Biol. Chem., 1923, Ivii, in press.

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J. B. Conant 403

FIG. 1. Titration of hemoglobin and methemoglobin in pH 6.8. Poten- tial against saturated calomel electrode plotted vertically; cc. of Na&.04 plotted horizontally from x to z; cc. of K3Fe(CN)a from z to x. Curve A titration of methemoglobin with Na&Oa; Curve B hemoglobin with KsFe(CN)a.

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404 Electrochemical Study of Hemoglobin

A great variety of samples was used; some freshly prepared con- sisted almost wholly of oxyhemoglobin, while others which had stood several months in the ice chest contained a large proportion of methemoglobin. Except for the varying content of methemo- globin no difference was observed in the behavior of the various samples.

Titrations can be carried out most conveniently by first adding small increments of ferricyanide to the hemoglobin solution (which usually contains varying amounts of oxy- and methemoglobin) until this reagent is present in excess as shown by the potential. Nitrogen is passed through the cell and then the titration with sodium hydrosulfite is commenced; the initial amount of ferricy- anide necessary to convert all the material to methemoglobin is only of significance in regard to the relative amounts of oxy- and methemoglobin in the original sample. After the completion of the titration with sodium hydrosulfite, the hemoglobin can be titrated back to methemoglobin with ferricyanide. This titration back and forth can be repeated a number of times with the same material. Table I summarizes the results of a number of experi- ments. A nitrogen analysis of the hemoglobin sample employed in each experiment furnished the necessary information for deter- mining the mols of hemoglobin present in the cell. (It was assumed that hemoglobin contained 17.3 per cent of nitrogen and that its molecular weight was 16,700.) From the amount of sodium hydrosulfite or ferricyanide required in a titration the number of mols of hemoglobin could be calculated. A good agreement was obtained between the amount of hemoglobin pres- ent according to the nitrogen analysis and according to the titra- tions calculated on the basis of 1 mol of Hb = 1 mol of K3Fe(CN)s and 2 mols of Hb * 1 mol of NazSz04. A comparison of the values in Column 2 and in Columns 5 and 9 of Table I, shows the agree-

-merit to be within our present experimental error (about 10 per cent). The change from reduced hemoglobin to methemoglobin involves only one hydrogen equivalent of oxidizing or reducing agent per gram- molecule, calculated on a molecular weight of 16,700.

Table II gives the actual potentials of the three electrodes in two of the titrations in Experiment 2. The curves in Fig. 1 are plotted from this table, the average potential being plotted against the amount of sodium hydrosulfite or ferricyanide added. It will be

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J. B. Conant 405

observed that except for a few cases near the end-point the poten- tials of all three electrodes are quite close together. The poten- tials enclosed in brackets are obviously in error and were not included in the averages.

TABLE I.

Titration of Hemoglobin at 19 f 1~.

1

2

3*

4

5

6

7*

8

9 -

(

(

(

(

C

(

C

(

c -

Nad3~0~ titrations.

j.000281

I.00252

j.00165

1.00067:

).00044i

). 000224

) ooow

I.000224

) .00099c

-

.-

0 .O

0 0

0

i0

‘0

LO

10

LO

10 -

.0330 4.00.00026E

.0285 4.50.00025$

.028544.00.00250

.032839.00.00256

.0320 19.0~0.00122

.0340 9.00:00061:

.0355 6.00.000427

.0280 4.00.00022:

.0375 10.00.00075C

.0360 3.50.00025C

.0224 21.0/O. 00094c I ’

c volts

i-0.06s to.081

to.124 to.104

to. 114

-0.01;

-0.031

to.116

to.024

t0.02c

to.029

)

.O

LO 10

LO

10

.O

1

LO

10

IO -

.109

,109 .109

.109

.109

.109

.109

.109

.lOO

KsFe(CN)s titrations. - -

j

5 -

cc.

2.8 )(

I0.C )( !l.E it

2.: i(

5.E i(

4 (

7.5 iC

2.c I(

9.5 ic L -

-

--

i-l

i -I

-I

)-

I-

i-l

I-

)- -

c o&s

).0003(H

).00219 j.00234

j.00136

).00060c

).00044

-0.08!

-0.11, -0.11,

-O.lO!

-0.021

-0.03.

).00081E

).00021E

).ooo95C

-0.02:

-0.08.

-0.01)

.

D

4 4

a

6

1

1

11

Y

m a

6.8

8.5

6.8

9.63

9.63

8.5

9.63

1.3

1.3

A phosphate buffer solution was used for pH 6.8; a borate-boric acid solution for 8.5; a sodium hydroxide-borate solution for 9.63, and 0.2 M

NHaOH forpH 11.3. The total salt concentration in each case was about 0.2M.

*Slight decomposition had occurred before the sample was used, judged by the odor.

The two curves (Fig. 1) showing the reduction with hydro- sulfite and the oxidation with ferricyanide are within 20 milli- volts of each other. Such relatively consistent results are best obtained by using rather concentrated solutions of hemoglobin (about 10 per cent) and by having reliable electrodes.

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406 Electrochemical Study of Hemoglobin

Since, for the transformation of methemoglobin (MHb) to reduced hemoglobin (Hb), only one hydrogen equivalent is used per mol calculated on a molecular weight of 16,700, which in

TABLE II.

Titration of 0.00248 Mot of Hb in pH 8.6. Potentials Against Saturated

cc.

0.00

2.00 4.00 6.00

10.00 14.00 18.00 22.00 26.00 32.00 36.00 40.00 41.00

0.00 0.56 1.00 2.00 3.00 4.00 8.00

13.00 17.00 19.00 20.00 21.00 22.00 22.50

Bright Pt.

-0.105 -0.116 -0.120 -0.125 -0.128 -0.121 -0.136 -0.154 -0.170 -0.190 -0.234 -0.628 -0.672

-

.-

-

Gold. Platinized Pt.

-

-0.107 -0.106 -0.120 -0.114 -0.120 -0.120 -0.128 -0.126 -0.138 -0.131 -0.131 -0.132 -0.141 -0.143 -0.166 -0.164 -0.170 -0.172 -0.198 -0.192 -0.237 -0.211 -0.607 [-0.3811 -0.665 [-0.4651

-

_-

- &Fe(CN)~O.lOS molar.

7

-0.672 -0.665 [-0.4651 -0.665 -0.532 -0.556 [-0.4411 -0.540 -0.307 -0.340 [--0.2941 -0.322 -0.207 -0.257 -0.222 -0.227

-0.189 -0.208 -0.185 -0.197 -0.163 -0.187 -0.164 -0.175 -0.141 -0.155 -0.142 -0.149 -0.132 -0.137 -0.129 -0.133 -0.118 -0.121 -0.117 -0.119 -0.110 -0.112 -0.110 -0.111 -0.099 -0.103 -0.103 -0.102 -0.084 -0.086 -0.096 -0.090

0.000 0.000 -0.033 -0.011 +0.079 +0.079 +0.045 +0.070

-

-

-

-

-

Average potential.

-0.106 -0.118 -0.120 -0.126 -0.132 -0.129 -0.140 -0.162 -0.170 -0.193 -0.225 -0.610 -0.666

turn is based on the assumption of 1 iron atom per molecule, there is every reason to believe we are dealing with the change of a ferri compound to a ferro compound. In this event the potential is given by the equation:

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J. B. Conant 407

(1) RT [MHbl

T = r. + T- In [~bl

The value for the normal potential (1~~) is most conveniently obtained by interpolating the titration curve to the mid-point where [MHb] = [Hb]. The values thus obtained, referred to the normal hydrogen electrode, are given in Columns 6 and 10 of Table I. The present results are not sufficiently accurate to allow of a significant comparison of the slope of the titration curve with the theoretical slope calculated from the above equation.

The variations in r,, with change in the pH of the buffer solu- tion require comment, although a more accurate determination of the values of r0 will be necessary before any very significant conclusions can be arrived at in this connection. If the activities of the oxidized and reduced compounds remain constant, the potential of the type represented by equation (1) should be inde- pendent of the hydrogen ion concentration. For the range pH 6.8 to 3.5 this seems to be the case for hemoglobin (within the present experimental error). There is a marked change in poten- tial, however, between pH 3.5 and 9.6, and the potential is approxi- mately constant again between pH 9.6 to 11.3. This change in potential at 8.5 to 9.6 may be caused by the formation of a salt by one of the acidic hydrogen atoms in the molecule which is pres- ent as the free acid in the range pH 6.8 to 8.5. If some such change takes place and the dissociation constants of this hydrogen in the reduced and oxidized form are different, a shift in the oxida- tion-reduction potential would take place. The following tabula- tion is a summary of the average values of H. (referred to the hydrogen electrode) at different values of pH.

PH I 7ro

volts

6.8 +0.092 Cf0.022) 8.5 +0.115 (f0.011) 9.63 -0.016 (fO.040)

11.3 -0.025 (f0.050)

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408 Electrochemical Study of Hemoglobin

E$ect of Oxygen and Carbon Monoxide on the Potential.

It is thus evident that the potential of an inert electrode immersed in a hemoglobin solution depends on the relative amounts of reduced hemoglobin and methemoglobin present. The effect of passing air or carbon monoxide into such a mixture would be to diminish the amount of reduced hemoglobin present by form- ing oxy- or carboxyhemoglobin, and to raise the potential (the concentration of methemoglobin being unaffected by the presence of the gas). Removing the gas by passing in nitrogen should lower the potential again to its original value. These qualita- tive predictions have been verified by a number of experiments of which the following is typical.

The cell contained 175 cc. of a solution of hemoglobin, containing 2.6 gm. of hemoglobin per 100 cc. and mono- and disodium phosphates correspond- ing to pH 6.8. Sodium hydrosulfite was added until present in slight excess (as shown by the potential), the cell having previously been swept out with nitrogen. Air was then passed in for some time, the excess of hydrosulfite being rapidly oxidized and the potential rising to a value some 100 millivolts higher than the mid-point of the normal titration curve as shown by the following figures.

Nature of g&8.

Equilibrium potential (referred

to the hydrogen electrode).

Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . Nitrogen .............................................. Carbonmonoxide ...................................... Nitrogen .............................................. Air ..................................................

o&s

+0.239 +0.149 +0.309 +0.139 +0.250

If the reduced hemoglobin (either freshly prepared or after reduction with sodium hydrosulfite) is oxygenated by passing air through the cell and then titrated with sodium hydrosulfite 2 mols of hydrosulfite are used up per mol of hemoglobin. This corresponds to the 1 molecule of loosely combined oxygen in oxyhemoglobin. The potential during this titration is some- what indefinite until the end-point is reached, but is usually at first some 100 or 200 millivolts above the normal titration curve. The dissolved oxygen in equilibrium with the oxyhemoglobin

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J. B. Conant

apparently oxidizes the hydrosulfite as fast as it is added and the potential corresponds to the ratio of the small amount of methe- moglobin to the relatively large amount of reduced hemoglobin.

The potentials first noted by Dr. Cohn and Dr. Ferry in ordinary hemoglobin-oxyhemoglobin solutions can be explained in the light of the experiments just mentioned. The potential is due to the oxidation-reduction equilibrium involving methemoglobin and reduced hemoglobin, since there is probably a small amount of methemoglobin present in all hemoglobin solutions due to a slight decomposition of the oxyhemoglobin. By varying the oxygenation of such a mixture, the amount of free reduced hemoglobin is varied, and thus the potential is changed. That the potential is not due directly to the oxyhemoglobin is evident from the fact that carbon monoxide has the same effect as oxygen on the potential of a hemoglobin solution.

It seems quite evident that the oxygenation of hemoglobin is not an oxidation in the electronic sense, whereas the formation of methemoglobin is. When one looks for an analogous pair of simple ferri and ferro compounds stable in alkaline solution and reversibly oxidized and reduced, one is immediately struck with the similarity between hemoglobin and methemoglobin on the one hand, and sodium ferrocyanide and ferricyanide on the other. The work of Manchot (4) and Baudisch (5) makes this line of speculation still more tempting. Manchot found that ferro com- pounds of the type Na3[Fe(CN)6NH2R] combine with carbon monoxide and nitric oxide in a fashion similar to hemoglobin. Baudisch believes the compound Naa [Fe(CN)s(02)] is formed under certain conditions, the oxygen being loosely held by one of the covalences of the iron in the same manner as the molecule of amine in Manchot’s compound. As Manchot pointed out, his compounds differ from hemoglobin in their action with carbon monoxide only in that the absorption is irreversible.

Nar IFe(CN)aNH2Rl $ CO -+ Naa [Fe(CN)&Ol + RNHz

If the amino group were part of a complex molecule joined at some other point to the central iron atom, the two products on the right of the above equation would be part of the same mole- cule and the reversible absorption of gas by a single molecule might be realized. The following formulas for hemoglobin, oxyhemo-

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410 Electrochemical Study of Hemoglobin

globin, and methemoglobin represent this idea. The four pyrrole rings (found in hematin) can be imagined as being the equivalent of 4 molecules of hydrocyanic acid,2 the corresponding ions (represented by Pr) replacing 4 cyanide ions in Manchot’s com- pounds. The fifth cyanide ion is replaced in these formulas by an acidic group of the globin molecule (G) while a free amino group of the same protein plays the role of the easily replaced amine.

[

(NIL) -G (Od ~Hz Na3 (Pr)dFe(OOC) - 1 + oae Naa [ (Pr)cFe(OOC) - G 1 Hemoglobin (ferro compound). Oxyhemoglobin.

II

(NIL) - G Naz (Pr)cFe(OOC) - 1

Methemoglobin (ferri compound).

While these formulas are, of course, speculative and incomplete it might be worth pointing out that they are in accord with several very different properties of hemoglobin. The relative ease with which the protein is split off from the hematin part of the molecule corresponds on the basis of these formulas to the breaking up of a complex iron salt and not to the hydrolysis of an amide linkage. The 3 hydrogen atoms of the complex ferro acid in hemoglobin (written above as the trisodium salt) by analogy with ferrocy- anic acid ought to be moderately acidic and their dissociation constants would be presumably greatly affected by the nature of the six groups attached to the central atom. The strength of the tri-basic acids corresponding to hemoglobin and oxyhemoglobin should differ. Henderson (6) as a result of his study of the absorp- tion of carbon dioxide by the blood believes that this must be the case. If the acidic hydrogen atoms involved in this change were part of ordinary acidic protein groups it is hard to see why the absorption of oxygen in some other portion of the molecule should materially affect their dissociation constants. The shift in the normal oxidation-reduction potential of hemoglobin referred to above, which seems to take place between pH 8.5 and 9.6, would be interpreted in terms of these formulas by the differ- ence in the dissociation constants of one of the hydrogen atoms of

* There is considerable chemical similarity between pyrrole and hydro- cyanic acid.

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J. B. Conant 411

the ferro and ferri complex acids. It can be shown that if this is the case the potential of the hemoglobin-methemoglobin system would be given by equation (2) in which

(2) RT [methemoglobin] RT

a = *O + F In [hemoglobin] ’ 7 In

K1 and Kz represent the dissociation constants of the weakest of the hydrogen atoms of the complex ferri acid and ferro acid, respectively. Such an equation as (2) would correspond to a shift in the oxidation-reduction potential over a rather small range of pH in which [Hf], Kl, and K2 were of the same order of magnitude; for all other ranges the potential would be independent of the hydrogen ion concentration.

Ferricyanide Method of Determining Combined Oxygen.

The action of potassium ferricyanide on hemoglobin solutions which is the basis of Haldane’s method for determining the combined oxygen, is readily interpreted now that the relation between hemoglobin and methemoglobin has been established. Reduced hemoglobin, oxyhemoglobin, and free oxygen are in equilibrium in the solution; the ferricyanide which is added oxi- dizes the reduced hemoglobin to methemoglobin, removing one of the components involved in the equilibrium. Oxyhemoglobin, therefore, dissociates into free oxygen and reduced hemoglobin as fast as the reduced hemoglobin is changed to methemoglobin, and the process continues until all the hemoglobin is converted to methemoglobin and the entire oxygen is liberated. Potassium ferricyanide is a peculiarly advantageous reagent for this oxida- tion as it is stable in alkaline solutions, has a high enough oxida- tion potential, and yet is not a sufhciently powerful oxidizing agent to attack the protein. The reaction between it and hemoglo- bin may be represented as follows:

Na9 [hemoglobin ion1 + NaJFe(CN) 6 -+ Nat [methemoglobin ion] + Na4Fe(CN) )

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412 Electrochemical Study of Hemoglobin

Experiments with Hematin.

Hematin dissolved in solutions of pH 8.5 or greater alkalinity is reduced by sodium hydrosulfite to hemochromogen which in turn is oxidized to hematin by air or ferricyanide. The reduction with hydrosulfite may be followed by the change in potential in exactly the same manner as hemoglobin. The normal potential of this reversible reduction, however, is much lower than that of hemoglobin, being about -0.153 (~0.030) in pH 8.5, -0.256 (f0.020)in pH 9.6, and -0.315(stO.O10) in pH 11.3. The experi- mental difficulties in this electrometric titration seem to be fully as great as those encountered with hemoglobin and the results may be in error by 30 or 40 millivolts. Between 70 and 80 per cent of 1 mol of sodium hydrosulfite per mol of hematin was used up in each titration. This indicates that the reaction involves two hydrogen equivalents though the results cannot be considered as conclusive, particularly as it was not possible to obtain satisfactory titrations of hemochromogen with ferricyanide. The progressive change in the normal potential with change in pH further indicates that 2 hydrogen atoms are involved in the reduction which would be represented thus:

Hematin + 2Hf + 2E k hemochromogen

The potential of such a change (which is the usual quinone reduc- tion) is given by the equation:

RT [hematinl RT ?r = ?r0 ’ %- log [hemochromogen] ’ 7 log IH+’

The term $ log [H+] in this equation corresponds roughly to

the change in potential observed with variations in pH values. Thus, the relation between hematin and hemochromogen seems

to be similar to that between quinone and hydroquinone, the unsaturated system involving the pyrrole rings probably being the point of reduction. If this is the case the reduction of hematin bears no relationship to the reduction of methemoglobin nor of oxyhemoglobin, but finds its parallel in the reduction of the iron-

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J. B. Conant 413

free hematoporphyrin to the leuco compound, which we have found can be brought about by vanadous chloride and is reversed by ferric chloride. This view of the structure of hemochromogen is contrary to Kuster’s ideas (7) according to which hemoglobin, oxyhemoglobin, and hemochromogen are ferro compounds, while methemoglobin and hematin are ferri compounds. Further inves- tigations along this line are planned, although it would appear that this problem is but distantly related to the oxidation and oxygena- tion of hemoglobin.

I am greatly indebted to Dr. Cohn and Dr. Ferry, not only for enlisting my services in the study of the oxidation of hemo- globin but also for much advice and assistance in carrying out the present work.

SUMMARY.

1. The oxidation of hemoglobin to methemoglobin by potas- sium ferricyanide and the reduction of methemoglobin to hemo- globin by sodium hydrosulfite can be followed electrometrically. The change involves one hydrogen equivalent and has a definite oxidation-reduction potential.

2. The potential of a mixture of hemoglobin and methemoglobin is raised by passing in oxygen or carbon monoxide, and lowered again by removing these gases completely. The potential of an inert electrode immersed in a hemoglobin solution varies with the partial pressure of the oxygen because the ratio of free hemoglobin to methemoglobin is varied by the degree of oxygenation. The change of hemoglobin to oxyhemoglobin is one involving oxygena- tion and not oxidation.

3. By representing hemoglobin as the sodium salt of a complex ferro acid, the relationships indicated by the present work can be adequately expressed and the analogy between hemoglobin and Manchot’s amino-ferrocyanides emphasized.

4. Preliminary experiments with hematin seem to indicate that the reduction of this substance to hemochromogen involves the addition of 2 hydrogen atoms. If this is so, the relationship between this pair of compounds has no bearing on the problem of the oxidation or oxygenation of hemoglobin.

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414 Electrochemical Study of Hemoglobin

BIBLIOGFtAPHY.

1. Conant, J. B., Kahn, H. M., Fieser, L. F., and Kurtz, S. S., Jr., J. Am. Chem. Sot., 1922, xliv, 1352.

2. Conant, J. B., and Fieser, L. F., J. Am. Chem. Sot., 1922, xliv, 2481. 3. Clark, W. M., J. Washington Acad. SC., 1920, x, 255. Clark, W. M., and

Zoller, H. F., Science, 1921, liv, 557. La Mer, V. K., and Baker, L. E., J. Am. Chem. Sot., 1922, xliv, 1954.

4. Manchot, W., Ber. them. Ges., 1912, xiv, 2869. Manchot, W., and Woringer, P., Ber. them. Ges., 1913, xlvi, 3516.

5. Baudiach, O., Ber. them. Ges., 1921, liv, 413; 1922, Iv, 2698. 6. Henderson, L. J., J. Biol. Chem., 1920, xli, 401. 7. Kiister, W., 2. physiol. Chem., 1910, lxvi, 244.

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James B. ConantHEMOGLOBIN

AN ELECTROCHEMICAL STUDY OF

1923, 57:401-414.J. Biol. Chem. 

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