THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 243, No. 12, Issue of June 25, pp. 33173326, 19G8

Printed in U.S.A.

Reaction of CO, with Human Hemoglobin Solution*

(Received for publication, November 27, 1967)

R. E. FORSTER, H. P. CONSTANTINE,~ MARGOT R. CRAW,§ H. H. ROTI\IAN,~ AND R. A. KLOCKE$

WITH THE TECHNICAL ASSISTANCE OF D. THYRUM

From the Department of Physiology, Graduate Division and Department of Anesthesiology, School of’Medicine1 University of Pennsylvania, Philadelphia, Pennsylvania 19104

SUMMARY 10-C M, the velocity constant for the dissociation, kd, ap-

We have measured the disappearance of dissolved COP with time after the mixing of dialyzed human hemoglobin solution (average 7.76 mM) at a COz partial pressure of nearly zero and water equilibrated with 7.5 torr of CO:! in a continuous flow rapid reaction apparatus with the use of a CO2 electrode to indicate its partial pressure. poz was zero in both solutions and the temperature was 37”. Acetazol- amide (1.1 InM) was included to inhibit carbonic anhydrase.

There was (a) a rapid initial fall in pcoz, complete within approximately 0.1 set and (b) a further slow fall continuing out to several seconds. The initial fall (Process a) is greater at greater pH and less when the hemoglobin is oxygenated. The decrement in pCoz at 0.1 set corresponds to the forma- tion of amounts of hemoglobin carbamate equal to those reported by others with different techniques, within experi- mental error. We conclude that Process a represents the formation of hemoglobin carbamate and Process b repre- sents the uncatalyzed hydration of CO*.

The velocity constant for the reaction of COz and hemo- globin-NHz (ka) was estimated from the relation between the over-all velocity of the reaction and [H+], which gives a value of 11,000 M-' set+. This value of k, is dependent upon the value chosen for the equilibrium constant (KZ) of the pertinent hemoglobin NH2 groups with H+ ions, which was considered to approximate 7.2 x 10-s M.

The equilibrium constant for the reaction of COZ and hemoglobin-NH*, K,, was calculated with less reliability from the change in pCcoz at 0.1 set, under the assumption that it represented equilibrium of carbamate and CO?. Values ranged from 2 X 10d5 to 8 X 10p6, as compared with an estimate of 2.4 X 10F5 in the literature. When this last value is assumed for K,, and a minimal value for the acid ionization constant of hemoglobin-NHCOOH is taken as

proximates 500 set-I. Calculations from previously~ reported measurements of

the rate of COZ uptake by suspensions of deoxygenated human red cells give a k, within the cell of approximately 5 X lo3 M-' set-I, which is probably not significantly differ- ent from that in solution.

In a previous communication, Constantine, Craw, and Forster (1) described the application of a pcOn (glass pH) electrode, in a continuous flow rapid reaction apparatus, to the measurement of the velocity of the reaction of dissolved CO2 with red cell suspensions. We thought that, by using this apparatus to follow the reaction of dissolved CO:, with hemoglobin solution in the presence of a carbonic anhydrase inhibitor, we could obtain a measure of the rate of this reaction separate from any hydration of COZ. The kinet,ics of this chemical process is of interest in COZ transport by blood. There was also the pos- sibility of calculating the total amount of any hemoglobin + COZ compound formed from the change in dissolved [COs] at the completion of this reaction, but at a timebeforeanysignificant hydrat.ion of COz had occurred.

EXPERIMENTAL PROCEDURE

All measurements were made on a continuous flow rapid reaction apparatus which had a CO2 electrode so placed that the flowing stream of reacting mixture impinged on it. The distance between the mixing chamber and the CO2 electrode could be varied by interchanging glass tubes of different lengths. The electrode itself consisted of a 3-mm diameter glass pH electrode (Instrumentation Laboratories, Boston, Massa-

* This work was supported in part by National Institutes of chusetts) placed vertically in a Lucite chimney. In the early

Health Grants 2-G-215 (C3) and He-04108. experiments, the glass electrode was covered with a 0.0012-cm $ Trainee in Physiology (HTS 5430). Present address, Depart- Teflon membrane held on with a rubber 0 ring. Later on,

ment of Medicine, Rhode Island Hospital, Providence, Rhode Island 02902.

a membrane of silicone rubber about 0.002 cm thick was used

§ Present address, Department of Anesthesiology, School of because of its much greater permeability to COz. The elec-

Medicine, University of Pennsylvania, Philadelphia, Pennsyl- trolyte consisted of 0.01 N KCl, 0.005 N NaHC03, and about vania 19104. 1 mg per ml of lyophilized carbonic anhydrase (obtained from

3317

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3318 Reaction of CO2 with Human Hemoglobin Vol. 243, No. 12

Nutritional Biochemicals). The output of the electrode was fed into a vibrat,ing capacitance electrometer (Applied Physics Corporat.ion, Monrovia, California) and recorded on a direct writing potentiometric recorder strip chart with l.O-mv full scale deflection. The average linear velocity of fluid down the observation tube was 280 cm per set, so that each centimeter of length corresponded to 0.0036 set of elapsed time. A more detailed description of the apparatus is given in the previous paper (1).

The calibration and calculation of the absolute value of the pcoZ to which the electrode is exposed is extremely critical, and some alterations in technique were made. The pcoz is an exponential function of the output of the recorder, namely

Pcoz,a - &EA-EB)

0) PCO2,B

~oo~,~ is the unknown COz tension, pC02,B is that of a known reference, X is the sensitivity of the electrode, E, is the out- put for Pco~.~, and EB is the output for the reference pco2. S is obtained during an experiment by measuring the outputs for two known gases containing about 10 and 5% CO*. Although not ideal, it is more convenient for calibration to use gases rather than fluid equilibrated with these gases. Moreover, the pco2 of such gases can be measured wit,h greater accuracy. The apparatus gives the same output for gas and liquid at the same pcoz, although there are occasionally pressure and flow effects. The reader should bear in mind that the datum sought is the di$erence between (a) the pcoz of the reacting mixture and (6) the pcoZ of this mixture before any COZ is absorbed. The latter was calculated from the proportion of the mixture which was contributed by the CO1 solution and its pco2. The contribution from each syringe could be calculated from the strip chart record upon which a signal was made every time another 1.84 ml had been delivered into the mixing chamber by each syringe. In this calculation it is assumed that the solubility of CO* in both solutions is the same. It was there- fore necessary to correct for the volume occupied by the hemo- globin. The average [Hb] in the mixture was 3.88 mM, or 66.0 g per liter. The density of hemoglobin in solution is about 1.33 g per ml (2), so that it would occupy 66/1.33 = 49 ml out of a liter of solution. Therefore, the pcoZ for the mixture calculated as above, was increased in each case by lOOO/(lOOO - 49) = 1.051, representing an average increment of 1.6 torr.’ The pcoz of the hemoglobin solution could not be measured on the apparatus as it was close to zero, so that only the COz solution, with a tension around 75 torr, is available as a ref- erence. The difference between this and the pcOa of the re- acting mixture, the latter bein, v less than half or as little as 15 torr, is so great as to increase the error in calculated pco2 of the reacting mixture to several torr. Therefore, when- ever possible, an auxiliary gas mixture with a pcoz of about 37 torr was used as a reference. The net error in determining a given decrease in pcoz was about 1 torr.

Hemoglobin solutions were prepared, by a method analogous to that of Adair and Adair (3), from 700 to 1000 ml of fresh human blood by first u-ashing and centrifuging three times with 0.9% sodium chloride and then slowly adding sufficient sodium

1 Gas pressure is stated in torr (1 torr = 1 mm of mercury pres- sure at 0”). The concentration of hemoglobin is expressed through- out this paper as the molar concentration of heme, or iron, and not as the molar concentration of tetrameric hemoglobin.

chloride to produce a 1.5 M solution. ,4 volume of toluene equal to that of the original blood was then added. The mixture was stirred gently and then centrifuged, and a middle layer of hemo- globin solution was separated from the upper layer of toluene and a lower layer of precipitate. This solution was then dialyzed against distilled water for 48 hours. The residual sodium was 11 and 22 meq per liter on two occasions. The concentration of hemoglobin ranged between 4 and 9 mM, in the final solution and the pH, from 6.6 to 7.1. ,4cetazolamide was added to a concen- tration of 1.1 mM. This hemoglobin solution was then deaerated under reduced pressure and flushed with nitrogen or helium at 37”. The final solution had a pcoZ of generally less than 1 torr and a poz equally low; it was stored in a 2-liter bottle at 37” before being placed in one syringe of the reaction apparatus.

The COs solution was prepared by equilibrating a 1.1 mM

solution of acetazolamide in distilled water with approximately 10% CO2 gas in nitrogen at 37”; it was then stored in a bath at the same temperature and, when needed, placed in the second syringe of the rapid reaction apparatus.

Total COZ content by the method of Van Slyke and Neil1 (4), pH, and usually pco3 by electrode were measured in the hemoglobin solution, in the CC, solution, and in a 1:l mixture of the two. Hemoglobin concentration was measured in the solution as cyanmethemoglobin. The poz values of the solutions were measured only occasionally to check the techniques of prep- aration, except in those several experiments in which 100% O2 was added to the gas phase over the hemoglobin solution.

One pint of fresh blood containing about 9 mM hemoglobin produced only about 350 ml of hemoglobin solution containing 5 to 9 mM hemoglobin. Owing to the slow response time of the CO:! electrode, it often required one syringe full of hemoglobin solution, 130 ml, to obtain a single data point. It is for this logistical reaction that only two points were obtained in some experiments. In later work, blood from two donors was mixed, permitting more points to be obtained, but the prime reason for the increased reliabilit’y of the later experiments was the more rapid response time of the improved CO? electrode, per- mitting markedly increased efficiency of reagent use.

RESULTS

The results are summarized in Table I. Every experiment that was done is included. Figs. 1 and 2 are graphs of the de- crease in pcoz from the calculated initial value in the mixture 2rer.su.s time. In each experiment there appears to be a rapid initial fall in pco2, completed in about 0.1 see, followed by a more gradual fall to the equilibrium value (see Table I). Ex- ponential curves were fitted by inspection to the individual points from 0 to about 0.1 sec. These curves (but not the data points) have had the uncatalyzed hydration rate subtracted from them (see Fig. 2), and the exponential constants are given in Table I.

Fig. 1 is an example of the early experiments in which it was not possible to get more than four data points in one ex- periment. The exponential curve is defined by points at only two different reaction times, in addition to zero. The particular experiment was designed to show that increasing the acetazol- amide concentration to 3 times that generally used led to no change in the rate of COZ disappearance and, therefore, that the action of carbonic anhydrase did not influence the process.

Fig. 2 is a graph from the data of the last three experiments in which a sufficient number of data points were obtained to

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Issue of June 25, 1968 3319 Forster, Constantine, Craw, Rotman, and Klocke

TABLE I Summary of results of reaction of hemoglobin solution with CO*

[CO,] is total CO2 in all forms. The pcos of the Hb solution was CO2 concentration or by both. All of the blood was obtained too low (less than 2 torr) to be measured by the electrode. ~~02 of fresh from healthy donors, save for three experiments in which the CO, solution was generally obtained in the rapid reaction ap- outdated blood bank blood was used, indicated with the experi- paratus by comparison with a standard gas, but was also checked ment number as “bank.” by pcaz electrode separately or by calculation from pH and total

Experiment

6-14-3, bank 7-3-3, bank l-27-5

Acetazolamide, 3 mMc l-28-5

HCl addedd 2-3-5, bank 3-10-5

~0.2 = 589 torr p02 = 589 torr + HCl

addedd 6-17-5

KOH addede p02 = 458 torr’

8-19-6 9-25-6 2-3-7

p02 = 620 torr

T Hb solution

[Hbl PH ICOZI

nzM

8.69 7.85 5.88

7.56

7.51 7.76

6.08

10.50 7.95 9.12

?%M

6.68 6.85 7.18 7.27 7.16 6.91 6.60 7.09 7.03 6.55

6.99 7.51 7.40 7.64 7.11 7.41 7.27

0.42 0.16 2.08 0.43

0.12 0.28

0.14

0.14 0.54 0.24 0.24

-

CO* solution

PH [CO21 Pcot PH

6.23 6.16 4.31 6.83 6.16 6.16 6.19 6.20 6.20 6.20

m‘?x tow

4.05 72 4.05 70 6.15 77 6.52 72 4.34 78 4.34 78 4.30 74 4.19 73 4.19 73 4.19 73

6.61 6.76 7.03

7.08 2.46 11 28 6.85 2.60 18 26 6.52 2.25 27 10 6.99 2.22 23 14

6.14 4.09 75 6.14 4.09 75 6.14 4.09 75 6.19 5.27 70 6.23 4.15 68 6.35 4.20 77 6.35 4.20 77

6.90 2.16

7.53 2.34 7.06 2.40 7.41 2.32

a The mixture (equilibrated) was a 1: 1 mixture of the Hb solu-

tion and the CO2 solution produced anaerobically in a syringe, and represents the completely equilibrated reaction.

b The decrease in ~~02 of the mixture at’equilibrium was ob- tained by subtracting from the calculated pco2 in the mixture, which was weighted according to the contributions of the indi- vidual syringes, the pco2 in the 1: I equilibrated mixture. The de- crease at 0.1 set was estimated by inspection from the graphs of pc02 versus time after the uncatalyzed rate had been subtracted.

c The data points for both concentrations of acetazolamide were

define the curves with more precision. These data indicate (a) that there is a rapid initial disappearance of CO2 followed by a much slower reaction, (b) that the decrease in pcoz at 0.1 see becomes greater as the pH increases, and (c) that at the same pH, the decrement in pcoz at 0.1 see is much smaller with OsHb than with reduced hemoglobin.

In Fig. 3 the exponential rate constants, for all experiments, are plotted against the pH of the hemoglobin in solution be- fore mixing. Owing to the relatively great buffering power of the hemoglobin solution, the pH at 0.1 set is little different from this value. The average exponential constant for all experiments with deoxygenated hemoglobin solution was 104 set-I. There is a large amount of scatter of those experiments ( 0) in which it was not possible to get more than one experi- mental observation under 0.040 set, presumably because the calculated exponential constant is extremely sensitive to small variations in this single measurement. In addition, as pH decreases, the amount of carbamate formed, and thus the total change in pcoZ at the completion of the reaction, both decrease, while the random errors in estimation of the pcoz at a given reaction time remain about the same, leading to a considerable increase in the variation of calculated fractional progress of the

Mixture (equilibrated)‘”

[CO21

nzM

2.39 2.38

PCO?

tow

15 20 12

I1

4 11 5

-

I kcrease in pcot of mixture a

e -

:quilibriuml 0.100 set

torr tow set-1

21 4.0 50 15 6.0 80 26 15.0 70

15.5 155 14.0 210 7.5 93

12.5 146 6.5 77

26 6.0 51 12.0 99

31 23 32

25.0 68 17.0 70 14.5 73

9.0 28

t Exponential

constant

considered together in calculating the velocity and extent of the reaction.

d The pH of the hemoglobin solution was adjusted to a lower value with 1 N HCl.

e The pH of the hemoglobin solution was adjusted to a higher value with 1 N KOH.

f One experimental point at 0.011 set and another at 1.4 set were obtained with the hemoglobin oxygenated. The early point showed a fall in pco2 only one-quarter of that at the same time in the same experiment but with the hemoglobin deoxygenated.

reaction. Therefore, we believe the greatest reliance should be placed on the three experiments in which several points were obtained at short reaction times ( 0). Neither these data nor the regression for all the points shows a significant dependence of exponential constant on pH. The average value of the exponential constant for the last three experiments was 70 set?.

In Fig. 4 the total decrease in pcoz at 0.1 set for all experi- ments is plotted, the decrement having been corrected to a standard pcoz of 37 torr at 0.1 set and a standard hemoglobin concentration of 3.88 InM.

DISCUSSION

A major problem in the early stages of the experimentation, when data at only two or three reaction times were available, was to be sure that the decrease in pcoz at 0.010 to 0.020 set was not an artifact, since there is no means of analyzing the calculated theoretical initial value of the mixture experimentally. However, measurements of pcoz in a mixture of water and a solution of COZ in water in the rapid reaction apparatus gave values that were within ~27~ of the calculated pcoz of the mix- ture. In this case there is no chemical reaction, but it validates

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3320 Reaction of CO% with Human Hemoglobin Vol. 243, No. 12

the pcoz analysis. I f the act,ual solubility of CO2 in hemo- globin solution is more than that assumed in the calculation of the initial peoz of the mixture, the time zero intercept of the experi- mental points will also be less. In making the calculation it was assumed that the solubility of the gas was the same in the

DECREASE

25

30 20 40 60 00 100

TIME IN MILLISECONDS

FIG. 1. Graph of Apcoz of the reacting mixture against time (Experiment l-27-5). Zero indicates the calculated pcoz of the reacting mixture before any CO2 had been absorbed. 0, data points obtained with the usual acetazolamide concentration of 1.2 mM; l were obtained with 3.6 mM acetazolamide in both solutions. t, indicates the total drop in pcoz at equilibrium, 26 torr. The smooth curve is a simple exponential with a rate constant of 70 set-i.

0 lm!NCAIALYLtD

DECREASE : IN

PCO,

IN TORR ‘c

a pH 7.64 Hb

40 80 120 160 200 240

TIME IN MILLISECONDS

FIG. 2. Graphs of the fall in poo, with time for the reaction of COz with deoxygenated hemoglobin solution at several pH values. The smooth curves are exponential fitted by inspection with the exponential constants given in Table I. X- - -X, Experiment 9-25-6; O---O, Experiment 2-3-7; O--O, Experiment 8-19-6. A graph of the change in pco, when the hemoglobin solution was oxygenated at pH 7.27, Experiment 2-3-7, is also shown (0-O). The fall in poo2 resulting from uncatalyzed hydration, assuming a rate constant of 0.12 set-1 at 37” and an initial poo, in the mixture of 37 torr, has been plotted for comparison. The data points have not been corrected for this hydration of COZ, although the smooth curves have been.

EXPONENTIAL 15c

CONSTANT t/1)

SEC-’

tot

5c

C

) I 6.4

o” I

0

0

i 0

0 0

0 0 /

l

X

6.6 6.8 7.0 7.2

INITIAL pH OF Hb

7.4 7.6 8

FIG. 3. The exponential constant (Equation 9) for the disap- pearance of COP with time as a function of the initial pH of the hemoglobin solution. l , the last three measurements as in Fig. 2. The arrows connect values obtained in the same experiment. X indicates that the hemoglobin solution was oxygenated. 0 in- dicates the first 10 measurements.

INITIAL pH OF Hb

FIG. 4. A graph of the decrease in pcoz at 0.1 set corrected to a final pco2 of 37 torr and a hemoglobin concentration of 3.88 mM, plotted against the initial pH of the hemoglobin solution before mixing. The arrows connect data points from the same experi- ment. 0 indicates the first eight measurements with deoxygen- ated hemoglobin solution, l indicates the last three, X indicates measurements with OsHb solution. The smooth curve was calcu- lated from Equation 3 assuming a pco, of 37 torr, a hemoglobin concentration of 3.88 mM, and the values for K, and K, indi- cated.

water of the hemoglobin solution as in the water alone, the actual volume of water in the mixture being corrected for the volume displaced by the hemoglobin. This calculation would lead to an error in the over-all solubility if hemoglobin altered the solubility of CO2 in the water around it, or if COz dissolved in the protein itself. However, Van Slyke et al. (5) measured the solubility of CO* in hemoglobin solutions of varying strengths at pH 3.5, at which acidity little bicarbonate or carbamate is formed. If one assumes a density of 1.33 for the hemoglobin, as we have here (2), the solubility of the gas in the water of the solution was the same as that in distilled water.

Theoretically there must be a stagnant layer of finite thick- ness at the surface of the electrode, and the chemicals within this layer must have had a longer time in which to react than those in the main stream. Therefore, in the reaction of CO2 with hemoglobin, the absolute change in pcoz registered by the electrode at any calculated time should be greater than the correct value, corresponding to that of the fluid in the central stream. This subtle error would shift the reaction curves (Figs. 1 and 2) to the left; the reaction would appear to pro- ceed more rapidly. We have devised no means of determin- ing the magnitude of this error except to measure the change in

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pcoz of a known rapid and approximately linear process and to have to be substituted for the 1.69-mm diameter glass tubes. extrapolate to an intercept. The reaction we have used is In these spirals, turbulent flow does not obtain, and the fluid the uncatalyzed displacement of CO2 from bicarbonate. Con- does not move with a square front. In view of this technical stantine et al. (1) found an intercept not different from the problem and the approximations involved, the agreement be- origin, with the apparatus used in the early part of the present tween theory and experiment is excellent and supports our sur- experiments. Because several new cuvettes were used, as well mise that the slow reaction is the uncatalyzed hydration of Con. as a different type of membrane, we have repeated these measure- The only other known chemical reaction which could be ments mixing an oxalate buffer (which produces minimal catal- consuming CO2 at this rate is the formation of hemoglobin ysis) and bicarbonate, both in 0.9% NaCl and in a mixture of carbamate, according to the reaction (6, 9-12) glycerol, dextran, and 0.9 % NaCl. This last was adjusted to give a viscosity, measured at 23” by a narrow bore glass tube,

COz + Hb-NH2 = Hb-NHCOO- + II+ (2)

equivalent to that of the hemoglobin solution, approximately Hb-NH2 is the unionized amine group, the only form of the 1.9 times that of distilled water. This reaction produced an amine with which the CO2 will combine. Hb-NHCOO- is the average increase of 440 torr at equilibrium and, when extrap- ionized carbamate, and almost all the compound is in this form olated, had an average ordinate intercept of +15 torr and an in the pH range under consideration (Reference 6, pp. 805, 12). abscissa1 intercept of -0.005 sec. No simple theoretical re- It is generally accepted that only the 4 terminal a-amino groups lationships are intuitively apparent; for example, at one extreme of valine and the 44 amino groups of lysine are able to form it could be considered that the electrode was exposed to a mix- carbamate compounds with COz (12). The pK for the Lu-amino ture contaminated with 15/440 = 3.3% of the completed re- groups should be in the 7.0 to 8.0 range, whereas the pK of the action mixture; at another extreme that there was a time error e-amino groups is about 10.5 (2). Therefore, it appears likely of -0.005 sec. Occasionally these intercept errors disappear that there is only one such NH2 group per Fe atom in hemoglobin, with slight changes in the apparatus, so that we are not con- appearing in the forms Hb-N&, Hb-NH3+, Hb-NHCOO-, vinced that this represents an insuperable limit in the accuracy and Hb-NH-COOH. The fraction of the available hemoglobin of the instrument. Increasing the viscosity of the reacting amine groups that is combined with COZ to form carbamate at mixtures produced nochange inthe intercept error. Fortunately, equilibrium, f, is described by the following equation (6, 9, 10, uncertainties of these magnitudes would not affect our present 12). results: an underestimate of time by 0.005 set would produce an increase in our estimate of the association velocity constant f=

[CO*1

of COn and hemoglobin, k,, of less than 10%; contamination [C(),+[Hfl+[H+lz (3) 2

of the mixture with 3% of the completed reacting mixture would Kc KJL

produce an insignificant artifact. There are several possible sources of this error in the apparatus, the most obvious of

[CO*] is the molar concentration of dissolved gas and [H+] is

which is the stagnant layer. It is therefore interesting that that of hydrogen ion. K, is the equilibrium constant for the

no augmentation occurred with increased viscosity, which ionization of the amine group and is defined by the relation

should have increased the depth of this layer. Since we can find no reason to doubt the correctness of the

K s

= lH+l Mb-N&l [Hb-NHs+l (4)

calculated initial pooZ in the flowing mixture, the rapid fall in pco2 with time must represent a chemical reaction. It cannot [Hb-NH3+] is the concentration of the pertinent ionized amine

be the uncatalyzed hydration of CO2 because this process is too groups.

slow. The velocity constant for this reaction at 37” is 0.11 K, is the equilibrium constant for the carbamate and ionization

set-1 (6, 7), and even if there was no significant amount of reactions and is defined by the reaction

carbonic acid present initially, at a pcoZ of 37 torr the maxi- mal rate of CO:! disappearance would be 0.4 torr in 0.1 sec.

K [Hb-NHCOOH] K, E

= [H+l [Hb-NHCOO-1 ~ = [CO,1 [Hb-NH,1 [Cod [Hb-NH,]

(5)

This process is plotted in Fig. 2 and is obviously negligible in comparison to experimentally determined rates of decrease in where K, is the acid ionization constant of Hb-NHCOOH.

PCOP The hydration reaction could not be catalyzed by car- These relationships can be derived from basic physicochemical bonic anhydrase (which is not entirely removed during the considerations (12) and have been verified in most respects by preparation of the hemoglobin solution) because of the acetazol- experiment. amide. A concentration of 1.1 mM should be enough to inhibit At a given pcoZ, the amount of carbamate formed at equi- all but 8 x 1OP of the carbonic anhydrase activity (8), and librium, corresponding to the drop in poo2 at 0.1 set, should tripling this concentration in one experiment (Fig. 1) did not rise as the pH rises. In Fig. 2 it can be seen that this is the reduce the intercept, supporting the conclusion that carbonic case, although the exact quantitative relationships will be anhydrare activity was not involved in the initial rapid dis- discussed in more detail later on. appearance of COZ. Oxygenating the hemoglobin should cause a decrease in the

On the other hand, in Experiment 2-3-7, measurements were carbamate concentration at the same pH and pcoz (6, 11, 12). obtained at 0.9 and 1.5 set in addition to those in Fig. 2. The In two experiments (Table I), oxygenating the hemoglobin average rate of fall of pooz beyond 0.25 set was 5.2 torr per solution caused a decrease of 5.5 and 6 torr in the drop in pcoz at set, as compared to the theoretically calculated value of 4 0.1 set, with slight changes in pH. We conclude that the results torr per set for the uncatalyzed hydration. The time resolution are qualitatively compatible with formation of hemoglobin of measurements at reaction times greater than 0.1 set is poorer carbamate, which is approximately complete by 0.100 set, than that for shorter times because large volume glass spirals and that this is the most probable explanation of our findings.

Issue of June 25, 1968 Forster, Constantine, Craw, Rotman, and Klocke 3321

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3322 Reaction of COz with Human Hemoglobin Vol. 243, iSo. 12

The reactions can be considered to occur in the following steps (9, 12, 13). Experiment 2-3-7 will be used as an example for computation.

Step 1: Physical Mixing-The fluids are mixed physically in the mixing chamber and the concentrations of the chemical con- stituents, before any reaction takes place, would be the weighted average of the concentrations of the two fluids. The pcoz would become approximately half that in the CO:! solution, 38.5 torr, equivalent to 1.23 mM. The [H+] in the hemoglobin solution at pH 7.41 is 0.39 x lo-’ M. The [H+] in the COZ solu- tion at pH 6.35 is 4.4 x lo-‘. The value in the mixture before any chemical reaction has taken place would be the average of these, or 2.43 x lo-‘, a pH of 6.61.

Step 2: .VeutraZixation-The buffer reactions should take place in far less than a millisecond (14) ; in fact, they will occur at least as fast as the mixing process itself. Proteins might react more slowly, but neutralization of acid by human plasma proteins and by hemoglobin is complete in several milliseconds,2 if not sooner. The hemoglobin, which binds 2.9 mmoles of Hf per mmole of protein for a change of one pH unit (15), is present in the mixture at 4.56 mM and is by far the dominant buffer pres- ent. The acetazolamide is in a concentration of 1.1 mM at an effective pK of 7.5 (16). The bicarbonate system is of no importance since its pK is 3.455 (6), and the COZ hydration is too rlow to be considered in this step. The neutralized mixture should have a pH only 0.01 less than that of the original hemoglobin rolution, in this example 7.40.

Step S: Formation of Carbamate-By 0.100 set the carbamate reaction appears to have been completed, but the uncatalyzed hydration of CO2 has consumed only 0.5 torr. The remaining drop in pco? by this time must represent the carbamate that is formed, 14 x 0.032 = 0.77 1nM. Almost all of this carbamate will ionize, liberating H+, and owing to interaction with the amine ionization equilibrium, additional amounts of H+ will be formed (I 2), the exact amount dependent upon the pH and pK of the amine group (see discussion related to Equation 16 later on). In this case, H+ equal to approximately 1.26 times the amomlt of carbamate formed nil1 be liberated, all of a-hich would be buffered by the hemoglobin. This would produce a further drop in pH of 0.04, to that the value at the end of this period would be 7.36.

Step /t: Formation of Carbonate-From 0.1 set on, the COz will hydrate without benefit of carbonic anhydrase, coming to equi- librium by about 15 sec. This formation of bicarbonate, with simultaneous release of protons, will reduce the pH and lower the pcop, both of which effects will reduce the [carbamate] according to Equation 3. The carbamate reaction, being much faster, will maintain equilibrium as the H+ and CO2 concentrations are continuously changed by the hydration reaction, until the final equilibrium is reached. The values of [HC03] were nob included in Table I because they were not believed sufficiently pertinent to m-arrant the additional com- plications. The [HCOZ-] value can be calculated either (a) from total [COJ less the dissolved [CO,], namely pcoz x 0.032; or (b) from the measured pH and total [CO& assuming the pK’ value is 6.31 (ionic strength, 1 mM) (Reference 13, p. 558). For example, in experiment 2-3-7, [HCO,]/[CO,] in the hemo- globin solution was 12.6 at pH 7.41, so that [HCO,]

2 I<. E. Forster, RI. Kokowsky, R. A. Klocke, and H. H. Rotman, unpublished observations.

was 0.22 InM. In the CO* solution, total [COZ] was 4.20 mM,

and dissolved [COZ] was 77 x 0.032 = 2.47 mM. Thus, [HCOa-] was 1.73 InM. In the mixture before chemical reaction took place, [HC03] was (1.73 + 0.22)/a = 0.93 mM. At final equilibrium, total [CO,] was 2.32 mM, dissolved [CO,] was 5 X

0.032 = 0.16 nlM, and therefore [HCOS-] was 2.16 mM.

Velocity Constant for Reaction of COz with Hemoglobin Amine (k,)

The rate of the reaction of CO* with Hb-NH2 should be pro- portional to the concentrations (activities) of the two reactant.s, although this has not been verified directly by experiment. The rate of dissociation has been shown to be proportional to the concentration of hemoglobin carbamate (11). We v-ould therefore assume that

dX - = -k,xy + kJHb-NHCOOH] dt

(6)

where z is the concentration of dissolved CO2 and y is that of Hb-NH*, k, is the association velocity constant in M-I set-I,

kd is the dissociation velocity constant in see-I, and t is time in seconds. A relation between k, and kd can be obtained at non- carbonate equilibrium as follows

kd[Hb-NHCOOH], = k,x,ym (71

Q is the concentration of dissolved CO*, yW is that of Hb-NHZ, and [Hb-NHCOOH], is that of the unionized carbamate, all at non-carbonate equilibrium. The maximum calculated increase in [H+] over the entire carbamate reaction is 12%. Therefore, pH may be assumed constant as a reasonable ap- proximation as will be [Hb-NHCOOH]/[Hb-NHCOO-1. Under these conditions (2, - X)/(X,, - XJ will equal [Hb-NHCOOH]/ [Hb-NHCOOH], and

[Hb-NH?] changes by a maximum of 15% during the entire reaction and will be considered constant, that is, y = y,. Sub- stituting Equation 8 in Equation 6 and integrating gives the following solution

x - x, - lc,yxot ~ = exp - [ 1 (9) x0 - x, x0 - x,

Xote that, since the solubility of COZ in the solutions does not. vary, the partial pressure of CO?, p, can be substituted throughout for x.

It was for these theoretical reasons that an exponential curve was fitted to the data and a value for the exponential constant extracted. The constant given in the last column of Table I (k) corresponds to the exponent in Equation 9, and equals kaypolbm. In Fig. 3 no apparent relation exists between k and pH. The true reaction velocity constant, k,, should, as a first approximation, be invariant with pH. The value of y should increase with increasing pH according to Equation 4. A;n, Fhould increase with increasing pH, according to Equation 3, because the amount of carbamate being formed would increase. Therefore, y/Ap, may vary little over the observed range of pH and the results, although variable, appear theoretically reason- able.

It is possible to obtain numerical estimates of K, and k, from these data. If we assume that a negligible amount of

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Issue of June 25, 1968 Forster, Constantine, Craw, Rotman, and Klocke 3323

carbamate is formed, in other words that (R-NH2) + (R- NH3+) is constant, and that there is one amine group available per hemoglobin iron atom to combine with CO*, we can re- arrange Equation 4 to obtain

In justification of this assumption, an estimate of the maximal error introduced can be obtained from a consideration of the experimental data in Table I below. The maximal amount of carbamate formed was in Experiment 8-19-6 and was equal to 25 torr X 0.032 mM/tOrr = 0.85 InM which, out of 5.25 mM hemog obin in the mixture, was 16%. This means that the value of y at carbamate equilibrium (0.1 set) was overestimated by IS%, but the correct mean value of y during the course of the reaction is overestimated by approximately 8%. As this is a maximum error and would be less in all the other experiments, it was considered acceptable in view of the greater simplification of the calculations.

Substituting this value for y into the expression for k and rearranging we obtain

[Total TTb]p”

[AP,] k = l/k, + g

z a

I f ae nom plot the experimentally determined values in the left hand term against initial [H+], the intercept on the ordinate equals the reciprocal of k,, and the intercept on the abscissa equals -K,. This has been done in Fig. 5 which, in order to present a complete summary, includes all of the experiments. Clearly there is too much Pcatter t’o define k, and K, with accuracy, but approximate values can be extracted. The regression line for all points m-as

(Total Hb)

ccc - 2,) x3 = 1620 [H+] + 4.7 X 1C5

from which k, is 2.1 X lo4 M-’ set-I, K, is 2.9 X 10-8, and pK, is 7.54. The regression line for t’he last three experiments (o), in which there were a greater number of experimental points at short times permitting a more reliable assessment of the exponential rate constants, was

(Total Hb)zo

(2” - z,)k = 1000(1-I’) + 1.02 x 10-h

from which k, is 9.8 X lo3 M-l set-I, K, is 1.02 X 10-7, and pK, is 6.99. Rossi-Bernardi and Roughton (12) have recently calculated values of pK, from older published data on hemo- globin carbamate at 37”, obtained by the barium carbonate precipitation method, varying from 7.58 for oxyhemoglobin (the data of Stadie and O’I3rien (17)) to 7.18 for human hemo- globin (the data of Ferguson (11)). These calculated values are more consistent with our estimate of pK, from the last three experiments, which should also be our most extensive experi- ments. For both these reasons we consider that the better estimate of k, is 9.8 X lo3 M-’ se0.

It is also theoretically possible to calculate k, starting with the initial slope of the pco2 disappearance curve. In this case, from Equation 6

- k, =

0.8

0.2

0

X

i

INITIAL [H’] OF Hb SOLUTION IN IO-*M

FIG. 5. The term [total Hb] pa/k up, according to Equation 11 plotted against the initial [H+] in the hemoglobin solution. 0, the first eight measurements; l , the last three measurements; and X, the measurements with oxygenated hemoglobin. The arrow.s connect measurements made in the same experiment. The line is the regression for l (see text,).

0.8

[TOTAL Hbl p.

dp/d* O.E

IN 10-3M SECONDS

0.4

0.2

-Id -5

1 -(. . INITIAL [Ii+] OF Hb SOLUTION IN lO*M

FIG. 6. The term [total Hb] po/dp/dt for the last three measure- ments plotted against [H+] according to Equation 13. The arrow

connects Experiment 2-3-7 on deoxygenated hemoglobin (0 ) with that on oxygenated hemoglobin (X). The line is the least mean square regression for the data on deoxygenated hemo- globin.

where (~Jp/dt)~ is the initial rate of change of pcoZ obtained by drawing a tangent to the reaction curves in Fig. 2 at time zero. pa 1s the total pco2 in the mixture before any reaction has taken place, assuming that the pcoZ in the hemoglobin solution before mixing is negligible. Here y is the [R--NH21 at the start of the reaction and can be obtained from Equation 10 by making the same assumptions as earlier. Substit.uting this expression for y into Equation 12 and rearranging

[Total Hb]po = ; + [H+]/K,k,

dp/dt a

I f we now plot the left hand term versus [H+] we can solve for k, and K, in a very similar manner. Fig. 6 is such a graph and when the line of best fit is plotted, we obtain k, = 11.7 X lo3 X M-’ see-l, K, = 4.5 X lo-‘, and pK, = 7.35.

The choice is limited between the two methods of analyzing the results. The first method, fitting a simple exponential curve to the data, requires t’he assumption that all of the chemical

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3324 Reaction of CO2 with Human Helnoglobin Vol. 243, No. 12

constituents except the CO2 stay constant, which is not exactly true, and the curves in Fig. 2 do not appear to be precisely of this form. The initial slope method, while having the advantage that the constituents are considered constant at time zero, has the technical disadvantage that it is difficult to fit a tangent to the early parts of the curves with any great precision. We therefore have combined the results and consider the best esti- niate of k, to be 11 X lo3 M-’ see-l. This is critically dependent on the estimated completion of the reaction at 0.100 sec. We place less reliance on the estimate of 7.35 for pK, because of the greater extrapolation and limited number of points, but consider it reassuring that this value is so close to the estimate of 7.18 calculated from the data of Ferguson (11) by Rossi- Bernardi and Roughton (12).

Roughton and Rupp (7), with a different method of analysis, estimated a value of 3 X lo3 M-l set-” for k, in methemoglobin solution. Bearing in mind the assumptions involved in all the calculations, this is not a serious disagreement. Values for k, for simple amines lie in the range of lo3 to lo5 M-l see-l, so that our present estimates are reasonable (18). Chipper- field (19) has measured the kinetics of the reactions of COz with glycylglycine and other amino acids, including valine, the terminal NH2 group of which is presumed to be reacting with CO* in the formation of hemoglobin carbamate. The value of k, at 25” was 4.9 X lo3 M‘-’ set-l. I f we assume that the activation energy for the reaction is the same as for glycine, namely 8.2 kcal, the value of k, at 37” for valine is 8.5 x lo3 M-~ see?. Our estimate of k, for hemoglobin carbamate of 11 X lo3 M-l see-l at 37” is very similar.

Amount of Carbamate Hemoglobin Formed

The concentration of carbaminohemoglobin at equilibrium is a function of [COP], total [Hb], and [H+], according to Equa- tion 3. Therefore, any comparison of theoretical and experi- mental values must take into consideration the variations in these variables. In addition, K, and K, are not known exactly; pK, for the dissociation of terminal a-NH* groups should be in the range of 7.4 to 7.9 (Reference 6, Table 5); that for the carbamino-COe equilibrium should be in the range of 4.0 to 5.2 (9). It is difficult to calculate any theoretical values for the [carbamate] without knowing the exact values for K, and K,, but it is possible to correct the measured decrements in pcoz at 0.1 set to a standard hemoglobin concentration and a standard total pcoz; this has been done in Fig. 4, according to the following argument. The fraction (f) of the total hemoglobin concentration that has reacted to form carbamate is given by the statement f = Ap- x O.O321/[total hemoglobin], where the solubility of COZ in mM/torr at 37” is 0.0321. Thus, Equation 3 can be rearranged to give

[H+l [H+P K+KK = s ([Total Hb] - 0.0321 p) (14)

c c D

We now wish to calculate another Ap with p at equilibrium equal to a standard value of 37 torr; [H+], K,, and K, are kept the same, so that the left hand side of the equation above is con- stant. We have therefore two simultaneous equations of the above form, which can be solved for the new Apcoz as follows:

3.88 &‘standerrd =

0.0321 + [Total Hbl

- 0.0321 p >

05)

AP 37

It would be more correct to plot Ap against pH in the solution at carbamate equilibrium, which was not measured experi- mentally, but could be approximated. However, the hemo- globin is by far the dominant buffer, and the error introduced by assuming that the initial pH in hemoglobin solution is equal to it would not alter the form of the graph. In Experiment S-19- 6, with the greatest change in pcoz of 25 torr, 0.032 x 25 = 0.85 mmole of COz have disappeared. The amount of Hf produced does not necessarily equal this value because, as pointed out by Roughton (6)) not only does the carbamate formed dissociate almost entirely giving an equivalent amount of H+, but the R-NH2 consumed also disturbs the amine ionization equilib- rium (Equation 4) which produces additional H+. The ratio of the H+ produced to the carbamate formed is

HH+l + Kz [H+l + Kz

(16)

Assuming a value for K, of 6.7 x lo-’ (12) at the pH of 7.64 in this experiment, the ratio is 1.26. The change in pH, as- suming that the hemoglobin is the only important buffer, would be 0.85 x 1.26/(5.25 x 2.9) = 0.071 pH unit. Since this is the largest change that ,occurred, it did not seem worth- while to add this complication. The least mean squares re- gression line, Ap = 29.7 pH - 192, which is not plotted in Fig. 4, shows a definite increase in carbamate with increase in pH at constant pco2 and total hemoglobin concentration. Also shown are the decreases in Apcoz with oxygenation at the same pcoz and nearly constant pH.

An estimate of K, theoretically can be obtained from the fall in pcoZ at 0.1 set in the following fashion. If Equation 14 is rearranged

- 0.0321 p = f + g 1 (17) E c B

The value of the entire left hand term can be computed for each data point, since all the parameters are known. If this function is now plotted as the ordinate with [H+] on the abscissa, it should give a straight line with the intercept on the ordinate equal to l/Kc and the intercept on the abscissa equal to K,.

Unfortunately, the variation in the points is so great that only a limiting value of K, can be obtained. The least mean squares regression line for Equation 17 has a negative slope, making the calculated value of K, negative and therefore chemically mean- ingless. The best that can be said is that the intercept on the ordinate must be less than the average value of the ordinate, 1.2 x 105, because the slope must be positive. Therefore, K, should be 28.3 x lOP, and pK, should be 15.32. The best available estimate of pK, for human blood at 37” is 4.62 (K = 2.4 x 1OP) calculated by Rossi-Bernardi and Roughton (12) from the published data of Ferguson (ll), which were obtained by the barium carbonate separation technique. Our “equilibrium” data are certainly compatible with this value. It is worth pointing out that our attention was focused pri- marily on the proportional and rapid changes in pcoz within the first 0.1 set, and that the equilibrated data are obviously not precise enough in the absolute sense for this particular type of analysis. This is particularly true of the values at low pH, where the total changes in pcoz are small. Moreover, in Fig. 4 we have plotted the decrease in pcoz calculated by means of Equation 3 for pK, = 7.18 and pK, = 4.62 at the standard

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Issue of June 25, 1968 Forster, Constantine, Craw, Rotman, and Kloclce 3325

values of 3.88 JXM for hemoglobin concentration and 37 torr for Pco~. Although there is considerable scatter, the fit is reasonable.

Since K, = K, (k&d), the dissociation rate constant, kd, can theoretically be calculated (see Equations 5 and 6). Al- though K, and k, are known, K,, the acid ionization constant of Hb-NHCOOH for hemoglobin, has not been measured, but it is probably greater than 1OP M (6). Therefore we can only set a lower limit on kd, equal to 1OP M X 1100 see-r/2.4 X 1O-5 M = 460 set-l.

Acetazolamide might alter the carbamate reaction, although there is no evidence for such an assumption. Other investi- gators have used acetazolamide, so at the least our data are legitimate for comparison. The concentration of electrolytes is probably less in these experiments (about 6 to 12 mM) than in the reported experiments of Ferguson (1 I), Stadie and O’Brien (17)) and their collaborators. Our method of estimating the total amount of carbamate formed might be considered an extrap- olation to shorter times of the method of Stadie and O’Brien (17), in which they measured the disappearance of COz as a gas into an amino acid solution; their earliest point was obtained at 15 sec. Constantine, Rossi, and Roughton (20) measured the pooz in a mixture of hemoglobin and CO2 solutions after 0.1 set and found that the decrement in dissolved COz cor- responded to the amount of hemoglobin carbamate that would have been expected on the basis of previous measurements at equilibrium with the use of the BaCL precipitation method.

E$ect of Oxygenation on Formation on Hemoglobin Carbamate

In two experiments the amount of carbamate present at 0.1 set decreased with oxygenation to 52 and 63% of that in deoxygenated hemoglobin (Table I and Fig. 2). This is to be compared with the data of Ferguson (11) which show a decrease of 61 70, a value in excellent agreement with the aforementioned.

The simple exponential rate constant k decreased to 53 and 38% of its value in deoxygenated hemoglobin. This could have occurred because of a decrease in k, or a decrease in [Hb-NH*] secondary to an increase in pK,, or both. In order to differ- entiate between these two possibilities, sufficient data for the reaction of CO:! and OzHb would be required to estimate K, and k, independently (as in Fig. 5). Such data are not yet available. Only one of the experiments (2-3-7) contains enough early data points to define the initial slope of the curve (Fig. 2). However, it seems probable that the dominant factor is a de- crease in K,, because oxygenation is generally known to alter the pK of various buffering groups on hemoglobin, although there are indications that the number of NH2 groups available to react with CO2 is less in OzHb than in Hb (see discussion in Reference 12).

Wyman (10) calculated that all the reduction in carbamino- hemoglobinate, which accompanies oxygenation of the hemo- globin, could be explained as secondary to the H+ liberated by the hemoglobin. However, in his argument he did not include the buffering action of the carbamate system (12). This is, in the end, tantamount to assuming that there is no direct action of oxygenation on carbamate formation. Rossi-Ber- nardi and Roughton (12) have determined carbamate in con- centrated hemoglobin solution at constant pH and poo,, and have found that it is reduced when the protein is oxygenated. Our data in Fig. 4 illustrate the same point.

TIME IN MILLISECONDS

FIG. 7. Graph of the decrease in pco2 with time after mixing of a 1:lO suspension of normal human blood in buffer at a pco2 ap- proximately zero and a bicarbonate solution with a pcol of 70 torr. ~0; was zero in both solutions. The solutions represented by the lower curve contained, in addition, 3.5 X IO+ M acetazolam- ide. (This graph was redrawn from Reference 1.)

Reaction of COZ and Hemoglobin in Red Cells

Constantine et al. (1) investigated the reaction of COZ with human red cell suspensions at 37”, both in the presence and absence of carbonic anhydrase inhibitor. An example of one of these experiments is presented in Fig. 7 (lower curve), which is a graph of the fall in pcoZ with time in a mixture of a 1: 10 dilution of normal human erythrocytes at a pcoz of 0 to 2 torr, and a solution of CO2 in 0.9% sodium chloride at a pcoz of 70 torr, both solutions being free of 02, at a concentration of 3.5 x lo-* M acetazolamide. The upper curve is an identical experiment but with the acetazolamide omitted. We presume that, in the presence of the carbonic anhydrase inhibitor, the upt,ake of COz is limited by the rate of the hydration within the cells. There is no apparent reason why the initial part of the lower curve should not be linear just as it is after 2 set, since the COZ would diffuse into the cell extremely rapidly, certainly within several milliseconds; in the absence of acetazol- amide (upper curve) the reaction was able to proceed at a more rapid and linear rate over this same range of pcoz decrement. In retrospect, it seems likely that the early part of the lower curve represents the disappearance of CO2 by some other mech- anism than that which dominates after 2 set, and the formation of carbamate is the obvious possibility. The amount of car- bamate formed by $ set should be represented by the intercept of the extrapolation of the later linear part of the curve, as shown by the dotted line, and the rate constant of carbamate formation should be calculable from Equation 9. This 3.8.torr drop in pcoz in the mixture corresponds to 0.12 mM carbamate. x0 is 35 torr, LZ~ is 31.2 torr, and ts is 0.060 sec. The pH in the solution was 7.5, so that within the red cell it should have been about 7.3, a hydrogen ion concentration of 5 X lo-’ M. The blood was diluted to 1:20 in the reacting mixture, so that [Hb] was 0.45 mM. The exponent in Equation 9 equals 0.693/0.060 = 11.6 set-I. [Hb-NH*] according to Equation 10, assuming K, = 6.7 X lo-’ M, is

0.45 x (3.7 x 10-8 6.7 X 10-S + 5 x 10-S

= 0.26 m&r

Therefore, according to Equat,ion 9,

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3326 Reaction of CO2 with Human Hemoglobin Vol. 243, No. 12

k, 11.6 X 3.8

= = M1 0.26 X lo-+ X 35

4.85 x 103 set-1

This is in reasonable agreement with the value calculated from our data in hemoglobin solution. According to calculations of chemical reaction + diffusion, similar to those of Roughton and Rupp (7), the rate of carbamate formation in the human red cell should be almost exactly the same as when this same hemoglobin is in solution.

AcknowledgmentsThe authors would like to thank Dr. Ricardo Puy, Mrs. Karen Creecy, Mrs. Mary Friedman, Mr. Irving Nagelberg, and Mrs. Beverly Florey for their help in performing these experiments; we thank Professor F. J. W. Roughton for letting us see some unpublished data at a critical stage in this work.

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With the technical assistance of D. ThyrumR. E. Forster, H. P. Constantine, Margot R. Craw, H. H. Rotman, R. A. Klocke and

with Human Hemoglobin Solution2Reaction of CO

1968, 243:3317-3326.J. Biol. Chem. 

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