Life Sciences

Vol. 7, Part I, pp . 1339-1343, 1968 .

Pergamon PressPrinted in Great Britain .

DIFFERENT EFFECTS OF 2 .3 DIPHOSPHOGLYCERATE AND ADENOSINE

TRIPHOSPHATE ON THE OXYGEN AFFINITY OF ADULT AND FOETAL

HUMAN HAEDfOGLOBIN

Ch . Bauer, I . Ludwig and Dt . Ludwig

Physiologisches Institut der Medizinischen Hochschule, }fannover

Germany(Received 5 September 1968 ; in final form 30 September 1968)

It is well known that the foetal blood of man has a higher oxygen

affinity at term than the blood of an adult (1) . If one examines

the OZ affinity of foetal (HbF) and adult (HbA) human haemoglobin

after haemolysis and dialysis in solutions with given molalities

and [ H+ ] concentrations, one observes in both haemoglobins an in-

crease of the oxygen affinity, which is more pronounced in HbA

than in HbF, so that the affinities become practically identical

(2,3,d) . This led to the conclusion that not the different struc-

ture of HbA and FIbF itself but environmental factors present in

the red cell are responsible for the different O Z affinities .

It has been found recently that organic phosphates and among

them especially the 2 .3 diphosphoglycerate (DPG) as well as the

adenosine triphosphate (ATP) considerably diminish the oxygen

affinity of 11bA (5,6) . These substances occur as substrates of

the erythrocyte metabolism in high concentrations in human red

cells . As the llPG and AfP concentration in the foetal red cell

is even higher than in the maternal red cell (7,8), the different

O Z affinities of foetal and adult blood can not be explained by

concentration differences of DPG and ATP . It might be possible

however, that DPG and ATP influence the O Z affinity of HbA and

1339

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OXYGEN AFFINITY

Vol. 7, No . 23

HbF to a different degree .

To clarify this question the specific effect of DPG and ATP

on the 02 affinity of HbA and HbF has been compared .

Methods

8 healthy mothers and their new-borns were examined . The mater-

nal blood was taken from an arm vein, the foetal blood from the

umbilical cord . To prepare haemoglobin solutions, erythrocytes

were washed three times in a bicarbonate buffer (0 .161 Mol/L H2 0 :

0.121 Mol/L KCL + 0.025 Mol/L NaCl + 0 .015 Mol/L NaHC03 ) . The

washed erythrocytes were haemolysed by thawing and freezing . To

remove membranes the haemolysate was centrifuged at 100 000 x g

for 3 hours at 20° C . The maternal and foetal haemoglobin solu-

tions were then dialysed in Visking sacks against large amounts of

bicarbonate buffer at 4 ° C for 3 days . 30 moles DPG/g Hb or 8

pmoles ATP/g Hb were added to the haemoglobin solutions, these con-

centrations being in the physiological range .

Analysi s

The 02 half saturation pressure at pH 7 .2o and 37 ° C (P50) as a

criterion for the oxygen affinity of the haemoglobin was measured

in whole blood and in haemoglobin solution according to the pro

cedure described by BARTELS and HARRIS (9) . Gas analyses were done

micro-manometrically (10) . The pH values of the samples (in whole

blood the intra-erythrocytec pHc value (11) ) were measured elec-

trometrically, and P50 was corrected to pH 7 .2o with a Bohr factor

of -0.48 .

Haemoglobin and methaemoglobin were measured photometrically

on the Zeiss spectrophotometer PMQ II .

Vol. 7, No . 23

OXYGEN AFFINITY

1341

Results and Discussion

TABLE 1

Half Saturation Pressures (P50) in mm Hg at pH 7 .20 (in Blood

pHc = 7 .20) and 37° C, in Maternal and Foetal Blood and Haemoglo-

bin Solutions+ without and with DPG or ATP . The following Symbols

were used : n = Number of Samples, x = Mean Value, sD = Standard

Deviation .

P50

Whole Blood

P50Dialysed Hb

P50Dialysed Hb + DPG

P 50Dialysed Hb + ATP

+The mean haemoglobin concentration of Hb-solutions was 12 g$,methaemoglobin was always lower than 2 .5 $ .

Results are summarized in Table 1 . P50 of maternal blood was

31 .o mm Hg, of foetal blood 24 .3 mm Hg . After dialysis the 02

affinity of HbA increased by 57$ and of HbF by 25$ : P50 was in

both solutions 19 .7 mm Hg . This observation is in accordance with

other authors (2,3,4) although our results can not be directly

compared because of different experimental conditions .

The effect of DPG on the 02 affinities of HbA and HbF was com-

pletely different .

Whereas P50 of HbA rose from 19 .7 to 29 .4 mm Hg by nearly 50$

thus confirming the results of other authors (5,6), P50 of HbF

Maternal Foetàl

x sD x sD31 .0 ± 1 .4 24 .3 ± 2 .4

19 .7 ± 1 .3 19 .7 ± 0 .9

29 .4 ± 1 .5 22 .4 ± 1 .4

21 .1 ± 1 .2 19 .6 ± 1 .8

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OXYGEN AFFINITY

Vol . 7, No . 23

increased only by 14$ from 19 .7 to 22 .4 mm Hg . Addition of ATP had

no influence on the oxygen affinity of foetal haemoglobin and lo-

wered that one of adult haemoglobin only slightly .

The binding of DPG to HbA shows an inverse relation to the bin-

ding of 02 , that is, the more DPG can be bound the more haemoglo-

bin is desoxygenated (12) . It seems that in foetal haemoglobin the

affinity for oxygen is definitely higher than for DPG . A hypothe-

sis,

to be work on, might be that the ~ 2 -chains the HbF tetramer

diminish the binding of DPG by the HbF tetramer .

It has been suggested recently that the different chemical com-

position of adult and foetal human erythrocyte membranes contri-

bute to the different 02 affinities (13) . However our results de

monstrate a different reaction between the two types of haemoglo-

bin and the two substrates influencing the oxygen affinity .

Summary

Blood from 8 mothers and their new-borns was examined . The oxy-

gen half saturation pressure (P50) as measure for the 02 affinity

of haemoglobin was determined in whole blood and in haemoglobin

solutions .

P50 of maternal blood was 31 .o mm Hg, P50 of foetal blood 24 .3

mm Hg . After haemolysis and dialysis P50 of adult (HbA) and foetal

haemoglobin (HbF) fell to 19 .7 mm Hg . Addition of DPG (30 .0 ~mo

les/g Hb) to the haemoglobin solutions raised P50 of HbA to 29 .4

mm Hg, of HbF only to 22 .4 mm Hg . ATP (8 .o umoles/g Hb) had little

influence on the 0 2 affinity of HbA (P50 rose from 19 .7 to 21 .1 mm

Hg) but not on the 02 affinity of HbF .

Vol. 7, No . 23

OXYGEN AFFII~TITY

1343

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