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Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes
ARCHIVES
OF
BIOCHEMISTRY
.\ND
Effect of Organic
BIOPHYSICS
and
Inorganic
Equilibrium ALFRED Biochemical
Laboratory.
Universily
Phosphates
of Human
CHANUTIX
Received
121, 96-102 (1967)
of Virginia
January
Erythrocytes’
RICHARD
AND
on the Oxygen
R. CURWISH
School of Medicine,
20, 1967; accepted
Febrrlary
Charlottesville,
Virginia
22903
4,1967
The effect of organic and inorganic phosphates on the functional properties of purified adult human hemoglobin has been investigated. Adenosine t,riphosphate, guanosine triphoaphate, and 2,3-diphosphoglycerate are particularly effective in decreasing the oxygen affinity of hemoglobin and are responsible for a small change in heme-heme interaction. The di- and mononucleotides have a neglibigle effect on the log PI/~ and 7~ values. Hexametaphosphate causes marked changes in both the oxygen affinity and the oxygen equilibrium curve, but tripolyphosphate, tetrametaphosphate, and pyrophosphate are less effective. The oxygen affinity of hemoglobin is decreased in the presence of 2,3-diphosphoglycerate over a wide pH range. At high concentrations of phosphate buffer, diphosphoglycerate has practically no effect on the log PI,? and II values. The result,s are discussed in relation to the changes in the oxyhemoglobin dissociation curve of stored blood.
This study was undertaken to determine t,he effect of phosphorylated carbohydrat,e intermediates and inorganic phosphates on the oxygen dissociation curve of hemoglobin. Electrophoretic analysis showed that a number of phosphate compounds reacted with hemoglobin to form a complex which was observed as a slow-moving boundary (designated as Component B) (1-3). The organic phosphorylated compounds which yielded the greatest concentration of Component B were 2, %diphosphoglycerate and trinucleotides; the respective dmucleotides were less effect,ive and the mononucleotides had a small or negligible effect,. Tripolyphosphate, tetra- and hexametaphosphates also formed hemoglobin-inorganic phosphate complexes. The results of the present, investigation show t,hat the organic and inorganic phosphates capable of yielding the great’est’ amount of t’he hemoglobin complex have the most pronounced effect’ on oxygen equilibrium.
EXPERIMENTAL
METHODS
Outdated blood, obtained from the University Hospital Blood Bank, was allowed t.o stand at room temperature for 3 days in order to reduce the concentration of red cell organic phosphate to trace amounts. The hemolyzate was prepared from red cells which were washed twice with cold isotonic saline. After three volumes of distilled water were added, the hemolyzate was centrifuged at high speed to remove practically all of the stroma. The clarified hemoglobin (Hb) solution was dialyzed against large volumes of distilled water for 24 hours, and small amounts of residual stroma were removed by centrifugation. Purified Hb was prepared from red cells of outdat,ed blood which had been stored at 4’ for 21-29 days. The washed red cells were hcmolyzed by addition of water and the stroma n-as removed by centrifugation. The clarified solution was dialyzed against neutralized ammonium sldfate according to the method of Chernoff and Liu (4). The Hb precipitate was dissolved in water and t,he solution was dialyzed in water nntil free of sulfate. The stock preparations of the hemolyzate and the purified Hb solution were adjusted to 2yG Hb. The solution was diluted with an equal volume of phosphate bufier, with or wit.hout a supplement, and clarified by filtration through a Millipore filter. The Hh concentration for all preparations was lro (1.55 X 1W5 M) and the final ionic strength
1 This work was supported by grant DA-MD49-193.66.G9206 from the U.S. Army Medical Research and Development Command, Office of the Surgeon General. 96
Ij:PFI
OF PHOSPH.4TF:S
O?i OSYGli;N
TABLE OXYGEN
kkjOILII3ILIIJ’LI
Conditiolls:
OF
Hb l..T.i X lo-”
I
IhYTHlWCY1.E
M (as Hh);
HEMOLYZ
\‘rW
.\SD
pH 7.0; 20”; KH2P04-KYIIP04
min.-max.
Hemolyzates (28) Purified Hb (11) error.
,\?J
07
Pc-~~~FIEIJ
Figltres
.81-.89 .74-.84 in parent.heses
.85 f .79 *
was 0.05, except when noted. The stock hemolgzate were st,ored at 4” for a and plu%ird Hb sol~~iio~~s maximum of olie week. Neither of these solutions contained methemoglobin when analyzed hi- t,he Evelyn-Malloy procedure (5). The spertrnphotometric method of RossiFanelli and hntnnini (6) was used for measuring the oxygen cqrlilibril~m. The procedl[re for deoxygenation was modified to prevent denaturat,ion of lib. Two ml of the bllffered 17, Hb solution was introduced into the tollometer wit.h an 8.mm spncer in place, thlls giving a 2.mm light, path. The solution was evacuated 3’5 minutes with a water vaclluni ptlmp :rlld argon was introduced into the lonometrr for 011c minute. Usrlally, four solirtions were prepared and were rot,ated ill a horizontal position at, about 2G revolrlt,ions per minllte for 10 mil1ute.a in a constant lemperatllre bath at 20”. After an additional lo-second evacuation of the tonometclr, argc,tt was again int,roduced and the solution was cqrlilibrated for 5 minutes. The tonometer was finally evacllatcd for 2 milllltfts before int,rodllcing air. Hemoglobin was completely reduced under these conditions and there was no turbidity. The loss of w-ater, as judged by weighing the tonometer or by determining Hh cnnrent ration, was negligible. The optical density was determined at, 540, 500, and 5% mp in a Beckman DU spectrophotometer; the temperat~ure of the solution was maintained at. 20” in :t ti1~1 thermospacer. When t,he tonometers were olltside the constant temperat,ure hath, the) were immersed in water at 20” in a Ucwar flask. After each addition of air, the sollttion was eqrrilibrated at 20” for 4 minLltes and the optical density determined. Hemoglobin was were readings assayed by the c~:~rl~~~ethcmoglobil~ procedltre of Crosby ct nl. (7). The oxygen pressllre in the tonometer and the percentagr oxygenation were calcrdated according to previously described procedclres (6). The csthe magnitltde of hem{>pressiotl n, represcllting hemc interactioll, was determined from the slope of the lilac obtaillrd by plottitlg log p (oxygen pressure) agailrst 2//l-!/, where y denotes percentage HbOt. The log of the oxygen pressure at which Hb u-w half-s:tt\lrated was designated as log pi:.
I’~oL[:,~I()N~
n Mean
represent
1~11)
bluffer, 0.023 1%.
log pi I’
S,wcimm
0 Standard
HUM
IQI~lI,lBl:IIr>I
min.-maT.
.ooQ .004”
hlenn
2. .i-3 .4 2.8-3.4
2.9 f 3.1 f
.oxP .023,1
nLlmher of samples. The organic phosphate compounds added to the Hb solutions were pllrchased from Schwarz BioItescarrh, Inc. Soditml triphosphate was donat,ed by the \.irgirria-Carolina Company and sodiltm tetra- and hexametaphosphat,es were provided by the Monsanto Company. Stock solutions of the compounds were adjrlsted to the desired pH before addition to the hemoglobin solutions. Srlpplements were added ahout 2 hollrs before analyses wpre begun. The cotlcentration of each supplement was expressed as ~molcs/gm Hb. Phosphat,e bluffer solrlt,iolls of kllowtl pH atrd ionic strength wprc prepared according to data presented by Green (8).
The n a~ttl log pi/g w~luos for red ccl1 hcmolyzates and for purified Ht) arc shown in Table I. Although the differcttccs t~etjw-eett the mean values are small, they arc significantl~ different (p < .Ol). The experiments descrthed hclow n-crc done with purified Hb solutions since they show smaller variations in the oxygen dis8oci3tiottcttrVCstl~:ltthenlolyzates during storage in the cold. RossiI’attelli and Antottini (9) noted no diffcronce in the n and log p1/2 values of freshly ptvpared hemolyzatw and crystallized Hh. Oxygen
equilibriwu
in
presence
oj orgmic
phosphates. The cffwt of different cortcwtr:ltions of :Ldcttositte tri-, di-, and monophosphwtcs (ATI’: ,L\I)I’. :ZRII’) and guanosinc tri-, di-, and ntottol,hosphates (GTI’, (+DI’, G.\IP) OIL thv oxygctt dissociation curvcx of purified hum:m Hh is showtt in I“@. 1 and 2. The pmolcs Hb ‘pmoles phosl)h:tto ratios for 2..i, -5.0, 10, :d 20 pmolw of 311compouttds added arcs 6.2, 3.1, 1 ..G, and 0.77.5, rwpcctivcl!.. Thwc data are typical of :I tiunitwr of cxprrimcnt~s cot~ducted \vith both hcmolyzatw and purified Hh solutions. It is ob scrvcd that the dissociat iott curve shift, apprcciahly to the right :w thv cottcwtrati(JlW (Jf AiTI’ and CT1 ar(’ itwtwscd. The
98
CHANUTIN
AND
CURNISH IOpmoles/gHb
AMP
54 .a
1.2
i
log P
4
.a
I.2
FIG. 1. Oxygen equilibrium of purified human Hb in presence of ATP, ADP and AMP. Hb (1%) in phosphate buffer r/2 = .05, pH 7.0,20”. 0, Control; V, 2.5pmoles/gm Hb; n , 5 pmoleslgm Hb; 0, 10 rmoles/gm Hb. pmoles/g
I
.4
.8
FIG. 2. Oxygen equilibrium Hb (lyO) in phosphate buffer
.-
12
4
.-
.8
Oxygen
equilibriunz
in presence
IOpmoledg Ht
o-10.0
1.2 4 log P
.8
12
of purified human Hb in presence r/2 = .05, pH 7.0,20”.
changes caused by the respective di- and mononucleotides are small or negligible. The data for n and log ~11~ for t,he phosphorylated compounds studied are shown in Table II. It is observed that, 2,3-diphosphoglycerate (DPG) has the same effect on the dissociation curves as the trinucleotides. Pyridoxal S-phosphate (Pyr-5-P) has an appreciable effect on the log ~11~ values while ribose 5-phosphate (R-*5-P) has practically no influence. The values for n decrease slightly in the presence of ATP, GTP, and DPG. of inorganic
Typical effects of varying concentrations of pyrophosphate (Pz), tripolyphosphate (P3), tet,ra- (PJ, and hexa-
phosphates.
Hb 0
4 of GTP,
.8 GDP,
I.2 and GMP.
metaphosphates (P6) on the oxygen equilibrium of purified Hb solutions are shown in Fig. 3 and Table III. The order of effectiveness in influencing log ~112and n values is P6 > Pp8> Pq > PB. Hexametaphosphate causes greater changes than are observed for any of the phosphates studied. Examination of the I’s dat,a shows that, the deviations from the straight line in the graphs are appreciable at low and high HbOz levels. It is assumed t’hat hexametaphosphate reacts with Hb to cause structural changes which are responsible for the results observed. E$ect of pH on oxygen equilibrium. The data presented graphically in Fig. 4 show the effect of pH on both the n and log pr/2 values of the control and DPG-supplemented
TABLE
II
OSYGES EQUILIBRIUM OF PURIFIED Hb IX PRESENCE OF ORGANIC PIIOSPHOI~TI..\,TIE~) Co~rwu~us Conditions: IIb 1.55 X lo-” M; pFI 7; 20”; phosphate buffer 0.023 M .AblP ATP ADP ~__ ~~ pmolessupplement __ ~---~ log PI/2 IL log PII2 n log PII2 w ~~-. __-~~ ..~ 0 2.5 5.0 10.0 10.0”
0.77 0.79 0.86 0.94 0.95
3.1 2.9 2.9 2.8 2.7
0.81 0.83 0.87 0.8i 0.87
GTP ~--0 2.5 5.0 10.0 10.0
0.78 0.82 0.90 0.97 0.91
0.78 0.81 0.85 0.93
0.81 0.82 0.84 0.79 0.82
3.0 2.9 2.8 2.7 2.7
0.82 0.82 0.86 0.86 0.86
2.9 2.9 2.7 2.7 2.9
0.78 0.80 0.83 0.84 0.81
0.80 0.82 0.84 0.84
(0.77
3.0)
2.9 2.8 2.7 2.7 3.0
(0.77
3.0)
Pyr-S-P
R-5-P 3.1 2.9 2.8 2.7
3. 1 3. 1 3.1 3.1 3.0 GMP
GDP
DPG 0 2.5 5.0 10.0
3.4 3.2 3.1 3.1 2.9
3.2 3.2 3.2 3.2
0 Figures in italics represent data for the same IIb solution; control data for log pm and n, respectively.
0.83 0.86 0.91 0.93 figures
2.9 2.9 2.9 2.8 in parentheses
represent
’ ’’ ’’
Hexametapho’
4
.e
1.2
log P FIG. 3. Oxygen eqiiilibrium of purified human Hb in presence of pyrophosphate, tripolyphosphate, and tetra- and hexametaphosphate. Hb (1%) in phosphate buffer 1‘/2 = .05, pH 7.0, 20”.
the
100
CHANUTIN
OXYGEN
EQUILIBRIUM
OF PURIFIED
AND
CURNISH
TABLE III Hb IN PRESENCE
OF INORG.\NIC
PHOSPHATES
Conditions: Hb 1.55 X IO-&M; pH 7; 20”; phosphate bnffer0.023 M pm&s
supplement 0 2.5 5.0 10.0 20.0
.77
3.1
.83 .87 .91
2.9 2.7 2.6
0 Pz, Pyrophosphate;
.77 .84 .89 .97
PI, tripolyphosphate;
.77 .80 .82 .91
3.0 2.9 2.5 2.4
3.3 3.1 2.9 2.9
P,, tetrametaphosphate;
.74 .81 .92 1.15
3.2 2.7 2.1 1.4
P6, hexametaphosphate.
I
6.0
6.5
7.0
7.5
8.0
PH FIG. 4. Effect of 2,3-diphosphoglycerate on Bohr effect and values of n. Hb (1%) in phdsphate buffers I’/2 = .05, 20”. The r/2 and molar concentrations of the buffers are shown in Table IV. (log P50 = log p&
Hb solutions. The mean value of n for the controls at different pH levels is 3.0 f .Oll (SE); that of the solutions supplemented with 10 pmoles DPG is 2.6 f .03. The difference between these two mean values is statistically significant’. The mean values of n for the control Hb solutions at different pH levels are consistentlygreaterthan those of the DPG-supplemented solutions. The characteristic Bohr effect is seen in both the control and DPG-supplemented Hb solutions. As the DPG concentration increases, the log ~112 values are elevated. Under these
experiment,al conditions, the HbOz is more readily dissociated over a wide pH range in the presence of DPG. The log p1/2 values for cont’rol and ARIPsupplemented (Ti pmoles/gm Hb) Hb solutions were identical at pH G.S, 7.0, 7.2, and
7.4. gffect of salt concentration and pH on oxygen equilibrium. Table IV shows the data for n and log ~111 of Hb in phosphate buffers with ionic strengths varying from .Oli to 2.0 at pH 6.2, 7.0, and 7.7. Results at pH 5.S were discarded due to the formation of
KFFECT
OF PHOSPHATES
Opi OSPGEN
TABLE EFFECT
I\’
AND pH ON OXYGEK EQUILIBRIUM OF C~NTROI, (lO~moles/gm Hb) I~EMOGLOBIN SOLUTIOSS Hb 1.55 X lo-” M; 20”.
OF SALT CONCENTRATION SUPPLEMENTED
Conditions:
pH 6.2
I r/2” Ma
-~
pH
DPG
Control n
log PI/t
?I
DPG .___._n log PII2
Control
I”
log PI/Z
?I
AND
DP(;-
pH 7.7
7.0
log PIi?
~..
Control
ila n
log PII2
DPG n log p,,s
~~.__.
.037 .070
0.05
0.10 0.20 0.40 0.50 1.00 1 ..50 2.00
101
EC~lJILIBI:IIDI
.13-l
,300 ..i83
.853
2.6 2.7 3.0
1.03 2.4 1.06 2.4 1.12 2.7
1.10 1.10 1.11
3.0 3.1 3.1
1.12 2.7 1.08 3.1 1.08 3.1
1.14 1.13 1.13
-I
,023 .045
2.9 0.82 3.0 0.91
,089 ,172 .213 ,425
3.1 3.0 3.0
,644 ,868
0.91 0.95 1.01 0.98 1.01
1.00 ---1.00 2.9
-1.02
0.96 0.99 0.99 1.00
3.0 3.0 3.0
2.4 0.94 2.6 2.9 2.9 2.9 2.9
,018 .036 .072
3.4 3.3 3.3
.46 2.6 .54 2.6 .56 2.9
.@2 .62 .59
.179 ,359
3.3
.70
3.1
.72
3.3 3.3 3.3
.7.5 3.2 .82 3.3 .84 3.3
.i.i .83 .83
.544 ,734
” Final ionic strength or molarity of phosphate lxlffers.
mcthemoglohin at high salt concent,rations. ,4t, pH 6.2 the rz values of the control solutions increase slightly at I’/2 = 0.2 and remain const,ant at higher ionic skengths; t,hese values are t)he same over the entire range of salt concentrations at pH 7.0 and 7.7. In the solutions supplemented wit’h DPG, the n values are consistently lower t,han those of the controls at low ionic strengths and approach or reach maximum levels as t,he salt concentrat,ion is increased. The log l)l/Z values for the control and the Dl’(:-supplemented Hb solutions arc about the same in t’he pH 6.2 and 7.0 groups; at pH 7.7 the values are higher in the DPG solut,ions at, lower ionic strengths. The inability of DPG to exert an effect on oxygen equilibrium at, high salt concentrations may bc due to t,he dissociation of the hemoglobinDI’G complex (10). The effect, of concentrated salt solutions on t,hr molrcular and funct,ional properties of Hb has been reviewed and discussed by Rossi-l’anelli ei al. (11) and Wyman (12). DISCUSSION
Two compounds, DPG and ATE’, which arc capable of increasing the dissociat’ion of Hb&, account for about 60 and 20 %, respectively, of the organic phosphate in the normal red cell. The molar concentrat,ion of Dl’cT is :rbout fourfold greater than ,4TP.
It is krlowvrl that Dl’(; part’icipates in the metabolism of t,he red cell, but its exact role is not understood or clearly defined. This compound readily forms a complex with Hb (1, 2) and also causes HbO:, to release 02 more readily. Valt’is and Kennedy (13) observed that the oxygen dissociation curves of blood shifted progressively to the left during storage in acid-&rate-dcxtrosc. It has been shown that DPG decreases rapidly during the first 2 weeks of storage nnd that ATP decreases slowly: (14). In view of the results presented in this paper, it is suggested t,hat the Valt,is-Kennedv shift may be due in part, to t,he decrease ‘in DPG concent,ration. If this hypot)hesis is valid, it should be possible to resynthesize DPG by incuhat8ing stored blood in the presence of inosine (15) at a proper pH and cause the oxygen dissociation curve to shift, back to the right. Sote added in proof: Benesch alld Benesch ohserved that 2,3 diphosphoglycerate and trinucleotides decreased the oxygen affinit,y of hwnan hemoglobin. They wggested that t,hese and ot,her phosphorylated componnds may fnnction as regrllators of oxygen transport. [Riochen2. Hiophus. Kes. Co,tzVLWL. 26, 162 (1967), Federation Proc. 26, fii3 (1967)l. REFERENCES 1.
SIJGITA,
&ptl.
Y ,, Biol.
AND
CIIA~TIN,
A., Proc. Sot.
Med. 112, 72 (1963).
102
CHANUTIN
2. CHANCTIN, A., AND CURNISH, 11. R., Arch. Biochem. Biophys. 106, 433 (1964). 3. CHANUTIN, A., AND CURNISH, 11. R., Proc. Sot. Exptl. Biol. Med. 120, 291 (1965). 4. CHERNOFF, A. I., AND LIU, J. C., Blood 17, 54 (1961). 5. EVELYN, K. A., AND MALLOY, H. T., J. Biol. Chem. 126, 655 (1938). 6. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 77, 478 (1958). 7. CROSBY, W. H., MUNN, J. I., AND FURTH, F. W., U.S. Armed Forces Med. J. 6, 693 (1954). 8. GREEN, A. A., J. Am. Chem. Sot. 66, 2331 (1933).
AND
CURNISH
9. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 80, 299 (1959). 10. LUDEWIG, S., AND CHANUTIN, A., Acta Biochim. Polonica 11, 85 (1964). Il. ROSSI-FANELLI, A., ANTONINI, E., AND CAPUTO, A., Advan. Protein Chem. 19, 73 (1964). 12. WYMAN, J., JR. Advan. Prolein Chem. 19, 223 (1964). 13. VALTIS, D. J., AND KENNEDY, A. C., Lancet 1, 119 (1954). 14. BARTLETT, G. R., AND BARNET, H. N., J. Clin. Invest. 39, 56 (1960). 15. BARTLETT, G. R., AND SHAFER, A. W., J. Clin. Invest. 40, 1185 (1961).
OF
BIOCHEMISTRY
.\ND
Effect of Organic
BIOPHYSICS
and
Inorganic
Equilibrium ALFRED Biochemical
Laboratory.
Universily
Phosphates
of Human
CHANUTIX
Received
121, 96-102 (1967)
of Virginia
January
Erythrocytes’
RICHARD
AND
on the Oxygen
R. CURWISH
School of Medicine,
20, 1967; accepted
Febrrlary
Charlottesville,
Virginia
22903
4,1967
The effect of organic and inorganic phosphates on the functional properties of purified adult human hemoglobin has been investigated. Adenosine t,riphosphate, guanosine triphoaphate, and 2,3-diphosphoglycerate are particularly effective in decreasing the oxygen affinity of hemoglobin and are responsible for a small change in heme-heme interaction. The di- and mononucleotides have a neglibigle effect on the log PI/~ and 7~ values. Hexametaphosphate causes marked changes in both the oxygen affinity and the oxygen equilibrium curve, but tripolyphosphate, tetrametaphosphate, and pyrophosphate are less effective. The oxygen affinity of hemoglobin is decreased in the presence of 2,3-diphosphoglycerate over a wide pH range. At high concentrations of phosphate buffer, diphosphoglycerate has practically no effect on the log PI,? and II values. The result,s are discussed in relation to the changes in the oxyhemoglobin dissociation curve of stored blood.
This study was undertaken to determine t,he effect of phosphorylated carbohydrat,e intermediates and inorganic phosphates on the oxygen dissociation curve of hemoglobin. Electrophoretic analysis showed that a number of phosphate compounds reacted with hemoglobin to form a complex which was observed as a slow-moving boundary (designated as Component B) (1-3). The organic phosphorylated compounds which yielded the greatest concentration of Component B were 2, %diphosphoglycerate and trinucleotides; the respective dmucleotides were less effect,ive and the mononucleotides had a small or negligible effect,. Tripolyphosphate, tetra- and hexametaphosphates also formed hemoglobin-inorganic phosphate complexes. The results of the present, investigation show t,hat the organic and inorganic phosphates capable of yielding the great’est’ amount of t’he hemoglobin complex have the most pronounced effect’ on oxygen equilibrium.
EXPERIMENTAL
METHODS
Outdated blood, obtained from the University Hospital Blood Bank, was allowed t.o stand at room temperature for 3 days in order to reduce the concentration of red cell organic phosphate to trace amounts. The hemolyzate was prepared from red cells which were washed twice with cold isotonic saline. After three volumes of distilled water were added, the hemolyzate was centrifuged at high speed to remove practically all of the stroma. The clarified hemoglobin (Hb) solution was dialyzed against large volumes of distilled water for 24 hours, and small amounts of residual stroma were removed by centrifugation. Purified Hb was prepared from red cells of outdat,ed blood which had been stored at 4’ for 21-29 days. The washed red cells were hcmolyzed by addition of water and the stroma n-as removed by centrifugation. The clarified solution was dialyzed against neutralized ammonium sldfate according to the method of Chernoff and Liu (4). The Hb precipitate was dissolved in water and t,he solution was dialyzed in water nntil free of sulfate. The stock preparations of the hemolyzate and the purified Hb solution were adjusted to 2yG Hb. The solution was diluted with an equal volume of phosphate bufier, with or wit.hout a supplement, and clarified by filtration through a Millipore filter. The Hh concentration for all preparations was lro (1.55 X 1W5 M) and the final ionic strength
1 This work was supported by grant DA-MD49-193.66.G9206 from the U.S. Army Medical Research and Development Command, Office of the Surgeon General. 96
Ij:PFI
OF PHOSPH.4TF:S
O?i OSYGli;N
TABLE OXYGEN
kkjOILII3ILIIJ’LI
Conditiolls:
OF
Hb l..T.i X lo-”
I
IhYTHlWCY1.E
M (as Hh);
HEMOLYZ
\‘rW
.\SD
pH 7.0; 20”; KH2P04-KYIIP04
min.-max.
Hemolyzates (28) Purified Hb (11) error.
,\?J
07
Pc-~~~FIEIJ
Figltres
.81-.89 .74-.84 in parent.heses
.85 f .79 *
was 0.05, except when noted. The stock hemolgzate were st,ored at 4” for a and plu%ird Hb sol~~iio~~s maximum of olie week. Neither of these solutions contained methemoglobin when analyzed hi- t,he Evelyn-Malloy procedure (5). The spertrnphotometric method of RossiFanelli and hntnnini (6) was used for measuring the oxygen cqrlilibril~m. The procedl[re for deoxygenation was modified to prevent denaturat,ion of lib. Two ml of the bllffered 17, Hb solution was introduced into the tollometer wit.h an 8.mm spncer in place, thlls giving a 2.mm light, path. The solution was evacuated 3’5 minutes with a water vaclluni ptlmp :rlld argon was introduced into the lonometrr for 011c minute. Usrlally, four solirtions were prepared and were rot,ated ill a horizontal position at, about 2G revolrlt,ions per minllte for 10 mil1ute.a in a constant lemperatllre bath at 20”. After an additional lo-second evacuation of the tonometclr, argc,tt was again int,roduced and the solution was cqrlilibrated for 5 minutes. The tonometer was finally evacllatcd for 2 milllltfts before int,rodllcing air. Hemoglobin was completely reduced under these conditions and there was no turbidity. The loss of w-ater, as judged by weighing the tonometer or by determining Hh cnnrent ration, was negligible. The optical density was determined at, 540, 500, and 5% mp in a Beckman DU spectrophotometer; the temperat~ure of the solution was maintained at. 20” in :t ti1~1 thermospacer. When t,he tonometers were olltside the constant temperat,ure hath, the) were immersed in water at 20” in a Ucwar flask. After each addition of air, the sollttion was eqrrilibrated at 20” for 4 minLltes and the optical density determined. Hemoglobin was were readings assayed by the c~:~rl~~~ethcmoglobil~ procedltre of Crosby ct nl. (7). The oxygen pressllre in the tonometer and the percentagr oxygenation were calcrdated according to previously described procedclres (6). The csthe magnitltde of hem{>pressiotl n, represcllting hemc interactioll, was determined from the slope of the lilac obtaillrd by plottitlg log p (oxygen pressure) agailrst 2//l-!/, where y denotes percentage HbOt. The log of the oxygen pressure at which Hb u-w half-s:tt\lrated was designated as log pi:.
I’~oL[:,~I()N~
n Mean
represent
1~11)
bluffer, 0.023 1%.
log pi I’
S,wcimm
0 Standard
HUM
IQI~lI,lBl:IIr>I
min.-maT.
.ooQ .004”
hlenn
2. .i-3 .4 2.8-3.4
2.9 f 3.1 f
.oxP .023,1
nLlmher of samples. The organic phosphate compounds added to the Hb solutions were pllrchased from Schwarz BioItescarrh, Inc. Soditml triphosphate was donat,ed by the \.irgirria-Carolina Company and sodiltm tetra- and hexametaphosphat,es were provided by the Monsanto Company. Stock solutions of the compounds were adjrlsted to the desired pH before addition to the hemoglobin solutions. Srlpplements were added ahout 2 hollrs before analyses wpre begun. The cotlcentration of each supplement was expressed as ~molcs/gm Hb. Phosphat,e bluffer solrlt,iolls of kllowtl pH atrd ionic strength wprc prepared according to data presented by Green (8).
The n a~ttl log pi/g w~luos for red ccl1 hcmolyzates and for purified Ht) arc shown in Table I. Although the differcttccs t~etjw-eett the mean values are small, they arc significantl~ different (p < .Ol). The experiments descrthed hclow n-crc done with purified Hb solutions since they show smaller variations in the oxygen dis8oci3tiottcttrVCstl~:ltthenlolyzates during storage in the cold. RossiI’attelli and Antottini (9) noted no diffcronce in the n and log p1/2 values of freshly ptvpared hemolyzatw and crystallized Hh. Oxygen
equilibriwu
in
presence
oj orgmic
phosphates. The cffwt of different cortcwtr:ltions of :Ldcttositte tri-, di-, and monophosphwtcs (ATI’: ,L\I)I’. :ZRII’) and guanosinc tri-, di-, and ntottol,hosphates (GTI’, (+DI’, G.\IP) OIL thv oxygctt dissociation curvcx of purified hum:m Hh is showtt in I“@. 1 and 2. The pmolcs Hb ‘pmoles phosl)h:tto ratios for 2..i, -5.0, 10, :d 20 pmolw of 311compouttds added arcs 6.2, 3.1, 1 ..G, and 0.77.5, rwpcctivcl!.. Thwc data are typical of :I tiunitwr of cxprrimcnt~s cot~ducted \vith both hcmolyzatw and purified Hh solutions. It is ob scrvcd that the dissociat iott curve shift, apprcciahly to the right :w thv cottcwtrati(JlW (Jf AiTI’ and CT1 ar(’ itwtwscd. The
98
CHANUTIN
AND
CURNISH IOpmoles/gHb
AMP
54 .a
1.2
i
log P
4
.a
I.2
FIG. 1. Oxygen equilibrium of purified human Hb in presence of ATP, ADP and AMP. Hb (1%) in phosphate buffer r/2 = .05, pH 7.0,20”. 0, Control; V, 2.5pmoles/gm Hb; n , 5 pmoleslgm Hb; 0, 10 rmoles/gm Hb. pmoles/g
I
.4
.8
FIG. 2. Oxygen equilibrium Hb (lyO) in phosphate buffer
.-
12
4
.-
.8
Oxygen
equilibriunz
in presence
IOpmoledg Ht
o-10.0
1.2 4 log P
.8
12
of purified human Hb in presence r/2 = .05, pH 7.0,20”.
changes caused by the respective di- and mononucleotides are small or negligible. The data for n and log ~11~ for t,he phosphorylated compounds studied are shown in Table II. It is observed that, 2,3-diphosphoglycerate (DPG) has the same effect on the dissociation curves as the trinucleotides. Pyridoxal S-phosphate (Pyr-5-P) has an appreciable effect on the log ~11~ values while ribose 5-phosphate (R-*5-P) has practically no influence. The values for n decrease slightly in the presence of ATP, GTP, and DPG. of inorganic
Typical effects of varying concentrations of pyrophosphate (Pz), tripolyphosphate (P3), tet,ra- (PJ, and hexa-
phosphates.
Hb 0
4 of GTP,
.8 GDP,
I.2 and GMP.
metaphosphates (P6) on the oxygen equilibrium of purified Hb solutions are shown in Fig. 3 and Table III. The order of effectiveness in influencing log ~112and n values is P6 > Pp8> Pq > PB. Hexametaphosphate causes greater changes than are observed for any of the phosphates studied. Examination of the I’s dat,a shows that, the deviations from the straight line in the graphs are appreciable at low and high HbOz levels. It is assumed t’hat hexametaphosphate reacts with Hb to cause structural changes which are responsible for the results observed. E$ect of pH on oxygen equilibrium. The data presented graphically in Fig. 4 show the effect of pH on both the n and log pr/2 values of the control and DPG-supplemented
TABLE
II
OSYGES EQUILIBRIUM OF PURIFIED Hb IX PRESENCE OF ORGANIC PIIOSPHOI~TI..\,TIE~) Co~rwu~us Conditions: IIb 1.55 X lo-” M; pFI 7; 20”; phosphate buffer 0.023 M .AblP ATP ADP ~__ ~~ pmolessupplement __ ~---~ log PI/2 IL log PII2 n log PII2 w ~~-. __-~~ ..~ 0 2.5 5.0 10.0 10.0”
0.77 0.79 0.86 0.94 0.95
3.1 2.9 2.9 2.8 2.7
0.81 0.83 0.87 0.8i 0.87
GTP ~--0 2.5 5.0 10.0 10.0
0.78 0.82 0.90 0.97 0.91
0.78 0.81 0.85 0.93
0.81 0.82 0.84 0.79 0.82
3.0 2.9 2.8 2.7 2.7
0.82 0.82 0.86 0.86 0.86
2.9 2.9 2.7 2.7 2.9
0.78 0.80 0.83 0.84 0.81
0.80 0.82 0.84 0.84
(0.77
3.0)
2.9 2.8 2.7 2.7 3.0
(0.77
3.0)
Pyr-S-P
R-5-P 3.1 2.9 2.8 2.7
3. 1 3. 1 3.1 3.1 3.0 GMP
GDP
DPG 0 2.5 5.0 10.0
3.4 3.2 3.1 3.1 2.9
3.2 3.2 3.2 3.2
0 Figures in italics represent data for the same IIb solution; control data for log pm and n, respectively.
0.83 0.86 0.91 0.93 figures
2.9 2.9 2.9 2.8 in parentheses
represent
’ ’’ ’’
Hexametapho’
4
.e
1.2
log P FIG. 3. Oxygen eqiiilibrium of purified human Hb in presence of pyrophosphate, tripolyphosphate, and tetra- and hexametaphosphate. Hb (1%) in phosphate buffer 1‘/2 = .05, pH 7.0, 20”.
the
100
CHANUTIN
OXYGEN
EQUILIBRIUM
OF PURIFIED
AND
CURNISH
TABLE III Hb IN PRESENCE
OF INORG.\NIC
PHOSPHATES
Conditions: Hb 1.55 X IO-&M; pH 7; 20”; phosphate bnffer0.023 M pm&s
supplement 0 2.5 5.0 10.0 20.0
.77
3.1
.83 .87 .91
2.9 2.7 2.6
0 Pz, Pyrophosphate;
.77 .84 .89 .97
PI, tripolyphosphate;
.77 .80 .82 .91
3.0 2.9 2.5 2.4
3.3 3.1 2.9 2.9
P,, tetrametaphosphate;
.74 .81 .92 1.15
3.2 2.7 2.1 1.4
P6, hexametaphosphate.
I
6.0
6.5
7.0
7.5
8.0
PH FIG. 4. Effect of 2,3-diphosphoglycerate on Bohr effect and values of n. Hb (1%) in phdsphate buffers I’/2 = .05, 20”. The r/2 and molar concentrations of the buffers are shown in Table IV. (log P50 = log p&
Hb solutions. The mean value of n for the controls at different pH levels is 3.0 f .Oll (SE); that of the solutions supplemented with 10 pmoles DPG is 2.6 f .03. The difference between these two mean values is statistically significant’. The mean values of n for the control Hb solutions at different pH levels are consistentlygreaterthan those of the DPG-supplemented solutions. The characteristic Bohr effect is seen in both the control and DPG-supplemented Hb solutions. As the DPG concentration increases, the log ~112 values are elevated. Under these
experiment,al conditions, the HbOz is more readily dissociated over a wide pH range in the presence of DPG. The log p1/2 values for cont’rol and ARIPsupplemented (Ti pmoles/gm Hb) Hb solutions were identical at pH G.S, 7.0, 7.2, and
7.4. gffect of salt concentration and pH on oxygen equilibrium. Table IV shows the data for n and log ~111 of Hb in phosphate buffers with ionic strengths varying from .Oli to 2.0 at pH 6.2, 7.0, and 7.7. Results at pH 5.S were discarded due to the formation of
KFFECT
OF PHOSPHATES
Opi OSPGEN
TABLE EFFECT
I\’
AND pH ON OXYGEK EQUILIBRIUM OF C~NTROI, (lO~moles/gm Hb) I~EMOGLOBIN SOLUTIOSS Hb 1.55 X lo-” M; 20”.
OF SALT CONCENTRATION SUPPLEMENTED
Conditions:
pH 6.2
I r/2” Ma
-~
pH
DPG
Control n
log PI/t
?I
DPG .___._n log PII2
Control
I”
log PI/Z
?I
AND
DP(;-
pH 7.7
7.0
log PIi?
~..
Control
ila n
log PII2
DPG n log p,,s
~~.__.
.037 .070
0.05
0.10 0.20 0.40 0.50 1.00 1 ..50 2.00
101
EC~lJILIBI:IIDI
.13-l
,300 ..i83
.853
2.6 2.7 3.0
1.03 2.4 1.06 2.4 1.12 2.7
1.10 1.10 1.11
3.0 3.1 3.1
1.12 2.7 1.08 3.1 1.08 3.1
1.14 1.13 1.13
-I
,023 .045
2.9 0.82 3.0 0.91
,089 ,172 .213 ,425
3.1 3.0 3.0
,644 ,868
0.91 0.95 1.01 0.98 1.01
1.00 ---1.00 2.9
-1.02
0.96 0.99 0.99 1.00
3.0 3.0 3.0
2.4 0.94 2.6 2.9 2.9 2.9 2.9
,018 .036 .072
3.4 3.3 3.3
.46 2.6 .54 2.6 .56 2.9
.@2 .62 .59
.179 ,359
3.3
.70
3.1
.72
3.3 3.3 3.3
.7.5 3.2 .82 3.3 .84 3.3
.i.i .83 .83
.544 ,734
” Final ionic strength or molarity of phosphate lxlffers.
mcthemoglohin at high salt concent,rations. ,4t, pH 6.2 the rz values of the control solutions increase slightly at I’/2 = 0.2 and remain const,ant at higher ionic skengths; t,hese values are t)he same over the entire range of salt concentrations at pH 7.0 and 7.7. In the solutions supplemented wit’h DPG, the n values are consistently lower t,han those of the controls at low ionic strengths and approach or reach maximum levels as t,he salt concentrat,ion is increased. The log l)l/Z values for the control and the Dl’(:-supplemented Hb solutions arc about the same in t’he pH 6.2 and 7.0 groups; at pH 7.7 the values are higher in the DPG solut,ions at, lower ionic strengths. The inability of DPG to exert an effect on oxygen equilibrium at, high salt concentrations may bc due to t,he dissociation of the hemoglobinDI’G complex (10). The effect, of concentrated salt solutions on t,hr molrcular and funct,ional properties of Hb has been reviewed and discussed by Rossi-l’anelli ei al. (11) and Wyman (12). DISCUSSION
Two compounds, DPG and ATE’, which arc capable of increasing the dissociat’ion of Hb&, account for about 60 and 20 %, respectively, of the organic phosphate in the normal red cell. The molar concentrat,ion of Dl’cT is :rbout fourfold greater than ,4TP.
It is krlowvrl that Dl’(; part’icipates in the metabolism of t,he red cell, but its exact role is not understood or clearly defined. This compound readily forms a complex with Hb (1, 2) and also causes HbO:, to release 02 more readily. Valt’is and Kennedy (13) observed that the oxygen dissociation curves of blood shifted progressively to the left during storage in acid-&rate-dcxtrosc. It has been shown that DPG decreases rapidly during the first 2 weeks of storage nnd that ATP decreases slowly: (14). In view of the results presented in this paper, it is suggested t,hat the Valt,is-Kennedv shift may be due in part, to t,he decrease ‘in DPG concent,ration. If this hypot)hesis is valid, it should be possible to resynthesize DPG by incuhat8ing stored blood in the presence of inosine (15) at a proper pH and cause the oxygen dissociation curve to shift, back to the right. Sote added in proof: Benesch alld Benesch ohserved that 2,3 diphosphoglycerate and trinucleotides decreased the oxygen affinit,y of hwnan hemoglobin. They wggested that t,hese and ot,her phosphorylated componnds may fnnction as regrllators of oxygen transport. [Riochen2. Hiophus. Kes. Co,tzVLWL. 26, 162 (1967), Federation Proc. 26, fii3 (1967)l. REFERENCES 1.
SIJGITA,
&ptl.
Y ,, Biol.
AND
CIIA~TIN,
A., Proc. Sot.
Med. 112, 72 (1963).
102
CHANUTIN
2. CHANCTIN, A., AND CURNISH, 11. R., Arch. Biochem. Biophys. 106, 433 (1964). 3. CHANUTIN, A., AND CURNISH, 11. R., Proc. Sot. Exptl. Biol. Med. 120, 291 (1965). 4. CHERNOFF, A. I., AND LIU, J. C., Blood 17, 54 (1961). 5. EVELYN, K. A., AND MALLOY, H. T., J. Biol. Chem. 126, 655 (1938). 6. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 77, 478 (1958). 7. CROSBY, W. H., MUNN, J. I., AND FURTH, F. W., U.S. Armed Forces Med. J. 6, 693 (1954). 8. GREEN, A. A., J. Am. Chem. Sot. 66, 2331 (1933).
AND
CURNISH
9. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Biophys. 80, 299 (1959). 10. LUDEWIG, S., AND CHANUTIN, A., Acta Biochim. Polonica 11, 85 (1964). Il. ROSSI-FANELLI, A., ANTONINI, E., AND CAPUTO, A., Advan. Protein Chem. 19, 73 (1964). 12. WYMAN, J., JR. Advan. Prolein Chem. 19, 223 (1964). 13. VALTIS, D. J., AND KENNEDY, A. C., Lancet 1, 119 (1954). 14. BARTLETT, G. R., AND BARNET, H. N., J. Clin. Invest. 39, 56 (1960). 15. BARTLETT, G. R., AND SHAFER, A. W., J. Clin. Invest. 40, 1185 (1961).