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Hemoglobin toulouse α2β266 (E 10) Lys→Glu
201
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35810 HEMOGLOBIN TOULOUSE adS266 (E 10) l~ys~mu STRUCTURE AND CONSEQUENCES IN MOLECULAR PATHOLOGY
D. L A B I E , J. R O S A ' , O. B E L K H O D J A AND l~. B I E R M E Institut de Pathologie Moldculaire, 24, rue du Faubourg Saint-Jacques, Paris I4 e, and Centre d'H&natologie, C.N.R.S., Toulouse (France) (Received N o v e n l b e r 4th, 197 o)
SUMMARY
Hemoglobin Toulouse a2f1266 Lysoalu is the first known abnormal hemoglobin in which the mutation involves the rupture of an ionic bond in the heme pocket. Some physicochemical properties of this hemoglobin have been studied in relationship with the structural abnormality. Besides a moderate instability the main feature of this hemoglobin is a tendency to form methemoglobin. This hemoglobin is then related not only to unstable hemoglobins, but also to hemoglobins M. The molecular basis of these abnormalities are discussed.
INTRODUCTION
A new unstable hemoglobin Hb (I) Toulouse /5~ (E 10)Lys-*mu has been described by ROSA et al. 1 in a man of French origin suffering from a chronic hemolytic anemia. A physicochemical study of this abnormal hemoglobin was made in an attempt to elucidate the influence of this Lys -~ Glu substitution which involves an heme 15 chain linkage. MATERIALS AND METHODS
Heat lability was explored by the method of DACIE et al. 2. Whole hemolysate or pure abnormal component were incubated in a o.oi M phosphate buffer (pH 7.0) at 5 °° for 2 h, or at 65 ° for various lengths of time within I h. Free sulfhydryl groups were titrated by p-mercuribenzoate (PMB) as described b y BENESCH AND BENESCH 3.
The reaction of the sulfhydryl groups with an excess of PMB was studied by A b b r e v i a t i o n : PMP, p - m e r c u r i b e n z o a t e . " P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , H 6 p i t a l H e n r i Mondor, (94) Cr6teil, France. Biochim. Biophys. Acta, 236 (1971) 2Ol-2O7
202
D. LABIE t'l al.
the m e t h o d of ROSEMEYER AND HUEHNS 4 in a p h o s p h a t e buffer (pH 6.o and ionic strength, o.I). The s t a b i l i t y of the h e m e - g l o b i n linkage was e x a m i n e d b y the m e t h o d of BUNN AND JANDL5 with m i n o r modifications. N o r m a l hemoglobin A a n d purified hemoglobin Toulouse were i n c u b a t e d s i m u l t a n e o u s l y w i t h an equal q u a n t i t y of hemoglobin F p r e p a r e d from cord blood a n d labelled with 5We. This i n c u b a t i o n was p e r f o r m e d at 37 ° in a 0.09 M p h o s p h a t e buffer (pH 7.19) in N a +, in the m e t h e m o globin form. The reaction was s t o p p e d b y a d d i t i o n of c y a n i d e a n d immersion in an ice-cold b a t h . Tile c o m p o n e n t s were i m m e d i a t e l y s e p a r a t e d b y starch block electrophoresis or A m b e r l i t e I R C 50 c h r o m a t o g r a p h y . A f t e r c o n c e n t r a t i o n b y dialysis u n d e r v a c u u m , the heine was s e p a r a t e d a n d cristallized b y c a d m i u m a c e t a t e in acetic acid b y the m e t h o d of LABBE AND NISHIDA6, a n d the specific a c t i v i t y was m e a s u r e d with a N u c l e a r Chicago a u t o m a t i c g a m m a counting system. I n one e x p e r i m e n t , the foetal hemoglobin was double-labelled with 5~Fe a n d [14C]glycine. A f t e r isolation of these components, heme was p r e p a r e d as described above, a n d globin was p r e p a r e d with HC1 acetone. The specific a c t i v i t y of the globin was m e a s u r e d in a N u c l e a r Chicago Mark one scintillation counter. O x y g e n affinity was d e t e r m i n e d on whole cells and on the purified c o m p o n e n t b y the m e t h o d of BENESCH et al. 7 as modified b y HUEHNS AND BELLINGHAM8. The rate of s p o n t a n e o u s o x i d a t i o n b y a t m o s p h e r i c oxygen was s t u d i e d b y controlled h e a t i n g of the h e m o l y s a t e or tile pure c o m p o n e n t s either in a p h o s p h a t e buffer (pH 6.0 at 5 o°) or in an a c e t a t e buffer (pH 4.7 at 37 °) for various lengths of t i m e within 2 h. The percentage of ferrihemoglobin was t h e n d e t e r m i n e d b y readings of a b s o r b a n c e at 577 and 630 n m as given b y HUNTER u. The final value of ferrihemoglobin was d e t e r m i n e d b y the m e t h o d of GUGGENHEIM 1°. The o x i d a t i o n was also s t u d i e d b y dialysis against a c e t a t e buffers of decreasing p H ' s from 5.5 to 4.7. The dissociation equilibrium into dimers was s t u d i e d b y a technique derived from GILBERT11 a n d GILBERT *'t a l ) ~. A column, 50 cm × i cm, of S e p h a d e x G - I o o was e q u i l i b r a t e d with a p h o s p h a t e buffer (pH 7.0) m a d e of I vol. isotonic sodium p h o s p h a t e buffer (pH 6.5), 2 vol. isotonic NaC1, 1.3 g/1 KCN, and a d j u s t e d to p H 7.0 with o r t h o p h o s p h o r i c acid. Two samples of hemoglobin A a n d h e m o g l o b i n Touhmse were d i l u t e d at e x a c t l y the same final c o n c e n t r a t i o n in the same buffer (absorbance at 420 n m = 0.5). The inlet of the column was connected with a t h r e e - w a y stopcock so t h a t b o t h solutions could be run a l t e r n a t i v e l y , and the effluent was m o n i t o r e d at 420 n m in a B e c k m a n D B s p e c t r o p h o t o m e t e r . Connections were m a d e through n a r r o w p o l y e t h y l e n e tubings. The e x p e r i m e n t was carried out at 4", and the flow rate m a i n t a i n e d c o n s t a n t at IO ml/h during the experiment. Tile solution of hemoglobin A was first run t h r o u g h the column for a long enough t i m e to ensure a p l a t e a u region. The inlet was t h e n shifted to the u n k n o w n hemoglobin. In this w a y a difference b o u n d a r y a n d a second p l a t e a u region were observed. The period of time between t h e i n t r o d u c t i o n of the u n k n o w n h e m o g l o b i n sample and the leading b o u n d a r y can be either the same, shorter or longer as c o m p a r e d to hemoglobin A, giving w a y to an horizontal, ascending or descending recording. This p h e n o m e n o n is r e l a t e d with the rate of the dissociation equilibrium. * Beside the hematological data already published 1, intra-erythrocytic hemolysis was looked for. 51Cr life span of erythrocytes was 13 days (normal values 27-3o (lays). No Heinz bodies, even after incubation with a reducing agent, were found. Biochirn. Biophys. Acta, 236 (1971) 2or-2o 7
HEMOGLOBIN TOULOUSE
~o ao[
/5 /
70~"
if
6
/
/ ~ 40~-
/
203
/
/ -
t
~3 2°I/
% lo 2b 3b do ~b Time (min)
Fig. I. Compared kinetics of d e n a t u r a t i o n of hemoglobin A ( 0 - - - - 0 ) ( O - - - O) b y controlled heating at 65 °.
and hemoglobin Toulouse
RESULTS
Heat lability At o ° no precipitation of hemoglobin Toulouse occurred for 2 h. At 65 ° where the reaction was more sensitive a complete precipitation occurred, which was much faster than the one observed with hemoglobin A (Fig. I). Hence hemoglobin Toulouse was proved to be more heat labile than hemoglobin A.
Study of sulJhydryl groups The determination of tile reactive - S H groups gave tile normal value of 2.5 per mole.
Instability during alkylation The alkylation This precipitate was (i.e. a chain with 9.6 of n ) corresponding
of the whole hemolysate by PMB gave rise to a precipitate. pure abnormal fl chain, as shown by the amino acid analysis residues of Lys instead of I i , and 12.2 residues of Glu instead to the mutation Lys --> Glu.
TABLE I HEME EXCHANGE AS MEASURED BY TRANSFER GLOBIN V TO UNLABELED HEMOGLOBINS
OF
RADIOACTIVITY
FROM
59Fe-LABELED
HEMO-
The t r a n s f e r s to hemoglobin A and hemoglobin Toulouse were studied in a parallel way.
Percentage of radioactivity transferred
H b A published found H b Toulouse found
3o rain
zoo min
-24.3 J2 1.2 3 °-1 • 3.5
46.2 ± 4.1 42.3 ~-- 5 48.0 ± 2
Biochim. Biophys. Acta, 236 (1971) 2Ol-2O7
D. LABIE et aI.
204
80
~ -x Hb Toulouse 60
~
40
//
C
~o "-HbA/Toulouse
xz
l/J"
/'
c~
//o
20 ,; I
b' / O
~
H bA
,9"/ //
/" 30 60 Time (rain)
9JO
12~O
Fig. 2. Compared kinetics of spontaneous oxidation of hemoglobin A, heterozygote hemolysate and hemoglobin Toulouse by controlled heating at 5o°, pH 6.o, for various lengths of time within 2h.
Study of heme-globin linkage ~, heine exchange rate In order to study heme exchange we used a 59Fe labeling. At two different times, 3 ° and IOO min, we were not able to find any significant difference, as compared with normal hemoglobin A, in the rate of heme exchange between adult unlabelled hemoglobin and foetal labelled hemoglobin (Table I). As a control, one experiment was performed using [14C]glycine as a label. No exchange of hemoglobin subunits was detected.
Oxygen affinity Oxygen affinity was found normal at physiological pH's. Oxygen equilibrium in whole cells was found normal at p H ' s between 6.5 and 7.5. Oxygen equilibrium of isolated hemoglobin Toulouse was found identical to t h a t of hemoglobin A. Hill's coefficient and the Bohr effect were normal. The enzymes of the glycolytie p a t h w a y were moderately increased. D P G content was normal.
Spontaneous oxidation The rate of formation of ferrihemoglobin by controlled heating was nmch higher for hemoglobin Toulouse than for hemoglobin A. Fig. 2 shows the kinetics at p H 6.0 and 5 °o of compared hemoglobin Toulouse and hemoglobin A. Similar results were found at other p H ' s and temperatures. It can be seen that both kinetics of oxidation have a different shape. For hemoglobin A it is an autocatalytic process. For hemoglobin Toulouse the curve is an hyperbolic one, which gives a linear curve in semi-logarithmic plotting. The final value of ferrihemoglobin is quickly obtained and calculated to be equal to 67} o. This tendency to fast oxidation was also found when hemoglobin is dialysed against buffers of decreasing p H ' s (Fig. 3).
Dissociation equilibrium Recording of the gel filtration experiment showed, from the fall in absorbance,
Biochim. 13iophys. Acta, 236 (i971) zo~-2o7
HEMOGLOBIN TOULOUSE Nb
Nb Toulouse
A
2.0
205
2.0 - -
...... ---
\\\
- --
5.6 pH 5.3 pHS.0 p H 4.7
pH
A420 nm
Shift
0.6 1.(
\\
'.\
\
,,.. - \ \ x \\'~
• "X\
"
from Toulouse
Hb A to Hb
1.0
l
0,5
0.4
630nm 600
630nm
t 6;0
600
t 6;0
Wavelength
Time
(h)
Fig. 3. Compared spontaneous oxidation of ferrihemoglobin A (left) and heterozygote hemolysate
(right) by dialysis against buffers of various pH's. Recording of the spectra between 60o and 7oo nm. Fig. 4. Study of the dissociation equilibrium of hemoglobin Toulouse and hemoglobin A, as described in MATERIALS AND METHODS. The first 2 h correspond to the plateau region of hemoglobin A; the arrow, to the shift from hemoglobin A to the unknown sample. The descending recording is from the end of the 4th h to the end of elution of hemoglobin A and the ascending part is to the leading boundary of the hemoglobin Toulouse.
that the elution of hemoglobin Toulouse was slightly delayed as compared to that of hemoglobin A. This should favor of an equilibrium of dissociation a little displaced towards the dimeric form. DISCUSSION
The lysine in the ioth position of the E helix is invariant in the normal hemoglobin of all the known species. According to the three-dimensional model of PERUTZ et al. la, it is in contact with the heme group. Besides the covalent bond between the iron and histidine 92 (F 8), there are about sixty interactions betweens atoms of the globin chain and atoms of the heme coming between 4 A- Almost all of them are nonpolar. Only two are polar. One of them is the ionic bond linking the e-NH 2 of the lysine 66 to the oxygen atom of the CO0- group of one propionic acid of the heme molecule. Hemoglobin Toulouse is the first mutation reported involving this kind of bond. It was of interest to compare the structural and functional abnormalities. Several mutations have been previously described 14 in the helix E opposite to heme iron. They are summarized in Fig. 5. Two mutants which had negatively charged amino acids, either strongly dissociated glutamic acid in the case of hemoglobin Milwaukee I or more weakly dissociated tyrosine in the case of hemoglobin M Saskatoon, gave rise to permanent in viva methemoglobinemia due to a stable bond with the heme iron. Two other mutants were described as unstable hemoglobins. The replacement in position 66 of a positively charged lysine by a highly dissociated and negatively charged glutamic acid revealed the following facts: Biochim.
Biophys. Acta, 236 (1971) 2 O l - 2 O 7
206
D. LABIE et al.
Haem
/ /Fe2~"~. ~ His ~, x , ~ --" (ET)
/
*r
( Tyr-: M Saskatoon \Arg+: Zi]rich (unstoble)
1', L ( 6 6 " ~ / - - cy s+ "-~
~
Lys+ Glu- : Toulouse (E 10)
Val (Glu- : M Milwaukee (E 11) ~ AIQ Sydney(unstable) Fi~. 5. S c h e m a t i c figure of p a r t of t h e / ~ chain in t h e zone o p p o s i t e t o hem2 iron. R i g h t : k n o w n m u t a n t s in t h i s zone.
(I) There is no in rive permanent bond with the heme iron. (2) The sensitivity to in vitro spontaneous oxidation is greatly increased. (3) The molecule itself is unstable. The following explanations can be brought: (a) The absence of a permanent bond can likely be explained in hemoglobin Toulouse by the fact that the mutant Glu 66 formed a salt bond with Lys 65 and could not be involved with heme iron. (b) Because of the disappearance of Lys 66 and the bonding of Lys 65 with Glu 66, the environment of the heme group will be much less basic. Acid p H accelerates oxidation of the Iron atom, and alkaline pH slows it down. The twolysine group, called the "basic center" of the heine pocket, probably has the function of providing an alkaline environment in the neighbourhood of the heme group, thus helping to stabilize the iron in the ferrous state. Substitution by an acid would, therefore, accelerate oxidation. (c) The instability of the molecule itself has normally three possible explanations: lability of the heine binding, lability of the subunit binding, and helical conformation of the globin itself. Since, from our investigations, the first two factors are at the upper limit of the normal range, one has to suspect that some change in globin conformation m a y result from the mutation itself in hemoglobin Toulouse. ACKNOXVLEDGEMENTS
The authors wish to thank Dr. M. F. Perutz, Dr. A. Hayashi, Dr. Bannerjee and Dr. M. Waks for their fruitful discussions. We thank Miss J. Pagnier for skilled technical assistance. This work was supported by a grant from ' T I n s t i t u t National de la Sant6 et de la Recherche M~dicale", "le Centre National de la Recherche Scientifique", "la D61dgation G6ndrale ~ la Recherche Scientifique et Technique et ses Comit6s Scientifiques (Fends de ddveloppement)". R I£FERENCES I J. R o s a , D. LaBIE, H. \¥AJCMAN, J. M. BOmNE, R. CABANN~S, R. BIERME AND J. RUV'F[E, Nature, 223 (1969) 52o2.
Biochim, Biophys. Acta, 236 (1971) 2Ol-2O7
HEMOGLOBIN TOULOUSE
207
2 J. V. DACIE, A. J. GRIMES, A. MEISLER, L. STEINGOLD, E. H. HEMSTED, G. M. BEAVEN AND J. C. WHITE, Brit. J. Haematol., IO (1966) 338. 3 R. BENESCH AND R. E. BENESCH, Methods of Biochemical Analysis, i o (1962) 43. 4 N. A. ROSEMEYER AND E. R. HUEHNS, J. Mol. Biol., 25 (1967) 253. 5 F. H. BUNN AND H. J. JANDL, Proc. Natl. Acad. Sci. U.S., 56 (1966) 974. 6 R. F. LABBE AND G. lX!ISHIDA, Biochim. Biophys. Acta, 26 (1957) 4377 F. BENESCH, G. MACDUFF AND R. E. BENESCH, Anal. Biochem., I I (1965) 81. 8 E. R. HUEHNS AND A. J. BELLINGHAM, Brit. J. Haematol., 17 (1969) I. 9 F. T. HUNTER, Quantitation of Mixtures of Hemoglobin Derivatives, T h o m a s , Springfield, Ill., 1951. i o FLOST AND PEARSON, Kinetics and Mec}~.anism, Wiley, N e w Y ork, 1961. i i G. A. GILBERT, 4th Meeting Federation European Biochem. Soes., Oslo, r967. Abstr. No. 555. 12 E. CHIANCONE, L. M. GILBERT, G. A. GILBERT AND G. L. KELLET, J. Biol. Chem., 243 (1968) 1212. 13 M. F. PERUTZ, H. MUIRHEAD, J. M. C o x AND L. C. G. GOAMAN, Nature, 219 (1968) 131. 14 M. F. PERUTZ AND H. LEHMANN, Nature, 219 (1968) 5157 . 15 P. S. GERALD AND M. L. EFRON, Proc. Natl. Acad. Sci. U.S., 47 (1958) 1758. 16 C. J. MULLER AND S. KINGMA, Biochim. Biophys. Aeta, 50 (1961) 595. 17 t{. W. CARRELL, ~t. LEHMANN, P. A. LORKIN, E. RAIK AND E. HUNTER, Nature, 215 (1967) 626. 18 P. S. GERALD AND P. GEORGE, Science, 129 (1959) 393. 19 K. IMAI, Arch. Biochem. Biophys., 127 (1968) 543. 20 A. HAYASHI, T. SUZUKI, I{. IMAI, H. MORIMOTO AND H. WATARI, Biochim. Biophys. Acta, 194 (1969) 6.
Bioehim. Biophys. Aeta, 236 (1971) 2Ol-2O 7
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35810 HEMOGLOBIN TOULOUSE adS266 (E 10) l~ys~mu STRUCTURE AND CONSEQUENCES IN MOLECULAR PATHOLOGY
D. L A B I E , J. R O S A ' , O. B E L K H O D J A AND l~. B I E R M E Institut de Pathologie Moldculaire, 24, rue du Faubourg Saint-Jacques, Paris I4 e, and Centre d'H&natologie, C.N.R.S., Toulouse (France) (Received N o v e n l b e r 4th, 197 o)
SUMMARY
Hemoglobin Toulouse a2f1266 Lysoalu is the first known abnormal hemoglobin in which the mutation involves the rupture of an ionic bond in the heme pocket. Some physicochemical properties of this hemoglobin have been studied in relationship with the structural abnormality. Besides a moderate instability the main feature of this hemoglobin is a tendency to form methemoglobin. This hemoglobin is then related not only to unstable hemoglobins, but also to hemoglobins M. The molecular basis of these abnormalities are discussed.
INTRODUCTION
A new unstable hemoglobin Hb (I) Toulouse /5~ (E 10)Lys-*mu has been described by ROSA et al. 1 in a man of French origin suffering from a chronic hemolytic anemia. A physicochemical study of this abnormal hemoglobin was made in an attempt to elucidate the influence of this Lys -~ Glu substitution which involves an heme 15 chain linkage. MATERIALS AND METHODS
Heat lability was explored by the method of DACIE et al. 2. Whole hemolysate or pure abnormal component were incubated in a o.oi M phosphate buffer (pH 7.0) at 5 °° for 2 h, or at 65 ° for various lengths of time within I h. Free sulfhydryl groups were titrated by p-mercuribenzoate (PMB) as described b y BENESCH AND BENESCH 3.
The reaction of the sulfhydryl groups with an excess of PMB was studied by A b b r e v i a t i o n : PMP, p - m e r c u r i b e n z o a t e . " P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , H 6 p i t a l H e n r i Mondor, (94) Cr6teil, France. Biochim. Biophys. Acta, 236 (1971) 2Ol-2O7
202
D. LABIE t'l al.
the m e t h o d of ROSEMEYER AND HUEHNS 4 in a p h o s p h a t e buffer (pH 6.o and ionic strength, o.I). The s t a b i l i t y of the h e m e - g l o b i n linkage was e x a m i n e d b y the m e t h o d of BUNN AND JANDL5 with m i n o r modifications. N o r m a l hemoglobin A a n d purified hemoglobin Toulouse were i n c u b a t e d s i m u l t a n e o u s l y w i t h an equal q u a n t i t y of hemoglobin F p r e p a r e d from cord blood a n d labelled with 5We. This i n c u b a t i o n was p e r f o r m e d at 37 ° in a 0.09 M p h o s p h a t e buffer (pH 7.19) in N a +, in the m e t h e m o globin form. The reaction was s t o p p e d b y a d d i t i o n of c y a n i d e a n d immersion in an ice-cold b a t h . Tile c o m p o n e n t s were i m m e d i a t e l y s e p a r a t e d b y starch block electrophoresis or A m b e r l i t e I R C 50 c h r o m a t o g r a p h y . A f t e r c o n c e n t r a t i o n b y dialysis u n d e r v a c u u m , the heine was s e p a r a t e d a n d cristallized b y c a d m i u m a c e t a t e in acetic acid b y the m e t h o d of LABBE AND NISHIDA6, a n d the specific a c t i v i t y was m e a s u r e d with a N u c l e a r Chicago a u t o m a t i c g a m m a counting system. I n one e x p e r i m e n t , the foetal hemoglobin was double-labelled with 5~Fe a n d [14C]glycine. A f t e r isolation of these components, heme was p r e p a r e d as described above, a n d globin was p r e p a r e d with HC1 acetone. The specific a c t i v i t y of the globin was m e a s u r e d in a N u c l e a r Chicago Mark one scintillation counter. O x y g e n affinity was d e t e r m i n e d on whole cells and on the purified c o m p o n e n t b y the m e t h o d of BENESCH et al. 7 as modified b y HUEHNS AND BELLINGHAM8. The rate of s p o n t a n e o u s o x i d a t i o n b y a t m o s p h e r i c oxygen was s t u d i e d b y controlled h e a t i n g of the h e m o l y s a t e or tile pure c o m p o n e n t s either in a p h o s p h a t e buffer (pH 6.0 at 5 o°) or in an a c e t a t e buffer (pH 4.7 at 37 °) for various lengths of t i m e within 2 h. The percentage of ferrihemoglobin was t h e n d e t e r m i n e d b y readings of a b s o r b a n c e at 577 and 630 n m as given b y HUNTER u. The final value of ferrihemoglobin was d e t e r m i n e d b y the m e t h o d of GUGGENHEIM 1°. The o x i d a t i o n was also s t u d i e d b y dialysis against a c e t a t e buffers of decreasing p H ' s from 5.5 to 4.7. The dissociation equilibrium into dimers was s t u d i e d b y a technique derived from GILBERT11 a n d GILBERT *'t a l ) ~. A column, 50 cm × i cm, of S e p h a d e x G - I o o was e q u i l i b r a t e d with a p h o s p h a t e buffer (pH 7.0) m a d e of I vol. isotonic sodium p h o s p h a t e buffer (pH 6.5), 2 vol. isotonic NaC1, 1.3 g/1 KCN, and a d j u s t e d to p H 7.0 with o r t h o p h o s p h o r i c acid. Two samples of hemoglobin A a n d h e m o g l o b i n Touhmse were d i l u t e d at e x a c t l y the same final c o n c e n t r a t i o n in the same buffer (absorbance at 420 n m = 0.5). The inlet of the column was connected with a t h r e e - w a y stopcock so t h a t b o t h solutions could be run a l t e r n a t i v e l y , and the effluent was m o n i t o r e d at 420 n m in a B e c k m a n D B s p e c t r o p h o t o m e t e r . Connections were m a d e through n a r r o w p o l y e t h y l e n e tubings. The e x p e r i m e n t was carried out at 4", and the flow rate m a i n t a i n e d c o n s t a n t at IO ml/h during the experiment. Tile solution of hemoglobin A was first run t h r o u g h the column for a long enough t i m e to ensure a p l a t e a u region. The inlet was t h e n shifted to the u n k n o w n hemoglobin. In this w a y a difference b o u n d a r y a n d a second p l a t e a u region were observed. The period of time between t h e i n t r o d u c t i o n of the u n k n o w n h e m o g l o b i n sample and the leading b o u n d a r y can be either the same, shorter or longer as c o m p a r e d to hemoglobin A, giving w a y to an horizontal, ascending or descending recording. This p h e n o m e n o n is r e l a t e d with the rate of the dissociation equilibrium. * Beside the hematological data already published 1, intra-erythrocytic hemolysis was looked for. 51Cr life span of erythrocytes was 13 days (normal values 27-3o (lays). No Heinz bodies, even after incubation with a reducing agent, were found. Biochirn. Biophys. Acta, 236 (1971) 2or-2o 7
HEMOGLOBIN TOULOUSE
~o ao[
/5 /
70~"
if
6
/
/ ~ 40~-
/
203
/
/ -
t
~3 2°I/
% lo 2b 3b do ~b Time (min)
Fig. I. Compared kinetics of d e n a t u r a t i o n of hemoglobin A ( 0 - - - - 0 ) ( O - - - O) b y controlled heating at 65 °.
and hemoglobin Toulouse
RESULTS
Heat lability At o ° no precipitation of hemoglobin Toulouse occurred for 2 h. At 65 ° where the reaction was more sensitive a complete precipitation occurred, which was much faster than the one observed with hemoglobin A (Fig. I). Hence hemoglobin Toulouse was proved to be more heat labile than hemoglobin A.
Study of sulJhydryl groups The determination of tile reactive - S H groups gave tile normal value of 2.5 per mole.
Instability during alkylation The alkylation This precipitate was (i.e. a chain with 9.6 of n ) corresponding
of the whole hemolysate by PMB gave rise to a precipitate. pure abnormal fl chain, as shown by the amino acid analysis residues of Lys instead of I i , and 12.2 residues of Glu instead to the mutation Lys --> Glu.
TABLE I HEME EXCHANGE AS MEASURED BY TRANSFER GLOBIN V TO UNLABELED HEMOGLOBINS
OF
RADIOACTIVITY
FROM
59Fe-LABELED
HEMO-
The t r a n s f e r s to hemoglobin A and hemoglobin Toulouse were studied in a parallel way.
Percentage of radioactivity transferred
H b A published found H b Toulouse found
3o rain
zoo min
-24.3 J2 1.2 3 °-1 • 3.5
46.2 ± 4.1 42.3 ~-- 5 48.0 ± 2
Biochim. Biophys. Acta, 236 (1971) 2Ol-2O7
D. LABIE et aI.
204
80
~ -x Hb Toulouse 60
~
40
//
C
~o "-HbA/Toulouse
xz
l/J"
/'
c~
//o
20 ,; I
b' / O
~
H bA
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/" 30 60 Time (rain)
9JO
12~O
Fig. 2. Compared kinetics of spontaneous oxidation of hemoglobin A, heterozygote hemolysate and hemoglobin Toulouse by controlled heating at 5o°, pH 6.o, for various lengths of time within 2h.
Study of heme-globin linkage ~, heine exchange rate In order to study heme exchange we used a 59Fe labeling. At two different times, 3 ° and IOO min, we were not able to find any significant difference, as compared with normal hemoglobin A, in the rate of heme exchange between adult unlabelled hemoglobin and foetal labelled hemoglobin (Table I). As a control, one experiment was performed using [14C]glycine as a label. No exchange of hemoglobin subunits was detected.
Oxygen affinity Oxygen affinity was found normal at physiological pH's. Oxygen equilibrium in whole cells was found normal at p H ' s between 6.5 and 7.5. Oxygen equilibrium of isolated hemoglobin Toulouse was found identical to t h a t of hemoglobin A. Hill's coefficient and the Bohr effect were normal. The enzymes of the glycolytie p a t h w a y were moderately increased. D P G content was normal.
Spontaneous oxidation The rate of formation of ferrihemoglobin by controlled heating was nmch higher for hemoglobin Toulouse than for hemoglobin A. Fig. 2 shows the kinetics at p H 6.0 and 5 °o of compared hemoglobin Toulouse and hemoglobin A. Similar results were found at other p H ' s and temperatures. It can be seen that both kinetics of oxidation have a different shape. For hemoglobin A it is an autocatalytic process. For hemoglobin Toulouse the curve is an hyperbolic one, which gives a linear curve in semi-logarithmic plotting. The final value of ferrihemoglobin is quickly obtained and calculated to be equal to 67} o. This tendency to fast oxidation was also found when hemoglobin is dialysed against buffers of decreasing p H ' s (Fig. 3).
Dissociation equilibrium Recording of the gel filtration experiment showed, from the fall in absorbance,
Biochim. 13iophys. Acta, 236 (i971) zo~-2o7
HEMOGLOBIN TOULOUSE Nb
Nb Toulouse
A
2.0
205
2.0 - -
...... ---
\\\
- --
5.6 pH 5.3 pHS.0 p H 4.7
pH
A420 nm
Shift
0.6 1.(
\\
'.\
\
,,.. - \ \ x \\'~
• "X\
"
from Toulouse
Hb A to Hb
1.0
l
0,5
0.4
630nm 600
630nm
t 6;0
600
t 6;0
Wavelength
Time
(h)
Fig. 3. Compared spontaneous oxidation of ferrihemoglobin A (left) and heterozygote hemolysate
(right) by dialysis against buffers of various pH's. Recording of the spectra between 60o and 7oo nm. Fig. 4. Study of the dissociation equilibrium of hemoglobin Toulouse and hemoglobin A, as described in MATERIALS AND METHODS. The first 2 h correspond to the plateau region of hemoglobin A; the arrow, to the shift from hemoglobin A to the unknown sample. The descending recording is from the end of the 4th h to the end of elution of hemoglobin A and the ascending part is to the leading boundary of the hemoglobin Toulouse.
that the elution of hemoglobin Toulouse was slightly delayed as compared to that of hemoglobin A. This should favor of an equilibrium of dissociation a little displaced towards the dimeric form. DISCUSSION
The lysine in the ioth position of the E helix is invariant in the normal hemoglobin of all the known species. According to the three-dimensional model of PERUTZ et al. la, it is in contact with the heme group. Besides the covalent bond between the iron and histidine 92 (F 8), there are about sixty interactions betweens atoms of the globin chain and atoms of the heme coming between 4 A- Almost all of them are nonpolar. Only two are polar. One of them is the ionic bond linking the e-NH 2 of the lysine 66 to the oxygen atom of the CO0- group of one propionic acid of the heme molecule. Hemoglobin Toulouse is the first mutation reported involving this kind of bond. It was of interest to compare the structural and functional abnormalities. Several mutations have been previously described 14 in the helix E opposite to heme iron. They are summarized in Fig. 5. Two mutants which had negatively charged amino acids, either strongly dissociated glutamic acid in the case of hemoglobin Milwaukee I or more weakly dissociated tyrosine in the case of hemoglobin M Saskatoon, gave rise to permanent in viva methemoglobinemia due to a stable bond with the heme iron. Two other mutants were described as unstable hemoglobins. The replacement in position 66 of a positively charged lysine by a highly dissociated and negatively charged glutamic acid revealed the following facts: Biochim.
Biophys. Acta, 236 (1971) 2 O l - 2 O 7
206
D. LABIE et al.
Haem
/ /Fe2~"~. ~ His ~, x , ~ --" (ET)
/
*r
( Tyr-: M Saskatoon \Arg+: Zi]rich (unstoble)
1', L ( 6 6 " ~ / - - cy s+ "-~
~
Lys+ Glu- : Toulouse (E 10)
Val (Glu- : M Milwaukee (E 11) ~ AIQ Sydney(unstable) Fi~. 5. S c h e m a t i c figure of p a r t of t h e / ~ chain in t h e zone o p p o s i t e t o hem2 iron. R i g h t : k n o w n m u t a n t s in t h i s zone.
(I) There is no in rive permanent bond with the heme iron. (2) The sensitivity to in vitro spontaneous oxidation is greatly increased. (3) The molecule itself is unstable. The following explanations can be brought: (a) The absence of a permanent bond can likely be explained in hemoglobin Toulouse by the fact that the mutant Glu 66 formed a salt bond with Lys 65 and could not be involved with heme iron. (b) Because of the disappearance of Lys 66 and the bonding of Lys 65 with Glu 66, the environment of the heme group will be much less basic. Acid p H accelerates oxidation of the Iron atom, and alkaline pH slows it down. The twolysine group, called the "basic center" of the heine pocket, probably has the function of providing an alkaline environment in the neighbourhood of the heme group, thus helping to stabilize the iron in the ferrous state. Substitution by an acid would, therefore, accelerate oxidation. (c) The instability of the molecule itself has normally three possible explanations: lability of the heine binding, lability of the subunit binding, and helical conformation of the globin itself. Since, from our investigations, the first two factors are at the upper limit of the normal range, one has to suspect that some change in globin conformation m a y result from the mutation itself in hemoglobin Toulouse. ACKNOXVLEDGEMENTS
The authors wish to thank Dr. M. F. Perutz, Dr. A. Hayashi, Dr. Bannerjee and Dr. M. Waks for their fruitful discussions. We thank Miss J. Pagnier for skilled technical assistance. This work was supported by a grant from ' T I n s t i t u t National de la Sant6 et de la Recherche M~dicale", "le Centre National de la Recherche Scientifique", "la D61dgation G6ndrale ~ la Recherche Scientifique et Technique et ses Comit6s Scientifiques (Fends de ddveloppement)". R I£FERENCES I J. R o s a , D. LaBIE, H. \¥AJCMAN, J. M. BOmNE, R. CABANN~S, R. BIERME AND J. RUV'F[E, Nature, 223 (1969) 52o2.
Biochim, Biophys. Acta, 236 (1971) 2Ol-2O7
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207
2 J. V. DACIE, A. J. GRIMES, A. MEISLER, L. STEINGOLD, E. H. HEMSTED, G. M. BEAVEN AND J. C. WHITE, Brit. J. Haematol., IO (1966) 338. 3 R. BENESCH AND R. E. BENESCH, Methods of Biochemical Analysis, i o (1962) 43. 4 N. A. ROSEMEYER AND E. R. HUEHNS, J. Mol. Biol., 25 (1967) 253. 5 F. H. BUNN AND H. J. JANDL, Proc. Natl. Acad. Sci. U.S., 56 (1966) 974. 6 R. F. LABBE AND G. lX!ISHIDA, Biochim. Biophys. Acta, 26 (1957) 4377 F. BENESCH, G. MACDUFF AND R. E. BENESCH, Anal. Biochem., I I (1965) 81. 8 E. R. HUEHNS AND A. J. BELLINGHAM, Brit. J. Haematol., 17 (1969) I. 9 F. T. HUNTER, Quantitation of Mixtures of Hemoglobin Derivatives, T h o m a s , Springfield, Ill., 1951. i o FLOST AND PEARSON, Kinetics and Mec}~.anism, Wiley, N e w Y ork, 1961. i i G. A. GILBERT, 4th Meeting Federation European Biochem. Soes., Oslo, r967. Abstr. No. 555. 12 E. CHIANCONE, L. M. GILBERT, G. A. GILBERT AND G. L. KELLET, J. Biol. Chem., 243 (1968) 1212. 13 M. F. PERUTZ, H. MUIRHEAD, J. M. C o x AND L. C. G. GOAMAN, Nature, 219 (1968) 131. 14 M. F. PERUTZ AND H. LEHMANN, Nature, 219 (1968) 5157 . 15 P. S. GERALD AND M. L. EFRON, Proc. Natl. Acad. Sci. U.S., 47 (1958) 1758. 16 C. J. MULLER AND S. KINGMA, Biochim. Biophys. Aeta, 50 (1961) 595. 17 t{. W. CARRELL, ~t. LEHMANN, P. A. LORKIN, E. RAIK AND E. HUNTER, Nature, 215 (1967) 626. 18 P. S. GERALD AND P. GEORGE, Science, 129 (1959) 393. 19 K. IMAI, Arch. Biochem. Biophys., 127 (1968) 543. 20 A. HAYASHI, T. SUZUKI, I{. IMAI, H. MORIMOTO AND H. WATARI, Biochim. Biophys. Acta, 194 (1969) 6.
Bioehim. Biophys. Aeta, 236 (1971) 2Ol-2O 7