Haemoglobin titusville: α94 Asp → Asn

Biochimica et Biophysica Acta, 400 (1975) 365-373

© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37106 H A E M O G L O B I N T I T U S V I L L E : a94 Asp ~ Asn A NEW HAEMOGLOBIN WITH A LOWERED AFFINITY FOR OXYGEN

R. G. SCHNEIDER', R. J. ATKINSb, T. S. HOSTY b, G. TOMLINb, R. CASEY c, H. LEHMANN °, P. A. LORKIN ~ and K. NAGAI c aDepartment of Pediatrics, University of Texas Medical Branch, Galveston, Texas 77550 (U.S.A.), bAlabama Department of Public Health, 434 Monroe Street, Montgomery, Ala. 36104 (U.S.A.) and CMRC Abnormal Haemoglobin Unit, University Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge CB2 2QR (U.K.)

(Received November 18th, 1974) (Revised version received April 10th, 1975)

SUMMARY A new haemoglobin variant with a decreased oxygen affinity is described, in which the substitution, a94 (G1) Asp ~ Asn, affects the alfl2 contact alGl-fl2G4. The relevance of this variant to our understanding of the importance of the hydrogen bond between a~G1 and f12G4 in Perutz's model of oxyhaemoglobin A is discussed.

INTRODUCTION We report here the identification and some physical properties of a new haemglobin variant, haemoglobin Titusville (a94 ( G 1 ) A s p ~ Asn). The variant, which has a mobility similar to that of haemoglobin S, was discovered in a healthy 3-yearold girl during the course of a voluntary screening programme for haemoglobinopathies. This programme, which was initiated in Alabama in 1972, now includes results from more than 160 000 Blacks. METHODS Isolation o f the haemoglobin

Haemoglobin electrophoresis was carried out on paper [1], starch gel [2], citrate agar [3] and cellulose acetate [4]; electrophoresis of globin chains was performed in the presence of 6 M urea [5]. Haemoglobin instability tests were carried out using isopropanol at 37 °C [6]. Haemolysates were fractionated and quantitated on DEAE-Sephadex [7] and the isolated fractions were further purified, for structural work, by paper electrophoresis [1]. For oxygen affinity studies, the variant haemoglobin was purified in the carbon monoxy (CO-) form on DEAE-Sephadex and concentrated to about 5-107o (w/v) by ultrafiltration at 4 °C.

366

Identification of the substitution The precipitation and tryptic digestion of globin and the preparation and staining of diagnostic "fingerprints" of the soluble portion of the tryptic digest were all carried out as previously described [8]. Preparative-scale "fingerprints" were stained with fluorescamine [9] (0.02 mg/100 ml of 1 ~o (v/v) pyridine in acetone). The abnormal peptide from haemoglobin Titusville was eluted from such preparative "fingerprints" and a portion hydrolysed for 18 h with 0.5 ml of constant boiling HCI at 108 °C in a sealed evacuated tube; after removal of excess HCt in vacuo, the amino acid composition of the dried hydrolysate was determined [10] by using a Locarte amino acid analyser. Two steps of Edman degradation were carried out [11] on the abnormal peptide and after each step the released thioazolinone derivative was converted to the phenylthiohydantoin (Ptc-) amino acid as described by Edman and Begg [12] but using aqueous 2 0 ~ (v/v) trifluoroacetic acid in place of 1 M HC1. Ptc-amino acids were identified by thin-layer chromatography against markers on polyamide sheets [13]. The net charge on the abnormal peptide at pH 6.4 was calculated from its electrophoretic mobility (at pH 6.4) relative to that of other peptides of known sequence [14].

Measurement of oxygen affinities The purified CO-haemoglobin solutions from both the Titusville and A fractions were equilibrated with 0.05 M KzHPO4, pH 8.6, by gel filtration on a column of Sephadex G-25 (fine grade) and converted into oxyhaemoglobin by illumination in the presence of oxygen [15]. The oxyhaemoglobin solutions were equilibrated with 1 mM K2HPO4 by gel filtration and then deionised by passage through a mixed-bed resin [16]. The oxygen dissociation curves were determined by the method of lmai et al. [17] using 0.1 ~ (w/v) haemoglobin solution in 0.05 M bis-Tris buffers containing 0.1 M NaCI at 25 °C in (a) the absence and (b) the presence of 2 mM inositol hexaphosphate. The solutions were incubated with a methaemoglobin reductase system [18] for 1-3 h at room temperature before making the measurements. Methaemoglobin concentration was determined from the change in absorbance at 540 nm on addition of dithionite after conversion to CO-haemoglobin. For studies on the concentration dependency of oxygen equilibrium curves, measurements were carried out at pH 7.4 and 10 °C, using a wavelength of 560 nm for 0.1 ~ haemoglobin solutions and 432 nm for 0.005~ haemoglobin solutions. Results were calculated from both of the closely concordant deoxygenation and reoxygenation curves.

Qualitative demonstration of the degree of dissociation of haemoglobin Titusville To determine the relative amounts of dimer and tetramer in preparations of liganded haemoglobin Titusville and haemoglobin A, CO-haemoglobin solutions were passed through a column (125 × 2.5 cm) of Sephadex G-100 equilibrated with CO-saturated 0.2 M Tris.HC1, pH 7.8, at 4 °C. The samples (0.9 ml o f a 5 . 2 ~ (w/v) CO-haemoglobin solution) were applied in, and eluted with the above buffer at a flow rate of 35 ml/h. The absorbance of the eluate at 280 nm was monitored continuously using a 1 cm path length flow cell, and 15-ml fractions were collected. The column was calibrated using Dextran blue 2000, haemoglobin which had been

367 uniformly labelled at leucyl residues with 14C (ref. 19), a-chain dimers which had been similarly labelled, and myoglobin. RESULTS The propositus had a near normal haemoglobin level and all routine haematological values were not noticeably abnormal (haemoglobin, 12.5 g/dl; red blood cells, 4.95. 106/#1; packed cell volume, 39 ~o; mean corpuscular volume, 78 fl; mean corpuscular haemoglobin, 25 pg; white blood cells, 7. 103//zl; reticulocytes, 1.5~; normal differential white cell count). Her haemolysate resolved into haemoglobin A and a slow-moving (HbS-like) component during electrophoresis on all media except paper, where it did not separate from haemoglobin A. A second, slow-moving, haemoglobin A2 was detected, which suggested that haemoglobin Titusville differed from haemoglobin A in its a-chains; this was confirmed by globin electrophoresis. Quantitation by column chromatography revealed the following proportions (average of two determinations): haemoglobin A2 plus Titusville2, 3.4~o; haemoglobin A, 61.9 ~ ; haemoglobin Titusville, 34.7~. Isopropanol stability tests showed haemoglobin Titusville to be no less stable than haemoglobin A. Comparison of the "fingerprint" obtained from haemoglobin Titusville with that from haemoglobin A (Fig. 1) showed a new positively charged peptide which

Fig. 1. 'Fingerprint'of the soluble tryptic peptides of the globin from haemoglobinTitusville.Electrophoresis at pH 6.4, 60 V/cm for 1 h; ascendingchromatographyin the upper phase of the solvent system pyridine/isoamylalcohol/water(6:6:7, by vol.) for 20 h. Peptides located using 0.2 ~ (w/v) ninhydrin in acetone containing 1~ (v/v) pyridine. gave no staining reactions for specific amino acids. The amino acid composition of this new peptide (Table I) was identical with that of normal aATpXI (a93-99), which latter is neutral at pH 6.4. The calculated charge of the new peptide was + 1.04, demonstrating that both of the residues of aspartic acid found on analysis of the abnormal peptide were derived from aspaiagine residues (which would have been con-

368 TABLE I HAEMOGLOBIN TITUSVILLE AMINO ACID ANALYSIS OF THE NEW ~tTpXl One residue is approx. 47 nmol. Amino acid

Expected for ~tATpXI

Asp 2.1 (2) Pro 1.1 (l) Val 1.9 (2) Phe 1.0 (1) Lys 1.0 (1)

2 1 2 1 1

verted into aspartic acid during the acid hydrolysis preceding amino acid analysis). Position a94 is normally occupied by a residue of aspartic acid, while position a97 is normally asparagine (Fig. 2). It follows therefore that the substitution in haemoglobin Titusville is a94 (F1) Asp ~ Asn; this was confirmed by the release of Ptc-Asn at the second step of degradation on the abnormal aTpXI. The absence of aATpXI is not apparent from Fig. 1 because it normally overlaps aATpt and gives no specific staining reactions. Helical No.

FG5

G1

G2

G3

G4

G5

G6

Residue No.

93

94

95

96

97

98

99

Haemoglobin A

Val

Asp

Pro

Val

Asn

Phe

Lys

Haemoglobin Titusville

Val

Asn

Pro

Val

Asn

Pb.e

Lys

Fig. 2. Amino acid sequence of aTpXI from haemoglobin A and haemoglobin Titusville. presents residues identified by sequential degradation. Table II and Figs 3-5 show that in the absence of inositol hexaphosphate, haemoglobin Titusville has a low oxygen affinity, very little co-operativity (n = 1.06 at pH 7.4 rising to 1.3 at pH 9.0) and a very small Bohr effect [A log Pso/A pH between pH 6.5 and p H 8.0 ---- --0.18; cf. a value of about --0.5 for haemoglobin A (Kilmartin, J. V., personal communication)]. In the presence of inositol hexaphosphate the affinity was further reduced (but by much less than is that of haemoglobin A); in contrast to haemoglobin A, where addition of inositol hexaphosphate results in an increase in the Bohr effect, the addition of inositol hexaphosphate to haemoglobin Titusville caused a further reduction in the Bohr effect (A log Pso/A pH between pH 6.5 and p H 8.0 = --0.05). Table II also shows that the Ps0 values of haemoglobin A and haemoglobin Titusville both show a small, but definite, concentration dependency, oxygen affinity increasing with decreasing concentration. The effect was somewhat more marked for haemoglobin Titusville than for haemoglobin A; unfortunately, lack of material precluded the examination of more than two different concentrations and of the effect of inositol hexaphosphate on the concentration dependence for haemoglobin Titusville. At pH 7.4 and 10 °C in the absence of inositol hexaphosphate, the Hills coefficient (n) for haemoglobin A was greater than 3 at 0.1 ~ and decreased to about

369 TABLE II OXYGEN AFFINITY DATA FOR HAEMOGLOBIN TITUSVILLE In 0.05 M bis-Tris + 0.1 M NaC1 at 25 °C. (1) Without inositol hexophosphate pH

Pso

n

6.5 7.O 7.4 8.0

22.2 19.5 14.5 12.1

1.06 1.07 1.03 1.17

--

A log Pso/A pH between pH 6.5 and pH 8.0 = 0.18

(2) With 2 mM inositol hexophosphate pH

Ps0

n

6.5 7.0 7.4 8.0

32.4 29.4 28.4 26.4

1.02 1.01 1.02 1.03

--

A log 1)5o//I pH between pH 6.5 and pH 8.0 = 0.05

(3) Pso values for Haemoglobin Titusville and Haemoglobin A at different concentrations Concentration

Ratio

0.1 ~ 0.005 % Pso (o.~%) Pso

Ps0 (mm Hg) Haemoglobin Titusville

Haemoglobin A

Deoxygenation

Reoxygenation

Methaemoglobin after

Deoxygenation

Reoxygenation

Methaemoglobin after

2.63 2.02

2.57 1.95

2~ (<9%)

1.51 1.32

1.48 1.22

1% (<2%)

1.30

1.32

1.14

1.21

(o.oos%)

100 9C

~P-

8C 7C

~ 6c i ~ o

5C

~

4C 3C 2(: 1C I

-0'4

0

0"4

0"8 log p

1"2

1'6

2"0

I

I

2'4

Fig. 3. Oxygen dissociation curves of haemoglobin A and haemoglobin Titusville under conditions described in the text, in the absence of inositol hexaphosphate at pH 7.4. A--&, haemoglobin A; 0--0, haemoglobin Titusville.

370

I m

0

I -1

P

o~

-1 Fig. 4. Hill plots for haemoglobin A and haemoglobin Titusville: A - - A , haemoglobin A in the absence of inositol hexaphosphate; ~ - - A , haemoglobin A plus inositol hexaphosphate: O---O, haemoglobin Titusville in the absence of inositol hexaphosphate; O - - O , haemoglobin Titusville plus inositol hexaphosphate.

1"5 1"4 1"3 log P5O 1"2 1"1 10 I 6"5

1 7"0

I 7'5

I 8'0

pH

Fig. 5. Plot of log Pso against pH for haemoglobin Titusville in the absence (tk---Q) and presence ( O - O ) of inositol hexaphosphate. Note the decrease in slope on addition of inositol hexaphosphate. Void

volume

Tetramer Dimer Monomer

I I

1

0'8 0.7 0.6 E c 0-5 <

f

,

0.4 0"3 0.2 0-1 I 200

I 300

400

Volume of eluate (ml.)

Fig. 6. Elution patterns of CO-haemoglobin A ( - -- --) and CO-haemoglobin Titusville ( from a column of Sephadex G-100, under the conditions described in the text.

-)

371 2.7 at 0.005 ~o, while n for haemoglobin Titusville was still close to unity at both concentrations. The elution pattern of CO-haemoglobin A from the Sephadex G-100 column was practically symmetrical, while that of CO-haemoglobin Titusville (Fig. 6) showed a marked heterogeneity with respect to molecular weight, an increased amount of material eluting in the position expected of dimers when compared with CO-haemoglobin A. DISCUSSION The substitution in haemoglobin Titusville (a94 (G1)Asp-+ Asn) is at the

a~fl2 contact [20], which is involved in the conformational changes associated with oxygenation and deoxygenation of the haemoglobin molecule; when haemoglobin dissociates into dimers, it does so across this contact region [21]. The interactions between a- and t-chains at this contact are largely non-polar, with the exception of the strong hydrogen bond which forms, in the quaternary oxy-, but not deoxy-, structure, between Asn G4 ill02 and Asp G1 a94, the residue affected in haemoglobin Titusville [20]. The importance of this inter-subunit hydrogen bond is illustrated by the abnormal properties of haemoglobin Kansas and haemoglobin Titusville. In the former, in which Asn G4 ill02 is replaced by threonine, the hydrogen bond cannot form. Haemoglobin Kansas has a very low oxygen affinity, a reduced Hill coefficient and dissociates more readily than haemoglobin A in the liganded form [22]; it exhibits a reduced Bohr effect under conditions similar to those used here [23], and in the presence of inositol hexaphosphate is in the permanent deoxy conformation [24]. alG1 Haemoglobin A Asp Haemoglobin Kansas Asp Haemoglobin Titusville Asn

flzG4 Asn Thr Asn

However, the abnormal properties of haemoglobin Kansas may not be due entirely to the change in quaternary structure because it has been reported that there are changes in the tertiary structure around the haem groups of the t-chains [25] (although this aspect is being re-examined), and that the isolated haemoglobin Kansas t-chains themselves have a reduced oxygen affinity [26]. It is therefore difficult to assess from haemoglobin Kansas the importance of the alGl-fl2G4 hydrogen bond to the structure and function of normal haemoglobin. This may prove feasible with haemoglobin Titusville, since the substitution in this variant represents an almost perfect isomorphous replacement (which would not be expected to disturb the tertiary structure of the a-chains) at the a-chain side of the same hydrogen bond which is broken in haemoglobin Kansas. In this context it is of interest to determine the affinity of the isolated a-Titusville subunits, since the haemoglobin Titusville tetramer, like haemoglobin Kansas, shows a low affinity for oxygen, little co-operativity, and a very small Bohr effect. Dr M. F. Perutz has shown us, on his atomic model of oxyhaemoglobin, that when a94 (G1) aspartic acid is replaced by asparagine, it is still possible to form a

372 h y d r o g e n b o n d between the side-chain a m i n o g r o u p o f this a s p a r a g i n e (c~94) and the side-chain c a r b o n y l g r o u p o f a s p a r a g i n e i l l 0 2 in the oxy-, but n o t the deoxy-, structure. This b o n d w o u l d not, however, be expected to be as strong as the hydrogen b o n d f o r m e d by the side-chain o f aspartic acid a94 in h a e m o g l o b i n A. The structure o f the l i g a n d e d f o r m o f h a e m o g l o b i n Titusville m a y thus be heavily biased t o w a r d s the q u a t e r n a r y deoxy-(T-) state, which effect w o u l d be strengthened further in the presence o f inositol h e x a p h o s p h a t e . S e p h a d e x G-100 elution profiles for C O - h a e m o g l o b i n A and C O - h a e m o g l o b i n Titusville i n d i c a t e d clearly that the liganded f o r m o f h a e m o g l o b i n Titusville, like that o f h a e m o g l o b i n K a n s a s , has an increased t e n d e n c y to dissociate into dimers. In this case, the oxygen affinity o f h a e m o g l o b i n Titusville should increase m o r e than that o f h a e m o g l o b i n A with falling c o n c e n t r a t i o n (which latter encourages d i m e r i s a t i o n ) ; this does seem to be so. L a c k o f material, however, has p r e c l u d e d m o r e than a single pair o f m e a s u r e m e n t s o f Ps0 as a function o f h a e m o g l o b i n c o n c e n t r a t i o n . When m o r e m a t e r i a l becomes available, it is h o p e d that the degree o f dissociation and the conc e n t r a t i o n d e p e n d e n c e will be e x a m i n e d in m o r e detail a n d a dissociation c o n s t a n t d e t e r m i n e d for the t e t r a m e r - d i m e r e q u i l i b r i u m in the presence a n d absence o f inositol hexaphosphate. The carrier o f h a e m o g l o b i n Titusville had a h a e m o g l o b i n level o f 12.5 g/dl o f b l o o d . In view o f the low oxygen affinity o f h a e m o g l o b i n Titusville, this h a e m o g l o b i n level should be a d e q u a t e for n o r m a l o x y g e n a t i o n o f the tissues and the carrier c a n n o t be considered " a n a e m i c " .

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