British Journal

of Haematology, 1975,30, 167.

Erythraemia due to Haemoglobin San Diego I. CHANARIN, D. SAMSON, A. LANG,"R. CASEY,*P. A. LORKIN*AND H. LEHMANN* Departmeiit of Haenzatology, Northwick Park Hospital and Clinical Research Centre, Harrow, Middlesex, and * M.R.C. Abnormal Haemoglobin Utiit, Deparhiietzt oj' Clinical Biochemistry, Addenbrooke's Hospital, Hills Road, Cambridge

(Received 22 Noveniber 1974; accepted for publicatioii

2.

Decewbcr 1974)

SUMMARY. A 63-year-old man with erythraemia was found to have a high affinity haemoglobin which was subsequently identified as Hb San Diego p 109 (GII) Val-tMet, recently described in a Filipino family (Nute et al, 1974). The oxygen affinity of the haemolysates was increased approximately two-fold at pH values bctwecii 6.04 aiid 7.90. The ii value was nearly normal (2.5-2.9) in the upper range of saturation (70~95%oxygenation) but was somewhat diminished (I .9-2. I) below this range. IIZvitro biosynthesis of globiii by reticulocytes showed that there was balanced synthesis of a aiid p chains (a : p ratio = I). Haciiioglobiii Sail Diego was first described in January 1974 by Nute ct a1 (1974) who reported erythraeniia inherited as an autosomal doniiiiaiit trait in six niciiibers of a Filipino family. The affected members had an abnormal liaenioglobiii characterized by high oxygen affinity with some loss of haem-liaem interaction aiid normal Bohr effect. Sequciice analysis demonstrated the amino-acid substitution to be p 109 (GI I ) Val-iMet. The present report describes erythraemia due to haemoglobin Sail Diego in a Caucasian (Anglo-Saxon), together with studies of the structure, function aiid biosynthesis of this unusual haemoglobin. METHODS

Structzrral Identification of Hb S a n Diego Globin was prepared from uiipurified haemolysate by precipitation from acid-acetone at - 20°C (Winterhalter & Huehns, 1964)) washed three times with cold acetone aiid dried in vactro over P,O,. The globiii was separated into a- aiid p-chains by chromatography on carboxymethylcellulose (Whatman CM23) using sodium phosphate buffers, pH 6.7, containing 8 M urea and 0.3 mM dithiothreitol (Clegg et al, 1966; Kilmartin & Rossi-Bernardi, 1971) ; the isolated chains were aminoethylated (Raftery & Cole, 1966). The amiiioethylated (AE) a- and P-chains were recovered by freeze-drying after removal of urea and salts by gel filtration on a roo x 2.5 cm column of Sephadex Gz5 (coarse) equilibrated with 0.5% (viv) formic acid in water, and digested with L-(I-tosyl amido-2-pheiiyl) ethyl chloroniethyl ketone (TPCK)-trypsin (I/SO, w/w) in 0.8 M-NH~HCO,for 2 h at 37°C. hisoluble material was removed by centrifugation aiid the supernatant was freeze-dried aiid fingerprinted as described previously (Beale, 1967). Diagnostic fingerprints were stained with 0.2% (w/v) ninhyCorrespondence: Dr I. Chanarin, Department of Haematology, Northwick Park Hospital and Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ.

167

168

I. Chanarin et a1

drin in acetone and with specific staining reagents for divalent sulphur, tyrosine, tryptophan, histidine and arginine (Smith, 1969). Peptides were located on preparative-scale fingerprints using fluorescaniine (Udenfrield et al, 1972); a stock solution (I mg/dl) of the reagent in anhydrous acetone was diluted teii-fold with I ”/, (v/v) pyridiiie in acetone immediately prior to dipping tlie papers, which were then inspected under a long wave-length ultraviolet light. The abnormal tryptic peptide from Hb San Diego was eluted with 0.5 M-NH,OH and a portion dried aiid hydrolysed with constant boiling HCl at 108°C for 18 11 in a sealed evacuated tube. The HC1 contained 0.01% (w/v) dithiotlireitol to protect sulphur-containing amino acids against oxidative losses during hydrolysis. After removal of the excess HC1 in vacua, tlie amino acid composition of the dried hydrolysate was determined (Spacknian et nl, 1958) using a Locarte amino acid analyser. The remainder of the abnormal peptide (160 nmoles) was subjected to two steps of dansylEdman degradation as described by Gray (1967) but with the following modifications: (a) prior to each daiisylation, the peptide was dried from 0.2 M trietliylainine (pH 11.8) to remove traces of ammonia; (b) daiisylatioii was carried out at 45°C for 40 inin in equal volumes (10 pl each) of dansyl chloride solution (2.5 mg/ml in dry dimethylformaniide) and 0.2 M triethylamine-acetic acid, pH 9.5 ; (c) daiisyl peptides were hydrolysed iii sealed tubes at 108°C for 4 h (Gros & Labouesse, 1969). Dansyl amino acids were identified by thin-layer chromatography on polyamide layer sheets (Woods & Wang, 1967) as described by Hartley (1970) except that benzene was replaced by toluene in the second solvent (Furth et al, 1974). To confirm the identification of the first amino acid residue of the variant peptide, the following procedure was adopted. The butyl acetate phase from the first step of Edman degradation on the peptide was dried using a rotary evaporator, transferred in 2 x IOO pl dry ethyl acetate to a 6 x 50 nim tube and dried once more in vacuo. (This phase contained the thiazolinone derivative of the variant amino acid residue from Hb Sail Diego, plus phenylthiourea (PTU) and diphenylthiourea (DPTU).) The thiazoliiione was converted to the phenylthioliydantoin (PTH) derivative (Edman & Begg, 19679, which was then identified by thin-layer chromatography on silica gel (containing a fluorescent indicator), using 2% (v/v) ethanol in chloroform to develop the plate. The presence of P T U and DPTU, both of which simulate PTH-amino acids during chromatography, did not interfere with the identification of the residue (PTH-methionine plus PTH-methionine sulphoxide) in this instance.

Oxygen Afinity Mcasurenzents of Haemolysates Haemolysates were prepared from the blood of the propositus and from a normal control using carbon tetrachloride aiid the haemoglobiii concentration adjusted to 10g/dl. Portions of tlie haemolysatcs (4 ml) were adjusted to pH 8.6 with 3 M tris solution and the haemoglobin was stripped of organic phosphates by gel filtration on a 2 x 30 cm columii of Sephadex G25 (fine) equilibrated with 0.1 M-KCl (Benesch et a!, 1968). Oxygen affinity curves were recorded by the automated method of Iniai ct al(1970) using 6 ml aliquots of haemoglobiii solution (c 0.1%) in 0.1M potassium phosphate buffers of various pH values containing 0.5 mM-EDTA. Measurements were made at 20°C using light of wave-length 474 nni. The methaemoglobin concentrations were determilied before and after each recording from the ratio of absorbancies at 576 nm and 508 iim.

Hb Sun Diego

169

In Vitro Biosynthesis of the Hb Sun Diego in Reticulocytes Blood was freed of plasma by centrifugation at 4°C and the packed cells washed three times with ice-cold isotonic saline (Lingrel & Borsook, 1963). A reticulocyte-enriched preparation of red cells was made (White et a!, 1971) and I in1 of the packed cells was preincubated for 15 min at 37°C in tlie medium described by Liiigrel& Borsook (1968). [‘4C]Leuciiie (10 pCi of sp.a. 270 mCi/mmolc) was added and the incubation continued for 2 h at 37°C. The cells were then washed free of excess isotope with ice-cold isotonic saliiic and hacmolysed by the addition of 5 vol distilled water at 4°C. Globin was prepared directly froin thc liaeinolysatc (Winterhalter & Huehns, 1964) and thc individual globin chains separated by ion-exchange chromatography (Clegg et ul, 1966). The fractions corresponding to the u- and fi-chains were pooled and the total volumes measured. Aliquots of I nil were removed to determine tlie total incorporation of [‘4C]leucine into each globin chain (total cpm). The remainder was dialyscd ovcriiiglit against 2 x 10 1. 0.5% formic acid at 4°C. The specific activities (sp.a.) were measured from the dialysates. [ ‘4C]Leucinc of tlie globin chains in cpm per OD280nm, deterininations were made with a ‘Tracerlab Coru-Matic 200’ liquid scintillation spectrometer using PPO-Triton X Ioo-toluene (Hunt ct d,1968) as scintillator. CASE REPORT The propositus, a 63-year-old man, was first noted to be ‘polycythaemic’ on routine examination in 1956, but no further investigations were performed at this time. His brother had also been known for some years to be ‘polycythaemic’. He first presented to Northwick Park Hospital in Julie 1973 following a fall from a loft when lie sustained a fractured liuiiierus and severe bruising. Subsequently he developed haematuria and an intravenous pyelogram was performed which showed a filling defect in the left kidney. At this time his haemoglobin was 15.3 g/dl. An arteriogram confirmed the presence of a lesion in the kidney and in October he was admitted for laparatomy; the haemoglobin was then 19.4 g/dl, with normal white cell and platelet count. At operation the spleen was found to contain a large liacmatoma and was removed. N o evidence of malignancy was found in the kidney but there was a softened area which was thought to be traumatic in origin. The post-operative course was complicated by bleeding from the splenic artery, an episode of jaundice and a rise of platelets to 900 x 10~11.with deep vein thrombosis and pulmonary embolism despite anticoagulant treatment. During this time his haemoglobin level was maintained by traiisfusioii and did not rise above 12-13 g/dl. The whole blood oxygen dissociation curve after massive transfusion showed a slight left shift (Ps023.3 mmHg) and the 2,3-DPG level was reduced (1.38 ymol/ml whole blood, normal range 1.6-2.6). Following his gradual recovery the haemoglobin level rose to 17-18 g/dl and in February 1974 he was referred for investigation of his ‘polycythaemia’. Hc was asymptomatic and did not complain of headaches, visual disturbance or dizziness. On examination the 011ly abnormalities were a plcthoric facies and a systolic cardiac murmur. At this time his haemoglobin was 17.6 g/dl, PCV 0.52 and the white cell count and platelet count were normal (5.2 x 10~11. and 300x 10~11.). The red cell mass, measured with 51Cr,was markedly elevated at 50.5 ml/kg (normal range 25-33 ml/kg). Sternal marrow aspiration was of normal cellularity but iron stores were

170

I. Chanarin et al

absent, though serum iron was normal (27.3 pmol/l., normal range 12-27 pmol/l.). Arterial oxygen tension was 80 mmHg and lung function tests showed mild reversible obstructive airways disease. The whole blood oxygen dissociation curve was now markedly shifted to the left (mean P,, 12.5 mmHg) and 2,3-DPG was normal (2.2 pmol/ml whole blood). The ii value was 2.2. Haemoglobin electrophoresis on agar gel at pH 6.2, starch gel at pH 8.6, aiid on cellulose acetate at pH 8.9 failed to demonstrate an abnormal haemoglobin, and the levels of Hb A, and F were normal (1.2% and 1.0%). The heat stability test was negative and no methaemoglobin was detected. Serum haptoglobins were 0.6 g/l. by the haenioglobin binding method. The serum erythropoietin was 1.84 units/ml, the upper limit of normal in inales being I unit/ml (Dr J. Napier, Department of Haematology, Welsh National School of Medicine).

F m d y Hisfory Thc brother of tlie propositus had been known to havc ‘polycythaemia’ for at least 12 years. He was under the care of another hospital where he was shown to have a high affinity liaenioglobin (Professor G. Wetherky-Mein, personal communication). There was one other surviving sibling, a sister, who had a normal blood count, as did their mother. Their father, who must be presumcd to have had Hb Sail Diego, had been killed at a young age and 110 information was available. The propositus had no children. RESULTS

Strirrfiiral Iderzfi$cation s f f i b Sail Diego The elution pattern of the globiii from CM-cellulose was normal, as was the fingerprint of the tryptic peptides of the AEa chain. The fingerprint of the AEP chain, however, showed (Fig I) an additional peptide which had a slightly higher chromatographic, and slightly lower electrophoretic, mobility than PATyXIIaz; the new peptide gave a positive divalent sulphur reaction and a transient yellow-brown colour with ninhydrin. In addition, PATpXIIa, gave a much stronger niahydrin reaction than normal. The peptides PATpXIIa, (P105-108) and PATpXIIaz(P109-I 12) arise froni a non-specific tryptic hydrolysis between Asn~108and Valjro9 (Labie et al, 1966), and tlie latter peptide is found only in digests of amiiioethylated P-chains. If the P-chain is not aminoethylated, the portion of the sequence containing P1o9-112 is precipitated in the ‘core’ (Sick ct al, 1967). The amino acid compositions of pATpXIIa, and flATpXIIa, are Asp, Gly, Leu, aiid Val, Leu, AE Cys (S-p-aminoethyl cysteine), respectively; that of the new peptide (Table I) was Val, Met, Leu, AE Cys, suggesting that one of the two valine residues (pro9 or 111, see Fig 2) normally found in PATpXIIazhad been replaced by one of methionine. Three steps of dansylEdinan degradation, combined with identification of PTH-methionine after tlie first step, confirmed that the sequence of the variant PTpXIIa, was Met-Leu-Val-AE Cys and that the variant was, therefore, Hb Sail Diego, Pro9 (GII) Val-Met (Nute et a/, 1974). Oxygen Afinity Measurements of Haeriiolysafes The ‘stripped’ haemolysate from the propositus showed an approximately two-fold increase in oxygen affinity at all pH values investigated (P5*apparently half its normal vahe-

Hb Sun Diego

,

ItF

FIG I . Fingerprint of the tryptic peptides of the AE D chain from Hb San Diego. Electrophoresis at pH 6.4, 3 kV for 70 min, ascending chromatography in thc upper phase of isoamyl alcohol-pyridincwater (6:6:7, by volume) for 20 11. Peptides located with 0.2% ninhydrln in acetone containing 1% vjv pyridine.

Residue No.

105

I06

107

108

109

110

Ill

I12

113

Helical No.

G7

G8

G9

GI0

GI1

GI2

GI3

GI4

GI5

Asn

Val

Residues Hb A

' L e u - Leu

Hb Son Diego

'Leu

-

-

Gly

-

Leu - Gly - Asn

-pTpXIIa,

-t

-

MET-

-PTpXIIa,

-PTpXIIa

t

indicates the points

-

Leu - Val Leu

-

-

t

AECys - Val

Val - AECys

Val

I

of hydrolysis by trypsin

FIG 2. Amino acid sequence of the residues 105-113

from Hb A and Hb Sail Diego AE

B chains.

see Table 2). The co-operative or allosteric interaction as measured by n, the exponent of the Hill equation (Hill, 1910)was nearly normal (2.5-2.9) in the upper range of saturation 70-90% oxygenation) but was somewhat diminished (1.9-2. I) below this range. The alkaline Bohr effect, defined as A log P5,JA pH, between pH 7.39 and pH 7.00 was near normal (Table 11, Fig 3).

I. Chanarin et a2 TABLE I. Amino acid composition of the peptide TpXIIa, (B1opr12) from Hb A and H b San Diego Residues Amino acid Hb A

H b San Diego

Val

2

1.1 (I)

Met LCU

0 I

AE Cys

I

0.9 (1) 1.1 (I) 0.9 ( I )

One amino acid residue is approximately 15 nmoles.

TABLE 11. Data on oxygen affinity, Hills coefficient and Bohr effect of haemolysates from the propositus

Saniple

PH

P5 0

10s 1’5.0

Hills coe&cierzt 12

Control Propositus Control Propositus Control Propositus Control Propositus Control Propositus

6.035 6.040 6.502 6.500 7.000 7.010 7.390 7.390 7.900 7.900

12.7 7.0 12.1

6.6 8.8 4.9 5.5 3.2 2.6 1.7

1.104 0.845 1.083

3.15 2.8-2.1 3.1

0.820

2.9-2.0

0.945 0.690 0.740

3.3

0.505

0.415 0.233

2.6-1.9

2.7 2.7-2. I 2.9 2.5-2.1

% Methaemoglobiu Befire

Aftcr

test

test

3.2 3.8 3.8 3.9 1.8 1.6

13.0 9.5 6.0 9.6 6.0 5.6 5-8 4.5 I .6

1.0

2.2

1.0

I .6 2.0

Bohr factor Alog Ps0/Ap H between p H 7.0 and p H 7.4 (A p H ) Control 0.54 Propositus 0.49

of Haenzoglobin by Reticulocytes Since a substitution of valine by methionine at residue 109of the P-chain does iiot alter the net charge of the protein, the P San Diego chain coelutes with the PA chain from CM cellulose. As a result, the total incorporation of [14C]leucine into lion a chains is the sum of the incorporation into both the PA and psanDiego chains. It is impossible, therefore, to quantitate directly the relative proportion of PSanDie g o chains synthesized during the incubation time. However, it is possible to isolate the abnormal peptide pSanDiego TpXIIa, (p109-112) and PTpXIIb (p113-120) which is reIeased from the mixture of the aminoethylated pSanDiego and PA chains by tryptic hydrolysis. From measurements of the specific activity of the leucine residues in both peptides information can be obtained about the biosynthesis of Hb San Diego per se. Thus the results (Table 111) clearly indicate that there is balanced synthesis of a- and P-chains in reticulocytes (a :P ratio is I). Furthermore, the specific activity data (Table 111) preclude the possibility that Hb San Diego is less stable in v i m than Hb A (a : P and PSanD i e g o : P A + Pan Diego ratios are I). Since the propositus was not iron deficient, it can therefore be infcrred from the data that the replacement of valine by methionine at residue 109 of the PA chain does not significantly alter the rate or extent of p-chain synthesis during erythroid cell maturation. In Vitro Biosynthesis

Hb San Diego

r

65

60

70

75

80

PH

FIG 3. The variation of log,, P,, with pH for normal hacmolysate (0) and haemolysatc containing Hb A and Sail Dicgo ( 0 ) .P,, is the oxygen pressure in nimHg at which the H b is 50% saturatcd with Oz

I Total cprri

---___ ____ aA chain

B chain

) chain

( ~ A + b S a n Diego

D""

D'eg"

34656 34170 -

-

Ratio a/B

1.01

1

Ratio SpeciJic Rctivity (cpm/OD 280 nm)

Ratio

1031 992

1.04

a/B

-

Specific actiuity S i n Diego/ dppm [ ' 4 C ] L e ~ i / p m o l e[LzC]Lc~i /3"+bsa"D ' e g o

746

1.Q9

814

DISCUSSION Several instances of non-specific tryptic hydrolysis at asparagine residues have been observed in both a- and P-globin chains (Beutler et al, 1974; Labie et al, 1966; A. Lang and R. Casey, unpublished data). It appears that the rate of hydrolysis at any given asparagine residue depends on the nature of the polypeptide sequence containing that residue. Thus peptides B"TpXIIa5 and PATpXIIa2are normally recovered in only trace amounts, while Psan D i e g o TpXIIaz was recovered in approximately 3 1% yield rclative to PTpXIIb isolated from the

I74

I. Chanarin et al

same fingerprint. Since a peptide corresponding to psanDiego TpXIIa could be detected on the fingerprint, it seems likely that the rate of tryptic hydrolysis of the Asn-Met (fl108-109) bond is comparable with that of the AE Cys-Val bond (p112-113). The percentage yield of the abnormal peptide does not, however, accurately reflect the proportion of Hb San Diego in the haemolysate since differential losses of the peptides during fingerprinting cannot be estimated. Nevertheless, the biosyiithesis data are consistent with the view that Hb Sail Diego represents 50% of the total haemoglobin in the erythrocyte. The usefulness of non-specific hydrolysis at asparagiiie residues is evident from this report. The characterization of the variant required one amino-acid analysis and two steps of Edinan degradation. This contrasts with the previous report of Hb San Diego (Nute et al, 1974) hi which the tryptic hydrolysis at Asn p108 was not observed. The fuiictional and structural properties of Hb San Diego have been discussed previously (Nute et al, 1974; Anderson, 1974). The present data confirm that haemolysates containing Hb San Diego have a two-fold increase in oxygen affinity compared with normal. However, iiieasuremeiits of the Hill coefficient, 11, on haeinolysates should be interpreted with caution. The reduced n value at low levels of oxygen saturation need not necessarily be interpreted as an indication of reduced allosteric interaction in the isolated Hb Sail Diego. Similar results to those given in Table I1 have been found (P. A. Lorkin, unpublished data) in haemolysates containing Hb Little Rock, which in fact has a normal n value when purified (Bromberg et at, 1973)Hb San Diego is the third haeinoglobiii variant to be described in which methionine replaces valine at a position in the p-chain. The others are Hb Olynipia p20 (B2) (Staniatoyaniiopoulos et al, 1973) and Hb Koln p98 (FG5) (Carrell et al, 1966). Hb San Diego and Hb Olympia both have increased oxygen affinities, are stable and produce erythrocytosis, while Hb Koln is unstable, producing a haemolytic anaemia. The biosyiithetic data for Hb Sail Diego clearly demonstrate that the mutation does not alter the rate of assembly of the p-chain on the polysomes. Similar results have been obtained for Hb Koln (Huehns & Steadman, 1970) but no studies have yet been reported for Hb Olympia, although from the available approximation of its proportion in haemolysates (Stamatoyannopoulos et al, 1973) the variant chain is most probably synthesized at the same rate as the PA chain. Finally, as originally pointed out for Hb Koln (Lehmann & Carrell, 1969), of the four available codons for valine, only GUG can be converted to the codon specifying inethionine (AUG) by a single point mutation. Thus GUG must be the sequence of the nucleotides in the mRNA for the PA chain at the positions corresponding to p20, p98 and p109. ACKNOWLEDGMENT

D.S. is an M.R.C. Clinical Research Fellow. REFERENCES ANDERSON, N.L. (1974)Haemoglobin San Diego (/I 109 ( G I ~ Val+Met). ) Crystal structure ofthe deoxy form. Journal of Clinical Investigation, 53, 329. BEALE,D. (1967) A partial amino acid sequence for sheep haemoglobin A. BiocherniculJournal, 103, 129.

BENESCH, R., BENESCH, R.E. & Yu, C.I. (1968) Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin. Proceedings of the National Academy of Sciences ofthe United States of America, 59, 526.

Hb San Diego BEUTLER, E., LANG,A. & LEHMANN, H. (1974) Haemoglobin Duarte: (a& 6 2 ( E 6 ) A i a + P r o ) : a new unstable hemoglobin with increased oxygen affinity. Blood, 43, 527. BROMBERG, P.A., ALBEN,J.O., BARE,G.H., BALCERZAK,S.P., JONES,R.T., BRIMHALL, B. 81 PADILLA, F. (1973) High oxygen affinity variant of Haemoglobin Little Rock with unique properties. Nafrrre: N e w Biology, 243, 177. CARRELL, R.W., LEHMANN, H. 81 HUTCHISON, H.E. (1966) Haeinoglobin Koln (8-98 valinc-tmethionine) : an unstable protein causing inclusion-body anaemia. Natrrre, 210,915. CLEGG, J.B., NAUGHTON, M.A. & WEATHERALL, D.J. (1966) Abnormal human haemoglobins. Separation and characterization of the a and B chains by chromatography, and the determination of two new variants, Hb Chesapeake and Hb J (Bangkok). Journal ofMolecular Biology, 19. 91. EDMAN,P. & BEGG,G. (1967) A protein sequcnator. Europearz Jounzal c~fBiocliemistry,I, 80. FURTH,A.J., MILMAN, J.D., PRIDDLE, J.D. & OPFORI), R.E. (1974) Studies on the subunit structure and amino acid sequence of triosc phosphate isomerasc from chicken breast muscle. BiochcrriicalJoirrrial, 139,

I75

chimica et Biophysica Acta, 127,428. J.M. & LEHMAN, H. (1972) Synthesis LANG,A., WHITE, of H b Lepore (a26/3,) : Influences of 6 and p nucleotide sequence on synthesis of 6p chain. Nature: New Biology, 240, 268. LEHMANN, H . & CARRELL, R.W. (1969) Variations in the structure of human haeinoglobin, with particular reference to the unstable haemoglobins. Brifish Medical Birlletirz, 25, 14. LINGREL, J.B. & BORSOOK, H. (1963) A comparison of amino acid incorporation into the hemoglobin and ribosomes of marrow erythroid cells and circulating reticulocytes of severely anemic rabbits. Biochemistry, 2 , 309. NUTE,P.E., STAMATOYANNOPOULOS, G., HERMODSON, M.A. & ROTH,D. (1974) Hemoglobinopathic erythrocytosis due to a new electrophoretically silent variant, Haemoglobin Sail Diego (B 109 (GII) ValMet). Joirrnal of Clinical Investigation, 53, 320. ItAFTERY, M.A. & COLE,R.D. (1966) On the aninloethylation of protcins.Joirrrral clfBiologica1 Chemistry, 2419 3457. SICK,K., BEALE,D., IRVINE, D., LEHMANN, H., GOODALL, P.T. & MACDOWALL, S . (1967) Haemoglobin C C and JCambridse. ~ ~T w o new ~ B-chain ~ 11. variants of haemoglobin A. Biochimica et Biopliysicn GRAY,W.R. (1967) Sequential degradation plus dansyArta, 140,231. lation. Methods in Bzzymology, 11, 469. SMITH,I. (1969) In : Chroriiatographic aud Electropliorefic GROS,C. 81 LABOUESSE, B. (1969) Study of the dansyTeclrriiqires (Ed. by I. Smith), 3rd edn, vol. I , lation reaction of amino acids, peptides and proteins. pp I 19-124. Heinemann, London. European Joirrnal of Biochemistry, 7, 463. SPACKMAN, D.H., STEIN,W.H. & MOORE,S. (1958) Automatic recording apparatus for use in the ehroHARTLEY, B.S. (1970) Strategy and tactics in protein matography of amino acids. Analytical Cheniirfry, 30, chemistry. BiochernicalJotrrnnl, 119,805. HILL,A.V. (1910) The possible effects of the aggregaI 190. tion of the molecules of haemoglobin on its dissociaSTAMATOYANNOPOULOS, G., NUTE, P.E., ADAMSON, tion curves. Jorimaf of Physiology, 40, iv. J.W., BELLINGHAM, A.J. & FUNK,D. (1973) HenioJ.H. (1970) Peptide chain HUEHNS, E.R. & STEADMAN, globin Olympia (820 Valine-t Methionine) : an synthesis in unstable haemoglobin diseases. Proceedclectrophoretically silent variant associated with irzgs of the 13th Congress Interrmtiorzal Society of high oxygen affinity and erythrocytosis. Joiirual qf Haeiizatology, Mirnich, p 7. Cliriical Investigation, 52, 342. HUNT,T., HUNTER, T. & MUNRO,A. (1968) Control of UDENFRIEND, S., STEIN,S . , BOHLEN,P., DAIRMAN, W., haelnoglobin synthesis: distribution of ribosomes on LEIMGRUBER, W. & WEIGELE, M. (1972) Fluorescaon the messenger RNA for a and 8 chains.Joournal oJ. mine: a reagent for assay of amino acids, peptidcs, A4olcc.irlar Biology, 36, 3 I. proteins and primary amincs in the picomole range. IMAI, K., MORIMOTO, H., KOTARI,M., WATARI,H., Science, 178, 871. HIRATA, W. & KURADO, M. (1970) Studies on the WHITE,J.M., BRAIN,M.C. & ALI, M.A.M. (1971) f~uictioiiofabnormal haemoglobins. I. An improved Globin synthesis in sideroblastic anaemia. I. a and b' incthod for automatic measurement of the oxygen peptidc chain synthesis. BritishJomrnal ofHaematology, equilibrium curve of hemoglobin. Biochimica ct 20, 263. Bioplrysica Acta, ZOO, 189. WINTERHALTER, K.H. & HUEHNS,E.R. (1964) Preparation, properties, and specific recombination of UPKILMARTIN, J.V. & ROSSI-BERNARDI, L. (1971) The binding of carbon dioxide by horse haemoglobin. globin subunits. Journal of Biological Cherriistry, 239, BiaclzemicalJoirrnal, 124, 3 I. 2699. LABIE,.D., SCHROEDER, W.A. & HUISMAN,T.H.J. WOODS,K.R.& WANG,K.-T. (1967) Separation of (1966) The amino acid sequence of the 6-8 chains of dansyl-amino acids by polyamide layer chromatohemoglobin Lepore = Lepore Washineton.Biography. Biochimica et Biophysica Acta, 133. 369.

~

~