The chemical heterogeneity of human hemoglobin F

421

Biochimica et Biophysica Acta, 579 (1979) 421--431

© Elsevier/North-Holland Biomedical Press

BBA 38232 THE CHEMICAL H E T E R O G E N E I T Y OF HUMAN HEMOGLOBIN F DIRECT EVIDENCE F O R THE EXISTENCE OF T H R E E TYPES OF CHAIN, THE G~fl, A~I, A N D A~/T CHAINS *

GEORGI D. EFREMOV, JERRY B. WILSON and TITUS H.J. HUISMAN Laboratory of Protein Chemistry, Department of Cell and Molecular Biology **, Medical College of Georgia, Augusta, GA 30912 (U.S.A.)

(Received October 19th, 1978) Key words: Hemoglobin; G~/] chain;

A~I

chain;

A,~T

chain

Summary Direct evidence is presented for the existence of three types of 9, chain of human hemoglobin F. A modification of a CM-cellulose chromatographic method has allowed the incomplete separation of these 9" chains while high pressure liquid chromatography and fingerprint analyses of tryptic peptides of zones of the isolated 7 chains, and amino acid analyses of isolated peptides were used to identify the chains. These studies have shown that the presence of a glycyl residue in position 136 (G~ chain) is directly related to that of an isoleucyl residue in position 75 (i7 chain), thus indicating the existence of an G~/I chain, and that the presence of an alanyl residue in position 136 (A7 chain) can be related to that of an isoleucyl residue in position 75, thus suggesting the existence of an A7I chain. When the isoleucyl residue at position 75 is replaced b y a threonyl residue, invariably it is related to the alanyl substitution at position 136 (Ag"T chain). These data support indirect evidence from case analyses and family studies which were published before, and indicate that the W7 chain is an allele of the A~, chain which should be renamed the A~/T chain.

Abbreviations: HPLC, high-pressure liquid chromatography. * This paper uses a tentative n o m e n c l a t u r e with s y m b o l s such as GT, AT, TT, and I~ which are explained separately in t h e t e x t . T h e G7 and A.y chains are p r o d u c t s of non-a].lelic g e n e s b u t data from this s t u d y show that the Ile-Thr substitution at position 75 results f r o m a m u t a t i o n o f t h e A ~ g e n e The chan~e is indicated as a postfixed superscript; thus, the A~I- (=7 75 h e - - 1 3 6 Ala) and A~fT ('=7 75"Thr--1-36Ala) c h m•n s are p r o d u c t s o f allelic g e n e s w h i"l e the GTI ( =T 75 Iie--136 G1y) c hain is t h e p r o d u c t o f a nonaUelic gene. ** Contribution No. 494.

422 Introduction Human fetal hemoglobin (Hb F) is heterogeneous in a vast majority of samples because position 136 of the 7 chain can be occupied by a glycyl residue (the G7 chain) or by an alanyl residue (the A7 chain) [1,2]. Rico and coworkers [3] discovered that the 7 chain of Hb F of m a n y samples can also be heterogeneous at position 75 in which either an isoleucyl residue (the ~7 chain) or a threonyl residue (the W~/ chain) can be found. This substitution was first reported as Hb F-Sardinia by Grifoni et al. who discovered this variation in the Hb F of a 59-year-old patient with fi-thalassemia [4]. Recent analyses of globin F samples from m a n y persons with different genetic disorders and from numerous newborn of various racial or ethnic origins, and family studies have shown that the T7 chain occurs with a frequency of 0.1--0.15 and is not related to any specific hematological disorder [5--7]. Indirect evidence has been provided that the W,~ and A~/ chains are linked and probably the same (the ATT chain) whereas the 17 and G7 chain are together as the G~,I chain. This probable relation is further supported by data from observations made on fetal globins from five orangutans showing that a v 7 chain (i.e. a chain with a valyl residue in position 75) is closely related to a A7 chain (i.e. a chain with an alanyl residue in position 135) while an IV chain (with an isoleucyl residue in position 75) is related to a W7 chain (with a threonyl residue in position 135) [8]. The present study attempts to obtain further, more direct, evidence for the existence of ATT, ATI, and GTI chains (thus, chains with Thr-75 and Ala-136; Ile-75 and Ala-136; Ile-75 and Gly-136, respectively) in h u m a n Hb F through analyses of sections of 7 chain zones which are isolated by CM-cellulose chromatography. The procedures used in this study concerned analyses of CNBr peptides and tryptic peptides from globin F samples by silica gel thin-layer fingerprinting, high pressure liquid chromatography and amino acid analyses. Materials and Methods

Materials. Blood samples from normal newborn and adults with elevated levels of Hb F were used. The donors included some patients with homozygous fl*-thalassemia, fl/Sfl-thalassemia, Lepore-fl-thalassemia, hereditary persistence of Hb F heterozygosity of different types, sickle cell anemia, and Hb Kenya heterozygosity. Since the origin of the samples are not pertinent to the technical aspects of this paper, this information is omitted. However, the presence or absence of specific types of ~, chains is included in the text or table when such samples were studied in detail. Hb F was isolated from red cell hemolysates by DEAE-cellulose chromatography [9], dehemed by the m e t h o d of Anson and Mirsky [10], and stored as globin F. Chromatographic separation of chains. The method of Clegg et al. [11] was used with modifications. The CM-cellulose column size was 0.9 × 20 cm, 40 mg of globin was applied to a column being equilibrated with 0.01 M sodium phosphate buffer, pH 6.7, with 8 M urea; a gradient was developed with the use of three cylinders of equal diameter, placed in series, each filled with 200 ml of the same sodium phosphate/urea, pH 6.7, developer but with 0.01 M, 0.0125 M and 0.015 M sodium phosphate, respectively; a flow rate of 24 ml/h was main-

423 tained throughout the experiment with fractions being collected every 10 min. Effluent containing a desired protein was dialyzed extensively against distilled water at pH 3.0, and the protein was recovered by freeze-drying. Digestion o f isolated chain. Treatment with cyanogen bromide followed the procedure of Schroeder et al. [1]. The ~, chain was also digested for 2 h at 37°C with trypsin in a trimethylamine buffer solution at pH 8.5 as described by Cohen-Solal et al. [12] or in an ammonium bicarbonate buffer, pH 8.5, as will be described elsewhere in this section. Each digest was concentrated by freezedrying and the dry material dissolved in a solution and concentration appropriate for the separation of the peptides. Thin-layer fingerprinting. The procedure is a slight modification of that described b y Braconnier et al. [13]. The thin-layer silica gel plates were 20 X 20 cm and were obtained from Schleicher and Schull (G1505 LS 254). Each plate was washed with pyridine/acetic acid/water ( 1 0 : 0 . 4 : 8 9 . 6 , by vol.; Ref. 14) or with pyridine/acetic acid/butanol/water (46 : 12 : 60 : 31.5, by vol.; Ref. 13) depending on whether electrophoresis or chromatography was used first. Next, the plates were dried for 1--2 h at 60°C and equilibrated for at least 12 h in a chromotank containing one of the two solutions listed above. Approximately 10--20 pl of a tryptic digest (containing digest of 0.5--1 mg ~, chain) was applied, whereafter electrophoresis was done in a, with tap water cooled, DeSaga tank for 7 h at 250--300 V and at 30--40 mA. Next, the plate was dried for 1--2 h at 60°C, equilibrated with the chromatographic solution in a chromotank for 12 h, and chromatography was started. Upon completion of this part of the experiment the plate was dried at 60°C for 2 h and stained b y dipping in a 0.01 g/100 ml Fluram solution (10 mg fluorescamine, Pierce and Co., in a mixture of 99 ml acetone and 1 ml pyridine). Within 10 min the plates were examined under ultraviolet light; the spots were traced with a pencil or a photograph was made (Polaroid, Type 52, Polapan 4 × 5 Land Film). Cyanogen bromide peptides were separated by electrophoresis only, using the same thin-layer procedure as described above. A limited number of peptides were examined by amino acid analyses. The silica gel containing the peptide was removed from the plate with a spatula and transferred into a test tube which contained 2 ml 6 M HC1. Hydrolysis was carried out in vacuo at l l 0 ° C for 24 h. After removal of the hydrochloric acid with a constant stream of filtered air at 40°C the residual material was dissolved in an appropriate volume of buffer used in amino acid analysis. Prior to application each sample was carefully filtered to remove the insoluble silica gel. Amino acid analyses were made with the Beckman Amino Acid Analyser, Model 121 M, equipped with a System AA computing integrator. Satisfactory elution of the intact peptide from the silica gel was not obtained despite the use of several solvents. HPLC o f tryptic peptides o f F globin or isolated 3, chain. Approximately 2--5 mg material was dissolved in 3 ml H20, 6 mg ammonium bicarbonate was added and the pH adjusted to 8.5. A small amount of trypsin (0.1--0.2 mg in 0.1--0.2 ml 0.001 M HC1) was addded and the mixture stirred for 4 h at 37°C. The pH was adjusted to 6.1 with dilute HC1 and the mixture stirred for an additional hour. Next, the precipitate (i.e the insoluble core) was removed at 10 000 rev./min for 20 min in a Beckman J-21C centrifuge with J-20 rotor. The

424 supernatant was filtered through a millipore FH 0.5 pm filter, lyophilized, and the residue dissolved in 0.1--0.3 ml starting developer (i.e. 10% acetonitrile/ 0.01 M a m m o n i u m acetate, pH 6.08; the a m m o n i u m acetate solution was filtered through a millipore type HA 0.45 pm filter prior to mixing with the acetonitrile) using a Vortex mixer. This solution was centrifuged in a micro tube at 2000 rev./min (Adams Sero-fuge) for 5 min prior to injection. Approximately 1--2 mg of digested material (in 0.1--0.2 ml) was analyzed on a Waters pBondapak C~s column using a Perkin Elmer Series 3 High Performance Liquid Chromatograph System equipped with a 7105 R h e o d y n e injector, 175 ~ loop, and LC-55 spectrophotometer with dual pen recorder. The peaks were detected at 220 nm using the next to the highest sensitivity setting. The chromatogram was developed with a gradient between 10% acetonitrile/ 0.01 M a m m o n i u m acetate, pH 6.08, and 30% acetonitrile/0.01 M a m m o n i u m acetate, pH 6.08. The program used adjusted the gradient from 1 to 100% in 80 min. The purge time was 10 min and that of equilibration was 20 min. Fractions were collected on an LKB 2070 fraction collector. Fractions containing a specific peptide were combined, freeze-dried, and hydrolyzed with 6 M HC1 for 24 h in vacuo at l l 0 ° C . Amino acid analyses used the Beckman Amino Acid Analyzer, Model 121 M. Results

CM-cellulose chrorna tography o f glob in F Fig. 1 illustrates separations obtained with the modified procedure. The G7 and h 7 chains had slightly different mobilities and resolved partially when in mixtures. Further modification of the gradient failed to improve the resolution.

Fingerprinting o f digests o f isolated 7 chains Zones A and B (chromatogram III, Fig. 1) have repeatedly been analyzed by this procedure; the study involved several samples of different origin. Fig. 2 gives examples; several peptides have been identified but only the T-9 and T-15 are indicated. Both fingerprints show one spot containing T-15. One T-9 peptide was also present in the fingerprint of zone A but two appeared in that of zone B. Table I lists the amino acid compositions of the spots indicated in the fingerprints of Fig. 2. The data show considerable contamination with other peptides or perhaps free amino acids. The T-15 peptide of zone B differs from that of zone A in that its glycine value is considerably less and the alanine value considerably more (the low recovery of valine remains unexplained). This observation, which was repeatedly made for other samples, indicated a partial resolution of the G7 (mainly in zone A) and the A7 (mainly in zone B) chains. Of considerable interest is the observation that the T7 T-9 peptide was only present in zone B of W~/chain-positive globin F samples and not in zone A. The amino acid composition data of Table I confirm the identity of the peptides; again the analyses are not very satisfactory (high glycine values, tow valine values) but differentiation between the W~ T-9 and 17 T-9 peptide could easily be made.

Thin-layer electrophoresis o f the CNBr peptides The 7CB-3 peptide was readily separated from the other fragments by elec-

425

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Fig. 1. C h r o m a t o g r a p h i c s e p a r a t i o n on a 0.9 X 20 cm column of CM-cellulose using a m o d i f i e d g r a d i e n t . S a m p l e I is f r o m G . J . w h o is a G 7 h e r e d i t a r y p e r s i s t a n c e o f H b F h e t e r o z y g o t e ( R e f . 1 6 , see a l s o T a b l e I V ) ; s a m p l e II is f r o m W.D.P. w h o is a n A 7 h e r e d i t a r y p e r s i s t a n c e H b F h e t e r o z y g o t e ( R e f . 1 5 , see also T a b l e IV); s a m p l e III is f r o m a p e r s o n w i t h sickle cell a n e m i a . Z o n e X l i k e l y is a c e t y l ated T chain.

trophoresis on silica gel (Fig. 3). Unfortunately we failed to isolate the peptide from the carrier material and analyses could only be made after hydrolysis with HC1. Some data, listed in Table II, suggest that the 6 3, chain is mainly present in the front zone A while the A? chain is present in the tail zone B.

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F i g . 2. T h i n - l a y e r f i n g e r p r i n t s o f t r y p t i c d i g e s t s o f 7 c h a i n s . ( A ) A t r a c i n g o f t h e f i n g e r p r i n t o f z o n e A o f e h r o m a t o g r a m I n s h o w n i n Fig. 1. (B) T h e s a m e b u t o f z o n e B.

426

TABLE I A M I N O A C I D C O M P O S I T I O N OF T-9 A N D T - 1 5 P E P T I D E S I S O L A T E D BY T H I N - L A Y E R F I N G E R P R I N T I N G FROM T H E Hb F OF A P A T I E N T WITH SICKLE CELL ANEMIA See chromatograzn III o f Fig. i a n d f i n g e r p r i n t s o f A a n d B of Fi~. 2. R e s i d u e s are c a l c u l a t e d a s s u m i n g L y s o r A r g as o n e r e s i d u e ; t h e n u m b e r s b e t w e e n p a r e n t h e s e s are t h e e x p e c t e d n u m b e r s o f r e s i d u e s . F o r I 7 T - 9 a n d 7 T - 9 d u p l i c a t e a n a l y s e s f o r e a c h z o n e are given. Amino acid

Lysine Arginine Aspartic acid Threonine Serine Glycine Alanine Valine Mcthionine Isoleucine Leucine

mnol recovered

T-15

17 T-9

Zone A

Zone B

1.00

1.00

(1)

1.00 2.72 1.52 1.89 I .42 0.42

1.00 2.96 0.64 2.82 1.28 0.62

(1) (3) (1 or 0) (2 or 3) (2) (l)

1.02

1.04

(1)

42

36

T~ T-9

Zone A

Zone B

Znne B

1.00--1.00

1 . 0 0 - - 1 . 0 0 (1)

1 . 0 0 - - 1 . 0 0 (1)

0.85--1.28 1.09--0.88 1.28--1.29 1.52--1.37 1.32--0.93 0.91--4).40

1.29--0.87 0.76--0.84 0.86--1.08 1.26--1.27 1.04--0.94 0.47-4).38

1.37~0.65 1.68--1.79 1 . 0 8 - 1.05 1.34--1.25 1.22--1.14 0.81--O. 53

0.89-4).72 2.14--1.96

0 . 7 8 - 4 ) . 9 3 (1) 1 . 7 0 - - 1 . 8 8 (2)

35--38

(1) (1) (1) (1) (1) (1)

(1) (2) (1) (1) (1) (1)

0--0 (0) 1 . 9 8 - - 1 . 6 9 (2)

42--40

25--35

HPLC analyses of tryptic peptides Fig. 4 illustrates the separation of the ~/chain of fetal globin from a newborn by CM-cellulose chromatography; the Hb F contained WT, 17, GT, and A7 chains. The 7 chain was heterogeneous and was subdivided into the three fractions labelled A, B and C. Protein in these fractions was isolated by freeze-drying after soluble material such as urea and salts were removed by extensive dialysis. Tryptic digests of this material were analyzed by HPLC chromatography; Fig. 5 illustrates parts of the three chromatograms that have been obtained. Amino acid analysis of four zones of chromatogram C identified these as the T-9 pep-

Fig. 3. Separation thin-layer plate.

of the CNBr

peptides

of sections

of the 7 chain

zone by electrophoresis

o n a silica g e l

427

T A B L E II THE GLYCINE AND ALANINE CONTENTS OF THE 7CB-3 PEPTIDES FROM SECTIONS OF THE 7 CHAIN OF THREE FETAL GLOBIN SAMPLES H P F H , h e r e d i t a r y p e r s i s t a n c e o f H b F. Case

Condition

P-48

5 j3-thalassemia Second sample Third sample HPFH-trait Newborn

P.S. CB-16

Zone A

Zone B

Glycine

Alanine

Glyeine

Alanine

1.14 0.98 0.88 1.02 1.07

2.12 2.11 2.35 2.43 2.08

0.46 0.57 0.42 0.47 0.48

2.70 2.64 2.78 2.85 2.(}6

tide of the W7 chain, the T-9 peptide of the I7 chain, the T-15 peptide of the G7 chain, and the T-15 of the Ag' chain, respectively. The analyses are not optimal and contamination with minor amount(s) of other, unidentified, peptides appears likely. Chromatogram A shows no Tg' T-9 peak, a small Ag' T-15 zone and two major zones being i9' T-9 and Gg' T-15. Chromatogram B is comparable except for a notable zone in the position of the Tg' T-9 zone, and a larger Ag' T-15 zone. Chromatogram C, however, shows the presence of a definite Wg' T-9 zone (the analysis of this peptide is given in Table III), a considerable Ag' T-15 zone, and the two major i9" T-9 and Gg' T-15 peptides. The chromatographic heterogeneity of the 9' chain is apparently caused by the presence of different types of 9" chains with the Gg' and i9' chains being present in the front section of the zone and the Ag' and Tg' chains in the tail section. The heterogeneity is most likely caused by the difference in glycine and alanine at position 136 and not by that in threonine and isoleucine at position 75, because a similar t y p e of chromatogram was obtained for the 9' chain of globin F in which the Wg'chain was n o t present. Fig. 6 illustrates similar HPLC chromatograms, but of digests of whole globin

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Fig. 4. Chromatographic heterogeneity of the ? c h a i n o f t h e H b F f r o m the newborn S.E. c o n t a i n i n g G?, AT, Iv, and T~, chains. The procedure is essentially the same as that used for producing the chzomatograms of Fig. 1.

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S.E. The zones are

Fig. 6. H P L c h r o m a t o g r a m s o f t r y p t i c p e p t i d e s o f p u r e f e t a l g l o b i n f r o m : ( A ) a n e w b o r n w i t h G T , A,y, T3,, a n d ~f c h a i n ; ( B ) a n A~f h e r e d i t a r y p e r s i s t a n c e o f H b F h e t e r o z y g o t e ; a n d (C) a G 7 h e r e d i t a r y p e r s i s tance of Hb F heterozygote.

F from a newborn being positive for the T~/ chain (chromatogram A), from an adult with an A7 t y p e of hereditary persistance of Hb F (chromatogram B; the person with this condition is described in Ref. 15; the T ~ / c h a i n is absent), and from an adult with a 6 7 type of hereditary persistance of Hb F (chromatogram

429 TABLE III A M I N O A C I D C O M P O S I T I O N OF P E P T I D E S I S O L A T E D BY H P L C C H R O M A T O G R A P H Y F R O M A T R Y P T I C D I G E S T OF Z O N E C O F T H E 7 C H A I N OF C A S E S.E. A s s u m i n g L y s or A r g as o n e r e s i d u e ; the n u m b e r s b e t w e e n p a r e n t h e s e s are t h e e x p e c t e d n u m b e r s o f residues. A m i n o acid

T 7 T-9

I 7 T-9

Lysine Arginine A s p a r t i c acid Threonine Serine Glycine Alanine

1.00 ( I )

1.00 ( I )

1.16 1.62 1.10 1.38 1.34

1.10 0.95 0.97 0.97 1.07

Valine Methionine Isoleucine Leucine

0.91 (I)

0.94 ( i )

1.71 (2)

nmol recovered

80

(1) (2) (1) (1) (1)

G 7 T-15

A7 T-15

1.00 ( 1 ) 0.79 (1) 2.61 (3)

1 . 0 0 (1) 0 . 8 6 (1) 2 . 6 0 (3)

1.00 (1) 2.33 ( 2 )

0 . 3 4 (0) 2.92 (3)

1.96 (2) 0.90 (I)

1.76 (2) 0.80 ( i )

0.63 ( i ) 1.69 (2)

1.12 (1)

0.95 (1)

320

230

165

(1) (1) (1) (1) (1)

T A B L E IV T H E R E L A T I V E Q U A N T I T I E S O F T 7 A N D G T C H A I N S IN T H E 7 C H A I N O F H b F F R O M D I F F E R ENT INDIVIDUALS T h e p e r c e n t a g e s are b a s e d o n the ratio o f the T 7 T-9 a n d the I7 T-9 a n d o f t h e G 7 T - 1 5 a n d A 7 T - 1 5 , resp e c t i v e l y . I o n - e x c h a n g e c h r o m a t o g r a p h y was c a r r i e d o u t bY the m e t h o d d e s c r i b e d in Ref. 5. H P L C 7CB-3 is H P L C using 7CB-3 isolated f r o m the Hb F b y S e p h a d e x c h r o m a t o g r a p h y [ 1 7 ] . H P F H , h e r e d i t a r y persistance o f Hb F. Case

A.P. T.P. CB-3 CB-4 CB-14 CB-15 * CB-16 W.J.P. G.J. A.P.

S.E. **

C.D.

Condition

Thalassemia major T h alassemia m a j o r Newborn Newborn S e c o n d analysds Newborn Newborn S e c o n d analysis Newborn S e c o n d analysis A7 H P F H t r a i t G 7 H P F H Wait Thalassemia major Zone A Zone C Newborn Zone A Zone B Zone C Newborn Zone A Zone B Zone C

% T7 c h a i n

% G7 c h a i n

HPLC tryptic peptides

Ion-exchange chromatography

HPLC tryptic peptides

0 4 22 18 19 0 10 13 0 0 0 0

0 0 20, 22 19 -0 19 -0 -0 0

80 77 73 68 65 76 69 70 74 76 15 92

0 0

0 ***

100 72

HPLC 7CB-3

70 72 70 71 -75 71 -66 -6 86 70 ***

0 13 26

14 ***

89 74 55

72 ***

2 11 16

16 ***

88 75 61

75 ***

* See e h r o m a t o g r a m A of Fig. 6. * * Z o n e s A , B a n d C w e r e i s o l a t e d b y CM-cellulose c h r o m a t o g r a p h y (Fig. 4); t h e s e p a r a t i o n o f t h e t r y p t i e p e p t i d e s is s h o w n in Fig. 5. * ** A n a l y s e s m a d e o n t o t a l fetal globin.

430 C; the carrier is described in Ref. 16; the T~ chain is absent). The chromatograms are much more complex, and contamination of the specific peptides with other fragments is considerable (amino acid analyses of several zones have confirmed this, b u t the contamination still allowed the identification of specific zones). Most notable is the presence of an additional peptide eluting in front of b u t close to the ~/T-9 peptide. A second peak is observed under the G,,f T-15 zone (chromatogram B of a Hb F in which the G~/ chain is absent), and similarly a small peak is present under the A.~, T-15 zone (chromatogram C of a Hb F in which the G ~/chain is absent). Despite the limitations of this procedure attempts have been made to calculate the percentages of the respective types of ~/chains (Table IV). Correlation of these data with results obtained b y other methods are rather satisfactory although some differences can be observed. Most interesting are the increases of the percentages of the T~, and the A~, chains in the 7 chain zones isolated by CM-cellulose chromatography, b u t none of these zones appeared to contain a single t y p e of 3' chain. The parallelism between the T,,~and A~/ chains is striking and was observed in experiments with two separate cases. Discussion

The modified procedure of CM-cellulose chromatography permitted the incomplete separation of G~/ and A~ chains. Numerous attempts to further improve this separation have failed and the use of other cation exchangers, anion exchangers and different developers was also not successful. Despite the incomplete separation the chemical analyses of zones of the isolated chain have shown that the presence of a glycyl residue in position 136 (the G7 chain) and that of an isoleucyl residue in position 75 (the '7 chain) are directly related, thus indicating the presence of an G~/I chain. A similar relation does exist between the t7 and A7 chains (thus the occurrence of an nTx chain) in globin F samples that are known to be negative for the Ile -~ Thr substitution at position 75. However, when this substitution is detected, it is invariably related to a Gly ~ Ala substitution at position 136 (the ATT chain). The most convincing evidence comes from the HPLC analyses of the tryptic peptides of zones of the isolated ~, chain. Through these analyses it was shown that in fetal globins containing the T~/ chain the increased presence of the h~/ T-15 peptide in digests was invariably accompanied by an increase in the relative amount of the W,~/T-9 peptide. Admittedly, the number of these sophisticated analyses was small; however, the final conclusions of the HPLC analyses were supported by the results from fingerprint analyses of similarly isolated ~, chain zones. Thus, the data obtained in these analyses strongly support the conclusion of previous studies [5,6] and indicate that the G~, chain has an isoleucyl residue in position 75 (the m~/chain) whereas the A7 chain either has an isoleucyl residue in position 75 (the G~,I chain) or a threonyl residue in this position (the A~/T chain). These results are contradictory to suggestions made b y Grifoni et al. [4] and by Ricco et al. [3] namely that the mutation had occurred in a G~/ structural gene. The data are also not supportive of the possibility that the W,~ chain is the product of a separate W~, structural locus. The

431 occurrence of the A•T chain appears to be rather universal although its indidence may vary from one ethnic or racial group to the other.

Acknowledgements The authors are indebted to Mrs. M. Stallings. Mrs. J. Henson, and H. Lam for capable technical assistance. This research was supported by U.S. Public Health Service Research Grants HLB-05168 and HLB-15158.

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