THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 246. No. 0, Issue of May 10, pp. 3001-3007, 1971

Printed in U.S.A.

Human Fetal Hemoglobin FI

RCETYLATION STATUS*

(Received for publication, December 7, 1970)

LEWIS D. STEGINK, PAUL D. MEYER, AND MARVIN C. BRUMMEL

From the Departments of Pediatrics and Biochemistry, The University of Iowa College of Medicine, Iowa City, Iowa 52240

SUMMARY

The y chains of human fetal hemoglobin occur in both acetylated and nonacetylated state in vivo. The acetylated fetal hemoglobin F1 has been suggested to be ~~~~~~~~~~ (Biochim. Biophys. Acfa, 63, 532 (1962)). The development of an electrophoretic assay capable of separating the cy, p, y, and yacetyl polypeptide chains in hemoglobins allowed us to show that highly purified hemoglobin F1 contained nearly equal quantities of o( chains and a polypeptide chain migrating more slowly than the y chain, and only trace quantities oi y chain. NHp-terminal and acetyl group analyses of globin prepared from hemoglobin FI indicated the presence of 2 NH2-terminal valine residues, 0.4 NH2-terminal glycine residue, and 1.6 N-acetyl groups per mole of hemoglobin (molecular weight of 66,000). The acetylated polypeptide chain was isolated from carboxymethyl cellulose columns in the presence of 8 M urea and shown to have a blocked NH2 terminus, to contain 0.76 mole of N-acetyl residue per poly- peptide chain (molecular weight of 16,500), and to migrate identically with the slow moving polypeptide chain of hemo- globin F1 in the electrophoretic assay. The acetylated NHz- terminal peptide was isolated from both FI globin and the isolated yacetyl chain, and shown to be N-acetylglycylhisti- dine. This structure is consistent with the NHz-terminal sequence of the y chain. These data indicate human fetal hemoglobin F1 should be designated ~~~~~~~~~~~

Human hemoglobins consist of four polypeptide chains, usu- ally two pairs of identical chains, attached to a heme group. During development from the embryonic to the adult state, at least six different polypeptide chains ((Y, ,8, y, 6, E, and {) are synthesized and incorporated in the various hemoglobin mole- cules, several of which may be present at a particular gestational age. For example, the erythrocytes of the term infant contain hemoglobins Fo,) F1, A, A?, and Bart’si

Allen, Schroeder, and Balog (1) first separated the minor

* This study was aided by a grant from Muscular Dystrophy Associations of America, Inc., and by a grant-in-aid from the Iowa Heart Association.

1 Assumed subunit composition of the various human hemoglo- bins: A, or&; AZ, 01282; Fo, 01~2; and Bart’s, ~4.

hemoglobin component designated F1 from umbilical cord blood hemoglobin, and Matsuda et al. (2) showed that the ratio of F1:Fo (the major fetal hemoglobin form) remained essentially constant during most of fetal development. Schroeder et al. (3) presented preliminary data suggesting the hemoglobin Fi con- tained two a! chains, a y chain, and a y chain acetylated at the amino terminus. Thus, human fetal hemoglobin appeared to occur both as an acetylated Pi (a!ZyyBcetyl) and a nonacetylated F0 (crzyZ) species.

The fetal hemoglobins therefore seemed to be an ideal model system for our studies investigating the biological function of the N-acetyl groups found on a number of proteins, and espe- cially muscle proteins. During these studies, we developed a rapid and sensitive electrophoretic assay which would dif- ferentiate y from yaeetyl chains. This method, which resolves ‘Y, ,8, y, and yaoetyl polypeptide chains without prior globin formation, indicated that highly purified fetal hemoglobin F1 contained equal quantities of the cy and yaeetyl polypeptide chains. The chemical data supporting this proposal, including the isolation of the acetylated amino-terminal peptide are presented in this paper.

MATERIALS AND METHODS

Isolation of Hemoglobins-Human fetal hemoglobins Fo and FL were isolated from human umbilical cord blood hemolysates by the method of Schroeder et al. (4). Hemoglobin F1 was rechromatographed on DEAE-Sephadex (A 50-120, Sigma) by a modification of the method of Dozy, Kleihauer, and Huisman (5). Both resin and hemoglobin sample were equilibrated with 0.05 M Tris buffer, pH 7.6, containing 5 InM KCN, and the hemoglobins were eluted from the column with 0.05 M Tris buffer, pH 7.4, containing 5 mM KCN. Fractions obtained from the chromatographic purification procedures were assayed with the acrylamide gel electrophoretic method of Stegink, Meyer, and Chalkley (6). Hemoglobin A was isolated from human umbilical cord blood hemolysates by chromatography on Bio-Rex 70 (Bio-Rad, Richmond, California, 200 to 400 mesh) with the method of Schroeder et al. (4), or by crystalliza- tion from human adult red cell hemolysates (7). Globin was prepared from hemoglobin solutions as described by Clegg, Naughton, and Weatherall (8). The precipitated globin was dried in a vacuum desiccator over sulfuric acid.

Isolation of Individual Hemoglobin Chains-The y and yaoetyl chains of human fetal hemoglobins were isolated by chroma-

3001

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tography on CM-cellulose2 columns with the method of Clegg, Naughton, and Weatherall (9). After the identity of each peak was confirmed electrophoretically (6) the appropriate fractions were pooled, and the urea removed by dialysis. The solutions were lyophiliaed and stored at -70”.

Amino-terminal and Acetyl Group Analysis-Amino-terminal and acetyl group analysis procedures were usually performed on globin or isolated polypeptide chains. Large variations occur when using intact hemoglobin because of the interference by the iron and heme portions of the molecule. Samples of the desiccated globin or polypeptide chain were weighed out by microbalance for the required assays. The quantity of fetal globin, y or yacetyl chain present was determined by isoleucine content after acid hydrolysis (22 hours, 6 N HCl) ; isoleucine is present exclusively in the y and yacetyl chains and is not found in QI and /3 chains. Acetyl group determinations were carried out with the microenzymic method of Stegink (10).

Quantitative amino-terminal analyses were carried out by the modified Sanger technique described by Fraenkel-Conrat, Harris, and Levy (11). Two samples of the protein (0.1 to 0.2 pmole) were used for each determination. One was assayed directly. The duplicate sample of the dinitrophenylated protein contained 0.2 pmole of each of the standard dinitrophenylamino acids ex- pected to be found and was carried through the appropriate steps to determine the correction factor for the loss of dinitro- phenylamino acids through hydrolysis and manipulation of the sample. These correction factors are shown in parentheses in Tables I and II. Since dinitrophenylglycine is destroyed under the usual hydrolysis conditions, it was quantitatively deter- mined by a modification of the method of Hanes, Hird, and Isherwood (12). The dinitrophenylated protein (0.1 pmole) was dissolved in 0.5 ml of a mixture of 90% (w/v) formic acid, acetic anhydride, and 60% (w/v) perchloric acid (20: 11: 3), and hydrolyzed in an evacuated hydrolysis tube (Kontes) for 16 hours at 105”. The hydrolysate was diluted to a final volume of 2 ml with water, and the dinitrophenylamino acids extracted with an equal volume of diethylether, the extraction being re- peated five times. Each ether extract was extracted with 2 ml of 6 N HCI, the ether layer transferred to another tube and evapo- rated. The residual liquid was then lyophilized to dryness. Since initial experiments had indicated the presence of only dinitrophenylvaline and dinitrophenylglycine, separation of the dinitrophenylamino acids was achieved by descending chromatography with the amyl alcohol-potassium phthalate buffer system of Blackburn and Lowther (13).

Synthesis of N-Acetylglycylhistidine-Glycylhistidine (Sigma) was dissolved in 5% sodium bicarbonate and reacted with a 30-fold excess of acetic anhydride for 3 hours at room tempera- ture. The reaction mixture was acidified to pH 2 with con- centrated HCl, the excess acetic anhydride extracted with an equal volume of diethylether, and the peptide desalted on a small Dowex 50-X8 column as described by O’Brien, Ibbott, and Rodgerson (14), eluting the peptide with 25 ml of 2 N am- monium hydroxide rather than the usual NaOH. The eluate was lyophilhed and the acetylated peptide dissolved in a minimal quantity of water before storing at -70”. Acetate, glycine, and histidine were obtained in equirnolar quantities after hy- drolysis, amino acid analysis, and acetyl group analysis.

2 The abbreviation used is: CM-cellulose, carboxymethyl cel- lulose.

3H-Acetylglycylhistidine was prepared by reacting 1 pmole of glycylhistidine with 60 &i of 3H-acetic anhydride (New England Nuclear) for 1 hour in 5y0 sodium bicarbonate, followed by a 30.fold excess of unlabeled acetic anhydride for an additional hour. The peptide was purified as described above, and had a specific activity of 25 $Zi per pmole.

Isolation of Acetylated Peptide from Fetal Hemoglobin FL-Iso- lated PI globin or yacetyl polypeptide chains (100 mg) were digested with 6 mg of Pronase (Calbiochem Grade B: 45,000 PUK per g) added in three batches of 2 mg each over the 22-hour period (pH maintained at 7.8, 40”, 1 mM CaC12, 50 ml). The reaction mixture was acidified to pH 4 with HCl, lyophilized, and stored at -70”.

The peptide was isolated by ion exchange chromatography on a Technicon NC-1 amino acid analyzer with the buffer system described by Efron (15). Synthetic N-3H-acetylglycylhistidine is eluted at 6% hours in this system, and appears between alanine and oc-aminobutyrate. The effluent from the amino acid ana- lyzer column was passed through a l-ml anthracene-packed flow-through cell in a liquid scintillation counter and was not reacted with ninhydrin, allowing detection and isolation of the radioactive fraction.

Portions of the Pronase digest calculated to contain 0.5 to 1 pmole of the acetylated peptide were dissolved in 2 ml of 3.5% sulfosalicylic acid. To this mixture was added 0.0002 pmole of synthetic 3H-acetylglycylhistidine (25 PC1 per pmole), and the mixture was placed on the amino acid analyzer. The eluate from the column passed through the flow-through cell of the scintillation counter and was collected in a fraction collector rather than reacted with ninhydrin. The appropriate fractions, containing the labeled tracer peptide were pooled, desalted on small Dowex 50 columns as described above, and the eluant lyophilized.

The isolated peptide was purified by chromatography on Rexyn 201 (Fisher). The Rexyn 201~Cl column (1 x 50 cm) was first equilibrated with water, and then washed with 200 ml of 10e4 M Tris buffer, pH 8.3, at a flow rate of 3 ml per min. The sample, dissolved in low4 M Tris buffer, pH 8.3, was applied to the column and eluted with a linear gradient of 200 ml of lo+ M

Tris buffer, pH 8.3, and 200 ml of 0.002 M HCI with a flow rate of 0.3 ml per min. N-Acetylglycylhistidine was eluted at ap- proximately 220 ml of effluent, well separated from both neutral and basic peptides which were eluted close to the void volume, and from the acidic peptides which eluted at considerably more acidic pH values. The column effluent was monitored for radioactivity and absorbance at 215 nm. The fractions con- taining the radioactivity and 215-nm absorbing peak were pooled, desalted on a Dowex 50 column, lyophilized, dissolved in a minimal quantity of water, and stored at -70”.

The peptide can also be detected in Pronase digests with high voltage electrophoresis on previously washed Whatman No. 1 paper (0.005 M phosphate buffer, pH 7.5, 15 volts per cm, 3 hours). Under these conditions, acetylglycylhistidine separates well from most peptides, behaving as if having a net charge of -0.6, and may be located by spraying with Pauly reagent (16).

RESULTS

Human fetal hemoglobin, present in viva in both acetylnted and nonacetylated forms, is an ideal protein for examining the addition, removal, and biological function of protein-bound acetyl groups. The principal polypeptide chains present in

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FIG. 1. Polyacrylamide gel electrophoresis of purified human hemoglobins F1, Fo, and A.

human umbilical cord blood in the various tetrameric mole- cules of native hemoglobin are OL, /3, y, and yacetyl. Only the y and yacetyl chains contain isoleucine (4 residues per chain). The y polypeptide chains have NHz-terminal glycine residues, the a! and p chains have NHz-terminal valine residues, and the yacetyl should terminate with N-acetyl glycine, although this had not been indicated prior to these experiments. This dif- ference in NHz-terminal residues and the recent development of a rapid polyacrylamide gel electrophoretic assay (6) which sepa- rates these chains from each other enabled us to examine the acetylation status of the fetal hemoglobins more easily.

In this study, hemoglobin F1 is the species designated olZ-y-racetY1 and F. the species designated a2y2 by Schroeder et al. (17). Electrophoretic examination of the F1 peak eluted from Bio-Rad 70 column chromatography of umbilical cord blood hemolysates indicated an unequal distribution of the y chain across the peak. Most tubes contained relatively little y chain, although some tubes contained both y and yacetyl chains, usually larger amounts of the latter. Evaluation of the exact chain composition of the F1 peak purified in this manlier with De- veloper 4 is difficult since the FL peak is immediately preceded by two peaks containing y chains and the trailing edges of the peak may contain some hemoglobin Fo. This F1 fraction also con- tained measurable quantities of p chain. Rechromatography of the pooled FL fraction on either Bio-Rad ‘70 with either De- veloper 4 (as in the first purification) or Developer 6 which better separates hemoglobin Bart’s from F1, or on DEAE- Sephadex yielded hemoglobin with only trace quantities of the y chain and nearly equal quantities of a and yaeetyl chains. A comparison of the electrophoretic patterns of purified hemo-

TABLE I

NH&rminal and N-acetyl group analyses of FO and F1 globin preparations

Sample assayed

j.cmole

F. globin, 0.077

F1 globin, 0.114

A globin, 0.090

a Recovery fact solute values.

Found Globin ~__

amole V&S/ mole

0.149 1.94 (91% )” 0.240 2.10

(89% ) 0.34 3.8

(87% )

Dinitrophenyl- glycine

Found Globin

p?de moles/ mole

0.152 1.98

(79% )” 0.045 0.40

(75% )

Acetate

Found Globin --

pWWle “.df;/

0.003 0.04

0.165 1.55

0.002 0.02

or for added standard used to determine ab-

globins A, Fo, and F1 (Fig. 1) shows that all species contain the fast migrating 01 chain, and that PI contains a polypeptide chain which does not correspond to either p or y chains.

Schroeder et al. (3) have reported that the amino acid com- positions of hemoglobins Fo and F1 were identical, and that tryptic peptide mapping of Fo and F1 was also identical, with the exception that these investigators were not able to locate the NHS-terminal acetylated peptide.

We have experienced difficulty in the determination of NH*- terminal and acetyl group content in purified intact hemoglobins Fo and F1 because of heme group interference with both assays, especially the microenzymic acetate assay. Our results from such studies resembled those reported by Schroeder et al. (3) with the exception that acetyl group content was always greater than 1 mole per mole (66,000) with F1 hemoglobin. To avoid these difiiculties, studies were carried out on globin prepared from human hemoglobins A, Fo, and Fi. The data shown in Table I indicate that theoretical values were obtained for globin prepared from hemoglobins A and Fa. Globin prepared from purified hemoglobin F1 contained 2 NHz-terminal valine, 0.4 NHz- terminal glycine, and 1.6 N-acetyl residues per 8 isoleucine resi- dues. In some preparations of FL globin, NHz-terminal glycine levels were lower, but most preparations contained this quantity despite the lower quantity expected on the basis of the electro- phoretic assay. A similar situation was noted when studying isolated yaoetyl polypeptide chains (see below).

Clegg et al. (9) noted the presence of a peak eluted just before the y chain upon chromatography of fetal hemoglobins on CM-cellulose in the presence of 8 M urea. This peak was re- ported to have a tryptic map identical with that of the y-peak. When fetal hemoglobins Fo and Fi, purified by a single chro- matographic step on Bio-Rad 70 with Developer 4, were chro- matographed on matched CM-cellulose columns in 8 M urea at constant flow rates, we found both preparations contained CY chains. The FL hemoglobin contained a polypeptide chain which was eluted more readily from the CM-cellulose column than the y chain of hemoglobin F. (Fig. 2), and which was identi- cal with the peak described by Clegg et al. (9). Polyacrylamide electrophoretic assay (6) of each tube in the yaCetyl region for the hemoglobin F1 chromatogram described in Fig. 2 is shown in Fig. 3. These data clearly indicate that the slowest moving electrophoretic band noted in hemoglobin FL is identical with

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3004 Petal Hemoglobin Fl Vol. 246, No. 9

the polypeptide chain eluted most readily from the CM-cellulose column, and is the polypeptide chain we have termed yacetyl.

The fractions from the CM-cellulose column fractionation of the various hemoglobin samples corresponding to yacetyl, y, and a chains were chosen so as to obtain maximally pure samples of the individual chains. The amino acid analyses of the y and yaoetyl chains were essentially identical. The results of NH2- terminal and acetyl group analysis carried out on the isolated,

1.6 -

1.4-

7 1.2-

c

g to- c!

$8-

5 m

5

0.6-

$4-

IO 20 30 40 50 60 70 80 90 FRACTION NUMBER

FIG. 2. Separation of the individual polypeptide chains of hemoglobins Fo (O---O) and Fl (O-O) on matched CM-cel- lulose columns in 8 M urea using the method of Clegg et al. (9).

o( marker-

FIG. 3. Polyacrylamide gel electrophoretic assay of the yacetyl peak of hemoglobin F1 shown in Fig. 2. Purified a chain has been added to each gel to serve as a marker. The gel labeled F. shows the electrophoretic pattern of purified hemoglobin Fo.

(Y, y, and yacetyl polypeptide chains are shown in Table II. As expected, the (Y chains contain 1 NHg-terminal valine residue per chain (molecular weight of 16,500). The y chains have 1 NHz-terminal glycine residue per 4 isoleucine residues (molecular weight of 16,500). The yacetyl chains have 0.16 NHz-terminal glycine and 0.76 N-acetyl residues per 4 isoleucine residues (molecular weight of 16,500). These data show that the yacetyl chain has a blocked NH2 terminus and contains an N-acetyl group, while the isolated y chain has the expected NH*-terminal glycine.

The position of the N-acetyl group was confirmed by isolation of the acetylated peptide from Pronase digests of both F1 globin and purified yacetyl chains with the basic technique described by Narita (18). When Pronase digests of either source were passed through an AG50 (H+) column at neutral pH, all of the acetyl groups were bound to the column, indicating the presence of a basic group on the NHz-terminal peptide. This was ex- pected since the NH,-terminal sequence of the y chain is Gly- His-Phe (4) and Pronase rapidly attacks the His-Phe bond while hydrolyzing the Ala-His linkage more slowly (19), suggesting that the Gly-His linkage is also hydrolyzed slowly.

High voltage paper electrophoresis of the Pronase digest at pH 7.5 yielded a spot which was negative to ninhydrin, positive to Pauly reagent and the Rydon-Smith colorination procedure, and which comigrated with authentic N-acetylglycylhistidine and contained bound acetate. Because of the poor recovery when isolated from paper, the acetylated peptide was isolated by chromatography on a Technicon single column NC-l amino acid analyzer equipped with a flow through cell for radioactivity analysis. A trace quantity of N-3H-acetylglycylhistidine (0.0002 pmole) was added to an aliquot of the Pronase digest calculated to contain 0.5 to 1.0 pmole of the naturally occurring acetylated peptide, and chromatographed on the analyzer. The fraction containing the labeled tracer peptide was collected, desalted to remove citrate, and assayed for acetate and amino acid content after hydrolysis. The isolated peptide contained approximately equal quantities of acetate, serine, glycine, and histidine, and smaller amounts of other amino acids. Since the fraction was shown to be contaminated with other peptides on high voltage

TABLE II NH&erminal and N-acetyl group analyses on isolated polypeptide

chains from human fetal hemoglobins

Sample Dinitrophenylvaline Dinitrophenylglycine Acetate

mole/mole folypeptide chain

y Chain.. . . 0.01 (89%‘, y&mtyl Chain. . . . . 0.00 (87%) 01 Chain.. . . . . . . . . . . 0.94 (92%)

; &I;Izi; ; pi

a Recovery of added standard dinitrophenylamino acid used to determine absolute values.

TABLE III Amino acid and acetyl analyses of isolated acetylated peptide

component

Glycine....................................... Histidine. _....... _, ., ,_. Acetate.................................,.....

Found

p?de

0.16 0.14 0.14

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electrophoresis, it was further purified on a Rexyn 201 (Cl-) column. The radioactivity was eluted coincidently with a 215-nm absorbing peak at about 220 ml of eluant. The acetyl group content and amino acid composition of the isolated peak are shown in Table III. The COOH-terminal amino acid, determined by Method C of Holcomb, James, and Ward (20) was shown to be histidine. Thus, the structure of the acetylated peptide is N-acetylglycylhistidine. Since the NH&erminal sequence for the y chain has been reported to be glycylhistidyl- phenylalanine, the isolated peptide is a reasonable one for the NHz-terminal acetylated peptide.

DISCUSSION

The changes occurring in the types of hemoglobin present in man during development, are excellent examples of gene activa- tion and repression. Human hemoglobins consist of four poly- peptide chains, usually two pairs of identical chains, attached to a heme group. During development from the embryonic to the adult state, at least six different polypeptide chains (o(, p, y, 6, E, and [) are synthesized. In the very young embryo, OL and e polypeptide chain syntheses predominate, leading to the forma- tion of ~4 and CQE~, the so-called Gower hemoglobins (21-23). By 3 months gestational age, E chain synthesis ceases and is replaced by increasing synthesis of the y chain, consistent with the ob- servation that hemoglobin Fo(azyz) is the major hemoglobin species found in fetal blood. The erythrocytes of the fetus also contain hemoglobin Bart’s (y4), hemoglobin A (a&), and hemo- globin Fi. Hemoglobin Portland 1 has been reported to be present in both normal newborn infants and those with several chromosomal abnormalities (24-26) and has a suggested struc- ture of lzyz.

At the time of birth, y chain synthesis decreases and p chain synthesis increases, so that by 3 to 6 months after birth only small quantities of hemoglobin F0 (less than 0.4$&) are found and hemoglobin A predominates. Adult erythrocytes containing hemoglobin A, small quantities of hemoglobin Az (&), and a number of minor hemoglobin components (Al,, AIs, A,,, Ald, and Al,) isolated by column chromatography or gel electro- phoresis. Hemoglobin A1, appears to contain two o( chains, and two /3 chains substituted at the amino terminus by a hexose component (27, 28). Hemoglobin Aa which may account for 10% of the total hemoglobin in aged erythrocytes appears to be formed by disulfide bond formation between hemoglobin A and glutathione on storage. The nature of the polypeptide chains present in hemoglobins Al,, Alb, Ald, and A,, has not been established. Two “different” y chains appear to be syn- thesized in the normal infant, differing only at position 136. One chain contains glycine at this position, the other alanine (29). The y chain is also unusual in that it exists in both an acetylated and a nonacetylated state (3).

Our highly purified human fetal hemoglobin FL contained only small quantities of the y polypeptide chain upon electro- phoretic analysis. Our NHn-terminal studies on the purified F, hemoglobin essentially confirmed the data reported by Schroeder et al. (3) indicating the presence of 2 NHz-terminal valine and 0.4 NHS-terminal glycine residues per 66,000 molecular weight. Our data however have consistently shown the presence of 1.4 to 1.7 N-acetyl residues per molecular weight of 66,000 in contrast to the 0.8 residue reported by Schroeder et ul. (3). The quantity of glycine found in our studies was greater than would have been expected on the basis of electrophoretic analysis,

which indicated 12% y chain. We have also noted this slight increase in NHt-terminal glycine content in our studies of the isolated yacetyl chain. The isolated chain appeared homogeneous upon electrophoresis, but contained 0.16 NHz-terminal glycine residue and 0.76 N-acetyl residue per 4 isoleucine residues. Thus, either the acetyl group is lost to some degree during the dinitrophenylation reaction, or another polypeptide chain is present which: (a) has an NHz-terminal glycine residue, (b) electrophoresis with the y acetyl chain on polyacrylamide gel, and (c) is eluted with the yacetyl chain in the CM-cellulose column chromatographic method of Clegg et al. (9).

Huehns and Shooter (30) were the first to conclude that the ~~~~~~~~~~ structure proposed for human fetal hemoglobin F1 was not correct on the basis of their elegant dissociation and recombination experiments. They suggested that its structure be designated as a!zyxyacetyl on the basis of their data and the acetyl group analysis data of Schroeder et al. (3), and showed that the net charge of the yx chain must be the same as the yacetyl chain in phosphate buffer at neutral pH and at pH 8.6. They also pointed out that either the acetyl group or the un- known x group, or both, must be relatively labile under certain conditions. Our data confirm the lability of the yacetyl chains either alone or in the intact F1 hemoglobin, especially under the acidic conditions used for globin formation. This lability undoubtedly accounts for the variable quantities of NH&erminal glycine residues found as noted above.

The peak fractions of hemoglobin F1 isolated by a single chro- matographic separation on Bio-Rad 70 with Developer 4 contain nearly equal quantities of o( and yacetyl chains, and smaller quantities of y and p chains when assayed either by polyacryl- amide gel electrophoresis or by CM-cellulose chromatography with the method of Clegg et al. (9) (see Fig. 2). These latter chains are largely removed by rechromatography on Bio-Rad 70 with either Developer 4 or 6, but are removed to a greater extent by DEAE-Sephadex chromatography. Similar chain composi- tion was noted in the peak tubes of the hemoglobin F1 fraction isolated by initial chromatography on DEAE-Sephadex. In both chromatographic separations the polyacrylamide electro- phoretic assay is essential to permit proper selection of fractions in order to avoid those containing substantial quantities of the y chain.

From the polyacrylamide electrophoretic separation of the y and yacetyl chains, it may be calculated that the separation between chains is compatible with a net charge difference of fl (6). The peptide mapping studies reported by Schroeder et al. (3) and by Clegg et al. (9) have shown that the tryptic peptide map of the intact F1 hemoglobin or the isolated chain now shown to be yacetyl have no differences except for the NHz-terminal peptide. Our own preliminary studies substantiate these find- ings. Thus, it may be concluded that fetal hemoglobin F1 contains equal quantities of o( and yacetyl polypeptide chains.

Interest in proteins cont,aining acetyl groups has grown rapidly since Narita (18) isolated and characterized the acetyl peptide from the NH2 terminus of tobacco mosaic virus protein. Since t,hat time, a number of proteins have been shown to contain such N-acetyl groups at the NH2 terminus (Table IV). A num- ber of other proteins contain such N-acetyl groups, including oc-glycerophosphate dehydrogenase (56)) low molecular weight fish muscle albumins (57), pyruvate kinase (10, 58), carbonic anhydrase (59), luteinizing hormone (60), tropocollagen (61), hyaluronidase (62), eel hemoglobin (63), larval and adult hemo-

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TABLE IV

Proteins acetylated at amino terminus

Protein Acetylated amino acid

Tobacco mosaic virus coat protein Turnip yellow mosaic virus protein Ovalbnmin Cytochrome c

Vertebrate Plant (wheatgerm, mung bean,

and sunflower) Histones (several species) Enolase (rabbit muscle) Myosin (rabbit muscle) Actin (rabbit muscle) Trypomyosin (rabbit muscle)

Serine 18

Methionine 31 Glycine 32, 33

Glycine Alanine

Apoferritin (horse spleen) a-Melanocyte-stimulating hormone a-Crystallin Mitochondria structural protein Lactate dehydrogenase

Mb (rat liver) Hq (bovine and chicken heart) Mq (dogfish muscle)

Hemoglobin Human fetal F,

Serine Alanine Serine Aspartate Aspartate or

methionine Serine Serine Methionine Serine

Alanine Alanine Threoninea

Glycine

Chicken Valine” Rana esculenta (frog) Alanine Carp Serine Bujo bufo (toad) Alanine

Reference

34 35-37

38 39 40

41, 42 43

44 45 46 47

48 49 50

(this

paper) 51 52 53 54

a Acyl grollp not identified b Some question has arisen concerning the acetylation status

of this hemoglobin (55).

globins of Rana catesbeiana (64), fish myoglobins (65), and thorax proteins of the honey bee (66), although the identity of the acetylated amino acid is not known.

tRNA’s may be involved, since several different N-acetyl amino

The mechanism by which such N-acetyl groups are added to the NH2 terminus is not clear. In 1963, Pearlman and Bloch (67) suggested that these groups might be involved in the initia- tion of protein synthesis. Since then, N-formylmethionyl- tRNA has been established as the initiator of protein synthesis in bacteria (68-71) and implicated in mitochondrial and chloro- plast protein synthesis (70-72). The initiation mechanism on the 80 S ribosomes in eukaryotic cells appears to involve a methionyl-tRNA species, (which although nonacylated, is capa-

ble of being formylated by the Escherichia co& transformylase), which incorporates a nonacylated methionine into the original NH&erminal position. This NH&erminal methionine residue t,hen appears to be removed during subsequent polypeptide chain elongation (73-77). However, this methionyl-tRNA species may not be the only mammalian initiator. Several ot.her N-acylated aminoacyl-tRNA’s have been suggested in various mammalian systems: NHz-blocked valyl-tRNA for hemoglobin (78)) pyroglutamyl-tRNA for immunoglobulins (79), and N-acetylseryl-tRNA for histone synthesis in regenerat- ing rat liver (80).

I f NHz-terminal acetyl groups are added to proteins only during initiation, at least four different N-acetylaminoacyl-

acids are found in acetylated proteins, even those isolated from a specific tissue of the same species. In rat liver at least three different NHz-terminal acetyl amino acids are noted: acetylserine in f2a histones (80), acetylalanine in Md lactate dehydrogenase (48), and acetylglycine is likely in cytochrome c (34). In rabbit muscle, at least four different N-acetyl amino acids are found: acetylalanine in enolase (39), acetylserine in myosin (40), acetyl- aspartate in actin (41), acetylglycine in cytochrome c (81), and possibly acetylmethionine in tropomyosin (43). Thus, either such tissues have more than one codon specific for acetylamino- acyl-tRNA-dependent initiation, or acetyl groups are added after polypeptide chain synthesis, or both. Marchis-Mouren and Lipmann (82) have reported the acetylation of chicken hemo- globin by ribosome-free erythrocyte lysates suggesting the latter possibility, although Moss and Thompson (55) have sug- gested that the acetyl groups are added at positions other than the amino terminus in this system.

It is not known whether changes in the acetylation status of the y chains involve the individual chain prior to aggregation to form hemoglobin, or whether these changes occur on chains already aggregated as intact hemoglobin. If the acetyl group is added either during initiation or polypeptide chain elongation, it must be removed in order to form the F. fetal hemoglobin which comprises 8O7o of total fetal hemoglobin. The mecha- nism may involve either deacetylation of the individual yacetyl chain or deacetylat,ion of these chains while present in the intact hemoglobin. Similarly, if groups are added after protein syn- thesis, they may be added to the individual y chain or to y chains in intact hemoglobin Fo. Additional studies differentiating these mechanisms are currently in progress.

Acknowledgments-We gratefully acknowledge the assistance of the delivery room staff of the University of Iowa Hospitals in obtaining umbilical cord blood specimens. We also wish to thank Dr. Andre Lascari for his assistance during our initial studies of the fetal hemoglobins.

REFERENCES

1. ALLEN, D. W., SCHROEDER, W. A., AND BALOG, J., J. Amer. Chem. Xoc., 80, 1628 (1958).

2. MATSUDA, G., SCHROEDER, W. A., JONES, R. T., AND WELIICY, N., Blood, i6, 984 (1960);

3. SCHROEDER. W. A.. CUA. J. T.. MATSUDA. G.. AND FENNINGER,

W. D., Biochim.‘BBiophys. A&, 63, 532’(1962). 4. SCHROEDER, W. A., SHELTON, J. It., SHELTON, J. B., CORMICI<,

J., AND JONES, R. T., Biochemistry, 2, 992 (1963). 5. DOZY, A. M., KLEIHAUER, E. F., AND HUISMAN, T. H. J., J.

Chromatogr., 32, 723 (1968). 6. STEGINK, L. D., MEYER, P. D., AND CHALKLEY, R., Anal.

Biochem., 41, in press (1971). 7. HINSON, J. A., AND MCMEEICIN, T. L., Biochem. Biophys. Res.

Commun., 36, 94 (1969). 8. CLEGG, J. B., NAUGHTON, M. A., AND WEATIIERALL, D. J., J.

Mol. Biol., 19, 91 (1966). 9. CLEGG, J. B., NAUGHTON, M. A., AND WEATHERALL, I). J.,

Nature, 219, 69 (1968). 10. STEGINK, L. D., Anal. Biochem., 20, 502 (1967). 11. FRAENKI&ON&AT, H., HARRIS, J. I., AND LEVY, A. L., Meth-

ods Biochem. Anal.. 2. 359 0955). 12. HANES, C. S., HIRD, k. i. R.,‘AND’~SHERVOOD, F. A., Biochem.

14. O’BRIEN, D., I~ROTT, F. A., AND RODGERSON, D. O., Labora-

J., Si, 25 (1952).

tory manual of pediatric microbiochemical techniques, Ed. 4,

13. BLACKBURN, S., AND LOWTHER, A. G., Biochem. J., 46, 126

Harper and Row, Publishers, New York, 1968, p. 22.

(1951).

by guest on July 1, 2018http://w

ww

.jbc.org/D

ownloaded from

Issue of May 10, 1971 L. D. Xtegink, P. D. Meyer, and M. C. Brummel 3007

15.

16.

17.

18. 19. 20.

21.

22.

23.

24.

25.

2G.

27.

28.

29.

30.

31. 32.

33. 34.

EFFRON, M. L., in L. T. SKEGGS, JR. (Editor), Automation in analytical chemistry, 1965, Mediad Inc., New York, 1966, p. 637.

SMITH, I., Chromatographic and electrophoretic techniques, Vol. 1, Interscience Publishers, Inc., New York, 1960, p. 96.

SCHROEDER, W. A., HCJISMAN, T. H. J., SHI~LTON, J. ^n., SHEL- TON, J. B.. KLEIHAUER. IX. F.. DOZY. A. M.. AND ROBBER- SON; B., Pkoc. Nat. Acah. Sci. ‘U. S. A., 60, i37 (1968).

NARITA, K., Biochim. Biophys. Acta, 28, 184 (1958).. TROP. M.. AND BIRIC. Y., Biochem. J.. 116. 19 (1970). HOI&MR; G. N., J.&E~, s. A., AND WARD, b. N:, B&chemistry,

7, 1291 (1968). HUEHNS, E. R., DANCE, N., BEAVEN, G. H., KEIL, J. V.,

HECHT, F., AND MOTULSICY, A. G., Nature, 201, 1095 (1964). HUEHNS, E. R., FLYNN, F. V., BUTLER, E..A., AND BEAVEN,

G. H.. Nature. 189, 496 (1961). HUEHN;, E. R., &&T, F.,‘K&, J. V., AND MOTULSKY, A. G.,

Proc. Nat. Acad. Sci. U. S. A., 1X,89 (1964). CAPP, G. L., RIGAS, D. A., AND JONES, R. T., Nature, 228, 278

(1970). WEATHERALL, D. J., CLEGG, J. B., AND BOON, W. H., Brit. J.

Haematol.. 18. 357 (1970). TODD, I)., L&, Y&. C. s., B~AVEN, G. H., AND HUEHNS, E. R.;

Brit. J. Haematol., 19, 27 (1970). HOLMQUIST, W. R., AND SCHROEDER, W. A., Biochemistry, 5,

2489 (1966). BOOICCHIN, R. M., AND GALLOP, P. M., Biochem. Biophys. Res.

Commun., 32, 86 (1968). HUISIXAN, T. H. J., SCHROEDER, W. A., DOZY, A.M., SHELTON,

J. R., SHELTON, J. B., BOYD, E. M., AND APELL, G., Ann. N. Y. Acad. Sci., 165, 320 (1969).

HUEHNS, E. R., AND SHOOTER, E. M., Biochem. J., 101, 852 (1966).

HARRIS, J. I., AND HINDLEY, J., J. Mol. Biol., 3, 117 (1961). JOHANSEN, P. G., MARSHALL, R. D., AND NEUBERGER, A.,

B&hem. J., 77, 239 (1960). N~RITA, K., AND ISHII, J., J. Biochem. (Tokyo), 62, 367 (1962). MBRGOLIASH, E., AND SCHEJTER, A., Advan. Protein Chem., 21,

113 (1966).

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60. 61.

62.

63.

64.

65.

66.

67.

68.

35. STEVENS, F. C., GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem., 69. 242.2764 (1967).

36.

37.

38. 39. 40. 41.

THOMPSON,‘E. %J., LAYCOCK, M. V., RAMSHAW, J. A. M., AND 70. BOULTER, D., Biochem. J., 117, 183 (1970). 71.

RAMSHAW, J. A. M., THOMPSON, E. W., AND BOULTER, D., Bio- 72. them. J., 119, 535 (1970). 73.

PHILLIPS, b. M. P., Biochem. J., 107, 135 (1968). WINSTEAD. J. A.. AND WOLD. F.. Biochemistru. 3. 791 (1964). OFFER, G.‘W., Biochim. Biophys. Acta, 90, 1% (i964).‘ ’ GAETJENS, E., AND BARANY, M., Biochim. Biophys. Acta, 117,

176 (1966).

74. 75.

42. 43.

44. 45. 46.

ALVING, R. E., AND LAICI, K., Biochemistry, 5, 2597 (1966). ALVING, R. E., MOCZAR, E., AND LAKI, K., Biochem. Biophys.

Res. Commun., 23, 540 (1966). SURAN, A. A., Arch. Biochem. Biophys., 113, 1 (1966). HARRIS, J. I., Biochem. J., 71, 451 (1959). HOENDERS, H. J., AND BLOEMENDAL, H., Biochim. Biophys.

Acta, 147, 183 (1967).

76.

2

79.

80.

47.

48.

GRIDDLE, R. S., EDWARDS, D. L., AND PETERSEN, T. G., Bio- chemistry, 6, 578 (1966).

81.

SANBORN, B. M., BRUMMEL, M. C., STEGINK, L. D., AND VEST- LING, C. S., Biochim. Biophys. Acta, 221, 125 (1970).

82.

BRUMMEL, M. C., SANBORN, B. M., AND STEGINK, L. I)., Arch. Biochem. Biophys., in press.

ALLISON, W. S., ADMIRAAL, J., AND KAPLAN, N. O., J. Biol. Chem., 244, 4743 (1969).

SATAI~E, K., SASAKAWA, S., AND MARUYAMA, T., J. Biochem. (Tokyo), 53, 516 (1963).

CHAUVET, J. P., AND ACHER, R., Fed. Eur. Biochem. Sot. Lett., 1, 305 (1968).

HILSE, K., SORGER, U., AND BRAUNITZXR, G., 2. Phys. Chem., 344, 166 (1966).

CAFFIN, J. P., CHAUVET, J. P., AND ACHER, R., Fed. Eur. Bio- them. Sot. Lett., 6, 196 (1969).

Moss, B. A., AND THOMPSON, E. 0. P., Biochim. Biophys. Acta, 188, 348 (1969).

VAN EYS, J., JUDD, J., FORD, J., AND WOMACI~, W. B., Biochem- istry, 3, 1755 (1965).

GERDAY, C., AND BHUSHANA RAO, K. S. P., Comp. Biochem. Physiol., 36, 229 (1970).

COTTAM, G. L., HOLLENBERG, P. F., AND COON, M. J., J. Biol. Chem., 244, 1481 (1969).

MARRIQ, C., LUCCIONI, F., AND LAURENT, G., Biochim. Bio- phys. Acta, 105, 606 (1965).

WARD, D. N., AND COFFEY, J. A., Biochemistry, 3, 1575 (1964). H~RMANN, H., AND JOSEPH, K. T., Biochim. Biophys. Acta,

100, 598 (1965). SORU, E., AND ZAHARIA, O., Arch. Biochem. Biophys., 119, 498

(1967). A~ANO, H., HASHIMOTO, K., AND MATSUURA, F., Bull. Jap.

Sot. Sci. Fish.. 34. 937 (1968). DEWITT, W., AND INGRAM~V. k., Biochem. Biophys. Res. Com-

mm., 27, 236 (1967). AMANO, H., HASHIMOTO, K., AND MATSUURA, F., Bull. Jap.

Sot. Sci. Fish., 34, 955 (1968). POLZ, G., AND KREIL, G., Biochem. Biophys. Res. Commun.,

39, 516 (1970). PEARLMAN, R., AND BLOCH, K., Proc. Nat. Acad. Sci. U. S. A.,

50, 533 (1963). ADAMS, J. M., AND CAPECCHI, M. R., Proc. Nat. Acad. Sci.

U. S. A., 55, 147 (1966). WEBSTER, R. E., ENGELHARDT, D. L., AND ZINDER, N. D.,

Proc. Nat. Acad. Sci. U. S. A., 66, 155 (1966). Cold Spring Harbor Symp. Quant. Biol., 31 (1966). Cold Spring Harbor Symp. Quant. Biol., 34, (1969). SMITH, A. E., AND MARCICER, K. A., J. Mol. Biol., 38,241 (1968). HOUSMAN, D., JACOBS-LORENA, M., RAJBHANDARY, U. L., AND

LODISH, H. F., Nature, 227, 913 (1970). SHAFRITZ, D. A., AND ANDERSON, W. F., Nature, 227,918 (1970). WILSON, D. B., AND DINTZIS, H. M., Proc. Nat. Acad. Sci.

U. S. A., 66, 1282 (1970). WIGLE, D. T., AND DIXON, G. H., Nature, 227, 676 (1970). JACKSON, R., AND HUNTER, T., Nature, 227, 672 (1970). ARNSTEIN, H. R. V., AND RAHAMIMOFF, H., Nature, 219, 942

(1968). MOAV, B., AND HARRIS, T. N., Biochem. Biophys. Res. Com-

mun.. 29, 773 (1967). LIEW, c. c., H~SLE~T, G. W., AND ALLFREY, V. G., Nature,

226, 414 (1970). NEEDLEMAN, S. B., AND MARGOLIASH, E., J. Biol. Chem., 241,

853 (1966). MARCHIS-MOUREN, G., AND LIPMANN, F., Proc. Nat. Acad.

Sci. 77. S. A., 53, 1147 (1965).

by guest on July 1, 2018http://w

ww

.jbc.org/D

ownloaded from

Lewis D. Stegink, Paul D. Meyer and Marvin C. Brummel : ACETYLATION STATUS1Human Fetal Hemoglobin F

1971, 246:3001-3007.J. Biol. Chem. 

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