THE JOURNAL OF l j 1 0 1 . 0 ~ 1 ~ ~ ~ CHEMISTRY Vol. 257, No. 16, Issue ofAugust 25. pp. 9406-Y415. 1982 Prrnted in 11 S A .

Effects of Fe3+ Binding on the Microenvironments of Individual Amino Groups in Human Serum Transferrin as Determined by Differential Kinetic Labeling*

(Received for publication, March 26, 1982)

Jaiprakash G. Shewale and Keith Brew From the Department of Biochemistry, University of Miami School of Medicine, Miami, Florida ,33101

The effects of Fe3+ binding on the structure of human serum transferrin has been examined by differential kinetic labeling with acetic anhydride. Equal amounts of apotransferrin (ATf) and diferric transferrin (FTQ were treated with trace amounts of r3H]acetic anhy- dride at pH 8.0 so that acetylation of less than one amino group per molecule of transferrin occurred. 28% less labeling was found in FTf than ATf. ATf that had been [14C]acetylated to a similarly low extent was added to each sample as an internal standard, and the 10 cyanogen bromide fragments were purified from each sample. Reduced 3H/’4C ratios, indicating the presence of residues perturbed on Fe3’ binding, were found in three fragments from FTf. Perturbed residues were identified by sequencer analysis of fragments and by complete acetylation, proteolytic digestion, and pep- tide separation, using various column chromatographic procedures. Peptides with altered 3H/’4C ratios were purified to homogeneity and identified by amino acid analysis and sequencer analysis. Only 12 out of a total of 59 amino groups were found to be significantly de- creased in reactivity as a result of iron binding. Most of these residues are in corresponding regions in the two homologous domains of transferrin, and all are con- fined to the COOH-terminal half of each domain. The strongest perturbations were in lysines 206 and 296 (NHa-terminal domain) and 534 and 626 (COOH-termi- nal domain) which decreased in reactivities by factors of 23.9, 9.9, 23.5, and 5.2, respectively. Other residues affected were lysines 276, 291, 511, 527, 545, 568, 639, and 656. Two residues showed small increases in reac- tivity, possibly reflecting conformational changes re- sulting from metal binding. The magnitude of the changes in the homologous lysines 206 and 534 suggests that the neighboring histidines (207 and 535) may func- tion directly in Fe binding.

Serum transferrin is responsible for the transport of iron in the circulation from the sites of absorption, storage, and hemoglobin turnover to iron-requiring tissues such as the erythropoietic tissue of the bone marrow. Transferrin is a monomeric glycoprotein of 80,000 molecular weight and pos- sesses two similar metal binding sites to which ferric ions bind in the presence of bicarbonate ions with association constants in the order of M” (1). Spectroscopic and chemical modification studies suggest that the Fe”’ ion at each site is

* This work was supported by Grant GM 21363 frorn the National Institute of General Medical Sciences, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertiserrent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

liganded with the bicarbonate ion, one or two histidine resi- dues, and two or three tyrosine residues; other unidentified groups may also be involved (2). On binding iron, the protein undergoes a conformational change to a more compact struc- ture (3, 4) that shows increased resistance to urea and heat denaturation and to proteolytic hydrolysis.

The complete primary structure of human serum transferrin has been reported recently from this laboratory (5). The 678- residue sequence contains two strongly homologous regions consisting of residues 1-336 and 337-678 in which 40% of the residues in corresponding positions are identical (5). A variety of evidence indicates that each region corresponds to a domain which contains a single iron-binding site (see Ref. 5 for dis- cussion). Comparisons with the sequence of chicken transfer- rin, deduced from the sequence of a cDNA prepared with ovotransferrin mRNA (6), permits the identification of a Iim- ited number of tyrosyl and histidyl residues that are conserved in corresponding positions in the two domains of both human and chicken transferrins and are therefore candidates for components of the iron-binding sites.

We report here a study of the effects of Fe:’+ binding on the reactivities of the individual amino groups of transferrin to- wards acylation with acetic anhydride. A kinetic labeling approach based on the “competitive labeling” procedure of Kaplan et al. (7) has been employed in which trace labeling with high specific activity [”Hlacetic anhydride is performed with samples of apotransferrin and diferric transferrin under conditions where, on the average, less than 1 amino group/ protein molecule is acylated. In this type of approach, the minimal level of modification ensures that the protein confor- mation is not perturbed by the chemical modification reaction. There is competition between the individual amino groups and the solvent for reaction with the radioactive reagent, and the final extent of reaction of each amino group is proportional to the second order velocity constant for the acylation at the particular pH employed (7). The aim of the experiment was to examine changes in the microenvironments of the individ- ual amino groups that occur on Fe”+ binding to determine if the conformational change accompanying metal binding in- volves localized regions of the protein or the bulk of the molecule and, by implication, to obtain information about regions that may function in Fe”’ binding. As the transferrin polypeptide chain is large and contains 59 amino groups (the a-amino group and 58 lysine e N H a groups), we have used a dual labeling approach involving the use of a transferrin sample that is trace labeled with [I4C]acetic anhydride in the apo state as an internal standard. This procedure permits the ready identification of fragments containing residues that are perturbed on iron binding so as to avoid the necessity of purifying peptides containing every amino group in the protein molecule.

Using this approach, we have shown that the labeling of

9406

Differential Kinetic Labeling of Transferrin 9407

amino groups in four CNBr' fragments is perturbed on Fe binding: CNl (residues 500-678), CN2 (residues 110-256), CN7 (residues 257-309), and CN9 (residues 310-313). Further analysis has shown that only 14 amino groups show significant changes. Most of these groups are located in similar regions in the two domains. Similar changes were observed in a confirm- atory experiment in which samples of diferric and apotrans- ferrin were trace labeled with [:'HI- and ['4C]acetic anhydride, respectively. I t is suggested that only limited areas in the COOH-terminal half of each domain are involved in the conformational change on metal binding. The very large per- turbations in labeling of some amino groups together with other information suggest that histidines 207 and 535 and aspartates 292 or 297 and 627 or 632 may be directly involved in binding ferric ions.

EXPERIMENTAL PROCEDURES~

RESULTS

Trace Acetylation of Apotransferrin and Diferric Trans- ferrin with ["HjAcetic Anhydride-To determine the changes in reactivity of the amino groups resulting from Fez+ binding, equal amounts of ATf and FTf were acetylated with equal trace levels of high specific activity ["Hlacetic anhydride. The precise experimental conditions are described in the Mini- print. The ["Hlacetylated proteins were separated from other products by gel filtration with columns of Sephadex G-50 in 5% formic acid (data not shown). The incorporation of radio- activity in FTf was 72% of the level obtained with ATf. The extent of chemical modification determined from the specific activity of ATf and FTf (2.47 X 10' counts and 1.67 X 10' counts, respectively) was found to be 0.49 acetyl groups/ molecule of ATf and 0.37 acetyl groups/molecule of FTf. As 30% of the total acetic anhydride was incorporated into pro- tein, and acetylation a t this level is kinetic one, the 28% lower incorporation of label into FTf directly reflects changes in the reactivity of amino groups resulting from iron binding.

Preparation of I4C Apotransferrin as Internal Standard- ATf was acetylated with trace amounts of I4C acetic anhydride and used as internal standard. 15% of the tctal acetic anhy- dride was incorporated to give a specific activity of 7.99 X 1 0 counts/mg of ATf, which is equivalent to 0.86 amino groups modified per molecule. The I4C-labeled ATf (70 mg) was divided into two parts, and 6.6 mgs each of ["HlATf and ["HI FTf were added. An additional aliquot of unlabeled transferrin was added as a carrier. The "H/14C ratio of ATf and FTf was 23.5 and 17.8, respectively.

CNBr Fragmentation-The polypeptide chain of human transferrin contains nine methionyl residues (5) and conse- quently is cleaved with CNBr to give ten fragments. When cleavage is performed on human transferrin prior to reduction and alkylation, two groups of disulfide bonded fragments (CNA and CNB) and three cystine-free fragments (CN7, CN8, and CN9) are obtained. CNA, CNB, and CN7 (CNC) can be purified by gel filtration with Sephadex G-75, while CN8 and CN9 are obtained as a mixture (CND); CN9 contains a single

' The abbreviations used are: CNBr, cyanogen bromide; ATf, apo- transferrin; FTf, diferric transferrin; KPLC, reverse-phase liquid chro- matography.

Portions of this paper (including "Experimental Procedures," part of "Results," Figs. 2, 3, 6-24, 27-31, and 33, and Tables 11-IX) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-784, cite the authors, and include a check or money order for $16.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

residue of lysine, but CN8 is devoid of lysine (8). Following reduction and alkylation with iodoacetamide, CN1, CN2, CN3, CN4, and CNlO are obtained from CNA and can be purified by gel fitration with Sephadex G-75, while CNB gives CN5 and CN6, which are separated with Sephadex G-50. Since the low levels of acetylation used in this study do not affect the gel filtration behavior of the CNBr fragments, the procedure summarized in Fig. 1 was used to separate the CNBr fragments in the labeled ATf and FTf samples. The separation profiles are shown in Figs. 2-7 and the ,'rH/L4C ratios of the individual CNBr fragments are summarized in Table I. It can be seen that the .'H/l4C ratios of CN1, CN2, and CN7 from FTf are considerably lower than that of the original protein sample, suggesting that the corresponding regions of the polypeptide chain (see Fig. 1) contain lysines that are perturbed on Fe binding. The corresponding fragments from the control ATf sample were essentially unchanged. The "H/14C ratios of CN3, CN4, CN5, CN6, and CNlO from FTf suggest that these regions do not contain perturbed residues. The '"H/l4C ratio of CN9 is increased by a factor of 1.36, indicating an increase in reactivity of lysine 312.

The further analysis of CNBr fragments to give 'H/I4C ratios in individual amino groups was performed in two ways: 1) automatic sequencer analysis of the partially acetylated fragments with the determination of "H/'4C ratios in residues released a t different cycles, and 2) reacetylation of fragments with unlabeled acetic anhydride to give chemically uniform (completely N-acetylated) fragments as described under "Experimental Procedures" followed by proteolytic cleavage and peptide mapping. As shown in Figs. 9,21, and 25, peptides containing perturbed residues (lowered "H/I4C ratios) are immediately recognizable in column profiles of such digests of fragments from FTf, whereas the corresponding peptides from ATf were not perturbed (e.g. Figs. 10, 22, 26). In general, only peaks with reduced "H/14C ratios from digests of fragments of FTf were selected for further purification to homogeneity, apart from all peptides from CN7 and unperturbed residues identified in sequencer analyses. In a few cases, peptides with altered :'H/I4C ratios contain multiple residues of lysine; ratios in individual lysines in these peptides were determined by sequencer analysis. As there were differences in the treatment of individual CNBr fragments, they are discussed separately below.

CNI"CN1 from FTf was analyzed by sequencer analysis (Table 11) and by examination of peptides from chymotryptic and Staphylococcus aureus protease digests after reacetyla- tion (Figs. 8, 9). A column profile of a S. aureus protease digest of ATf is shown for comparison (Fig. 10). It should be noted that the S. aureus protease digestion was performed with CN1 from the initial experiment but proved unsatisfac- tory for identifying certain perturbed residues. The chymo- tryptic digestion was performed on a similar CNl sample obtained from a separate labeling experiment (see below). Two peaks from the chymotryptic digest of FTf CN1 showed decreased ratios (C2 and C5, Fig. 8). Further fractionation of these peaks by RPLC and gel filtration (Figs. 11-14) yielded a number of peptides with lowered "H/"C ratios: C2D, C5B, C5C, and C5D, which were identified by amino acid analysis as arising from residues 655-661, 622-630, 533-550, and 522- 532, respectively (Table 111). These peptides contained single residues of lysine, except for C5C, which contained lysines 534 and 545. Peptide C5C was therefore subjected to sequencer analysis (Table IV) to give separate isotope ratios for lysines 534 and 545. These results indicated that lysine 534 has a greatly decreased ratio (LO), but lysine 545 showed little perturbation. It should be noted that the content of both I4C and "H in the latter residue is low, and consequently the ratio

9408 Differential Kinetic Labeling of Transferrin

TRANSFERRIN , 1 :N;; Cleavage

CN A CN 8 CN C CN D

Reduction

c 75

Reduction 1 CAM C 50

CN 1 CN 2 CN 3 CN 4 CN 10 CN 5 CN 6 CN 7 CN 8 CN 9 (500-678) (110-256) (390-464) (314-382) (465-499) (27-109) (1-26) (257-309) (383-389) (310-313)

C SP C SP Th C SP Th SP-fh SA SA SA S A

FIG. 1. Scheme depicting the fragmentation procedures used in analysis of labeled human transferrin samples. The following abbreviations are used: CAM, carboxamidomethylation; C, chymotryptic digestion; SP, digestion with S. aureus protease; Th, thermolysin digestion; SA, automatic sequencer analysis.

I I

* u 2004 - I 1

e 150

CN 1 I .. p: CN2

I 100- m F ' 0

50- I e 0 I I I I +o

30 50 75 100 125 150 F R A C T I O N N O

FIG. 4. Separation of reduced, carboxamidomethylated FTf CNA (Fig. 2) by gel filtration with a column (2.5 X 95 cm) of Sephadex G-75 (SF), equilibrated and eluted with 5% (v/v) formic acid. Fractions of 3.0 ml were collected end 203~1 aliquots used for determination of radioactivity. ... ., :'H; -, I4C; -.-. , ALLYO nm.

has a high degree of uncertainty. Peak C10 also appeared to be somewhat decreased in "H/I4C ratio and was further puri- fied to give a peptide (ClOC) identified as residues 551-561 (Fig. 15, Table 111). As this region contains two residues of lysine, it was also analyzed by automatic Edman degradation (Table V). However, the two lysines in this case, residues 552 and 557, showed insignificant changes and are therefore con- sidered to be unperturbed.

From the S. aureus protease digest of FTf CN1, three peaks were identified with reduced ratios: G9, G10, and G12, while the corresponding digest of the control sample (ATQ gave uniform 3H/14C ratios in the column profile (Figs. 9, 10). G9, G10, and G12 from FTf CN1 were separately digested with thermolysin and refractionated (Figs. 16-20). Pure peptides corresponding to residues 505-512 (G9Th3), 565-572 (G9Th4A), and 637-639 (G12Th2) were isolated (see Table I11 for amino acid compositions and ratios). In addition, the digests contained peptides with highly perturbed 'H/I4C ra- tios, (e.g. GlOTh2 and G12Th3). Amino acid analysis indicated the presence of small amounts of impure peptide in these

; 200 1 ! ! C N 2 . . . . - : : :: i . . . . . . .

f) I ""1 '% 5 "'1 50

. . _ i : : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

: : : :

I I I 1 30 50 75 loo 125 150

FRACTION NO

FIG. 5. Separation of reduced, carboxamidomethylated ATf CNA (Fig. 3) by gel filtration with a column (2.5 X 95 cm) of Sephadex G-75. Other details are the same as for Fig. 4.

TABLE I 'H/"C ratios of CNBr fraRments

Fragment "H/W ratio

. -

Protection ATf FTf factor R

Before fragmentation 23.5 CN1 21.2 CN2 21.9 CN3 20.7 CN4 18.5 CN5 22.1 CN6 25.3 CN7 24.3 CN9 23.2 CNlO 23.6

17.8 10.8 14.0 22.5 21.1 21.4 21.7 14.4 30.1 20.0

1.32 2.17 1.67 1.04 1.11 1.09 1.08 1.63 0.77 1.17

pools; the composition of G12Th3 was consistent with that of an impure form of residues 530-537. However, after further purification by gel filtration and RPLC with buffers of differ- ent pH, insufficient material was recovered for identification. Based on the results of the chymotryptic digest, it appears likely that these pools (G9, G10, and G12) contained multiple forms of a peptide derived from the region around lysine 534.

Differential Kinetic Labeling of Transferrin 9409

r-

l Z 0 1

1000

zu

x L 400 Y

zoo

0

0 c

I I I 0 40 60 120 l i e 200

F R A C T I O N NO

FIG. 25. Separation of a thermolysin digest of acetylated FI'f CN7 (Fig. 2, fraction C ) by RPLC as described under "Experimental Procedures." Fractions of 2.0 ml were collected and aliquots of 100 p1 taken for radioactivity measurements. . . . . , "H counts; -, "C counts.

In summary, the combined results from two proteolytic digests and a sequencer analysis show that the following lysines in CN1 are perturbed on iron binding: residues 511, 527, 534, 545, 568, 626, 639, and 656 (see Table 111).

CN2"Perturbed residues in FTf CN2 were identified by sequencer analysis (Table VI) and by examination of a S. aureus protease digest of the reacetylated peptide (Fig. 21). Two peaks in the chromatographic separation of this digest showed changed ratios, G8, which was markedly reduced, and G10, which was slightiy increased. In contrast, the correspond- ing digest of ATf contained no perturbed peaks (Fig. 22). FTf CN2 G8, and G10 were further purified (Fig. 23, 24) to give peptides corresponding to residues 202-212 (lysine 206) and residues 238-240 (lysine 239). The compositions of these pep- tides and their corresponding ratios are given in Table VII.

CN7-FTf CN7 was examined by sequencer analysis (Table VIII) and by peptide mapping of a thermolysin digest of the reacetylated peptide using RPLC (Fig. 25). Sequencer analysis indicated a significantly decreased 'H/I4C ratio in lysine 276. From the thermolysin digest of FTf CN7, five perturbed peptides were identified (Th3, Th8, ThlO, T h l l , and Th12) while in the similar separation profile of the corresponding digest of ATf (Fig. 26) the corresponding peptides had 'H/I4C ratios similar to that of the initial protein sample (23.5). Amino acid analysis showed that peptides Th3 (residues 295- 298) and T h l l (294-301) contain the same lysine residue (296) while Th8 (residues 285-292) and Th12 (284-292) both contain lysine 291. Th6 contained three r5sidues of lysine (276, 278, 280) and was further analyzed by sequencer analysis (Table IX) to show that, of these, only lysine 276 is perturbed. The amino acid compositions and 'H/I4C ratios of these peptides are summarized in Table VII.

CN3, CN4, CN5, CN6, and CNlO-The :'H/'4C ratios of these fragments (Table I) suggest that they do not contain residues that are affected by Fe binding. However, since it is

possible that residues with compensatory perturbations may be present in the same fragment (i.e. residues with increased and decreased ratios), the CNBr fragments from FTf were reacetylated with unlabeled acetic anhydride and digested with appropriate proteases (chosen to give as many lysines as possible in separate peptides, based on the known sequence). The column profiies of these digests are shown in Figs. 27-31. Perturbed peaks were absent from all digests, confirming that these regions are devoid of perturbed residues. A sample of CN5 was also subjected to sequencer analysis (without re- acetylation) to confirm that lysines 27,41, and 42 were unper- turbed.

Reproducibility ofperturbations-To check that the obser- vations are reproducible, the trace labeling of FTf and ATf was repeated. However, since the 'H/I4C ratios in the ATf sample in the f i s t experiment were uniform, the labeling of ATf with ["Hlacetic anhydride was not repeated. Equal sam- ples (70 mg in 5 milliliters of 0.1 M Tris buffer, pH 8.0) of FTf and ATf were reacted with ["]acetic anhydride (0.7 pmoles, 8.7 Ci/mmol) and [I4C]acetic anhydride (4.3 pmol, 115 mCi/ mmol), respectively. Extents of modification determined from the incorporation of label were 0.09 acetyl groups/molecule for FTf (3H label) and 0.16 acetyl groups/molecule for ATf (I4C label). The two samples were mixed to give a 'H/I4C ratio of 24.4 and subjected to CNBr cleavage and separation as described in the prior study. Essentially identical separations of CNBr fragments were obtained (data not shown) but CNB (i.e. CN5 plus CN6) was not subjected to reduction, alkylation, and reseparation. 'H/I4C ratios in the CNBr fragments were as follows: CN1, 13.4; CN2, 17.3; CN3, 25.2; CN4, 24.9; CN10, 19.6; CNB, 19.8; CN7, 16.5; CND (CN8 and CN9) 36.G. Thus, as in the first experiment, major decreases in 'H/I4C ratios were present in CN1, CN2, and CN7 while CN9 (since CN8 is devoid of lysine) shows an increased ratio. The sample of CN1 was subjected to chymotryptic digestion and separation as described above, since analysis of this region by digestion with

1200-

1000-

tu E e 800

,x 600

r ' 0

v-

I- .3 n 1

m E

0 40 60 120 160 zoo F R A C T I O N NO

. : . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I I

. . I

i : i : / j

/ j . . . . i [ / I

. . : : i

ATf CN7 (Fig. 3, fraction C) by RPLC. The details are as for Fig. FIG. 26. Separation of a thermolysin digest of acetylated

25.

9410 Differential Kinetic Labeling of Transferrin

S. aureus protease proved unsatisfactory. CN2 and CN7 were used to confirm the perturbations observed in the initial experiment. CN2 was reacetylated with unlabeled acetic an- hydride, digested with chymotrypsin, and separated by ion exchange chromatography with DEAE-cellulose, as described. Six major peaks were obtained of which one had a reduced "H/'4C ratio. The perturbed peptide was purified by HPLC and identified as residues 205-211 (sequence: Val-Lys-His- Ser-Thr-Ile-Phe, composition Thr, 0.96; Ser, 1.07; Val, 0.88; Ile, 0.93; Phe, 0.95; His, 1.11; Lys, 0.90), produced by expected chymotryptic cleavages at Phe 204 and Phe 211. The ,'JH/'4C ratio in this peptide was 0.75. Sequencer analysis confirmed the expected sequence in this region (data not included).

CN7 was also reacetylated, hydrolyzed with thermolysin, and the digest fractionated by RPLC. Ratios in the peaks corresponding to Th3, Th6, and Th7 were 1.66, 15.5. and 10.6, in agreement with the results from the initial experiment. The relatively high ratio in CND (presumably reflecting the pres- ence of lysine 312 of CN9) is also consistent with the results of the first experiment (FTf ratio 28.9, ATf ratio 21.2, an increase of 1.36 in reactivity on Fe b'inding).

DISCUSSION

The differential kinetic labeling technique has proved useful for identifying regions of proteins in a number of different systems that undergo changes in reactivity as a result of protein-ligand, protein-protein, and protein-RNA interactions (9-11). In the most widely used procedure, samples of protein in two states (e.g. free and complexed states) have been trace- labeled with ["Hlacetic anhydride and the labeling patterns in the two samples compared by reacetylating with unlabeled reagent to produce a chemically uniform derivative, mixing with a sample of the same protein that has been completely acetylated with [I4C]acetic anhydride under denaturing con- ditions, to give an appropriate "H/"C ratio followed by cleav- age to give peptides containing preferably single, (but in some cases, multiple) labeled residues, that can be purified, identi- fied, and whose relative reactivity will be apparent from the "H/14C ratio. Comparison of this ratio for every amino group in the two samples will then identify residues that have undergone changes (e.g. see Ref. 9). Because of the large size of the polypeptide chain of transferrin and multiplicity of amino groups (59), this approach would be extremely time- consuming. However, by using a sample of ATf that had been trace-acetylated with [ 14C]acetic anhydride as an internal standard and performing an initial cleavage with CNBr and separation of the 10 fragments prior to reacetylation we ob- tained 2 main advantages: 1) as trace acetylation did not affect the gel filtration procedures developed for isolating these fragments from the unmodified protein, and 2) peptides de- rived from regions whose reactivities were modified by Fe- binding become immediately apparent in column separations by changed "H/'4C ratios in the sample that was acetylated in the diferric state. These perturbed regions are apparent both at the CNBr fragment level (CN1, CN2, CN7) as well as in peptides derived from these regions. In contrast, the sample that was ["Hlacetylated in the apo state did not show these pertubations. The absence of multiple residues with compen- sating (2.e. both positive and negative) perturbations in CNBr fragments showing no overall change in ratio (CN3, CN4, CN5, CN6, and CNlO is readily confirmed by the uniform "JH/ 14C ratios in column profiles of peptides from proteolytic digests of these fragments (Figs. 27-31). Reproducibility of the changed labeling pattern at the level of some peptides has been demonstrated.

Previous workers have investigated the effects of chemical modification of amino and other groups on the Fe-binding

properties of transferrin. Thus, modification of a large pro- portion of the amino groups of apotransferrin had only slight effects on the Fe-binding properties (12, 13); chemical modi- fication of tyrosines by iodination and acetylation with N- acetylimidazole (14-16) and of histidines by ethoxyformyla- tion (17, 18) suggested that two to three tyrosines and two histidines are involved in Fe"' binding at each site. A review of chemical and physical evidence leaves a wider range of uncertainty in the conclusion that one or two histidines and two or three tyrosines are involved in Fe binding together with an arginyl residue, which probably functions as a binding site for the synergistic anion (19). An unknown protein func- tional group or groups may also coordinate with the metal ion (see Ref. 2).

Although the €-amino groups of lysyl residues are not di- rectly involved in metal binding, our data show that changes in the microenvironments of these groups, as determined by rates of acetylation, are a sensitive index of the conformational changes that occur on metal binding. Fe binding to the two sites of transferrin affects the reactivities of only 14 out of 59 amino groups; the quantitative changes are summarized in Table X and displayed graphically in Fig. 32 in relation to the complete amino acid sequence of human tranferrin which is given in Fig. 33. The sequence is arranged to show the presence of two homologous metal-binding domains, containing 404, identical residues (5). It is immediately apparent that the regions of the molecule perturbed by iron binding are localized in the COOH-terminal half of each domain (residues 180-336, 510-678). More residues in the COOH-terminal domain are affected than in the NHr-terminal domain (8 as compared to 6).

The greatest changes in reactivity are observed in lysines 206 and 534, which are in precisely corresponding positions in the two domains (see Figs. 32 and 33). The magnitude of these changes suggests that these lysines are very close to the metal- binding site in each domain. I t is, therefore, of considerable interest that the histidyl residue adjacent to each of these lysines (histidines 207 and 535) is one of only three histidyl residues that are invariant in both domains of human trans- ferrin and are also preserved in chicken ovotransferrin (resi- dues 210, 542) (see Refs. 6 and 20). We conclude, therefore, that histidines 207 and 535 probably provide a nitrogen ligand to the bound ferric iron in the NH2-terminal and COOH- terminal domains ( 9 , respectively, of human transferrin. An- other conserved histidine (119 and 451) is separated from a pair of lysines in each domain (residues 115 and 116, 447 and

TABLE X .'H/''C ratio and protection factor ofperturbed residues in

transferrin Lvsine resi- due No.W

206 239 276, 278, 280 276

29 1 296 312 511

527 534,545 534 545 568 626 639 656

Source of data ,H,lIC ratio Protection factor K

CN2G8C, Fig. 23 0.98 23.9 CNZGlOB, Fig. 24 33.4 0.70 CN7Th6, Fig. 25 16.9 CN7Th6, Table IX 9.47 2.48 CN7, Table VI11 9.4 CN7Th8, Fig. 25 8.44 2.78 CN7Th3, Fig. 25 2.37 9.91 CN9, Fig. 2 30.1 0.77 CNlGSTh3, Fig. 17 8.1 2.90 CN1, Table I1 8.8 CNlC5DP2, Fig. 14 9.23 2.54 CNlCsC, Fig. 13 1.61 CNlC5C, Table IV 1 .o 23.5 CNlC5C, Table IV 14.5 1.62 CNIG9Th4A, Fig. I8 13.2 1.78 CNlC5B, Fig. 12 4.51 5.21 CNlGIZTh2, Fig. 20 11.2 2.09 CNlCLD, Fig. 11 6.83 3.44

Differential Kinetic Labeling of Transierrin 9411

24

16

+ 50 100 250 300 336

2 4-

20-

16-

R 1 2 -

8 7

4-

RESIDUE NUMBER

FIG. 32. A graphical display of the effects of iron binding on the rates of acetylation of individual amino groups in trans- ferrin in relation to their positions in the sequence (Fig. 33). R denotes the protection factor for each residue (rate of acetylation in ATf/rate of acetylation in FTO. Each line represents a domain of transferrin. Gaps have been placed to align corresponding residues in the domains.

448) by only two amino acids, yet these lysines are clearly unperturbed on iron-binding and these histidines, therefore, are less likely to be directly involved in metal binding.

A second focus of perturbation in each domain is in the corresponding sections: 291-296 and 626-639. In the amino terminal domain, residue 296 is changed in reactivity by 9.9 and 291 by a factor of 2.7. In the COOH-terminal domain, the equivalent residue to 296 is arginine 631, however, the closest observable residue, lysine 626, undergoes a 5.2-fold change in reactivity. Although this area is the main focus of perturba- tion, residues around this region in both domains are also affected by Fe binding, i e . residue 276 (decreased) and 312 (increased) in the NH2-terminal domain and residues 639 and 648 (both decreased in reactivity) in the COOH-terminal domain. Therefore, although a region encompassing residues 291-296 and 626-639 is the main site of change, minor confor- mational affects appear to occur in a more widespread area. A similar conformational adjustment appears to extend around residue 534, although the effect in the NH2-terminal domain is localized in residue 206 alone. The apparently greater con-

formational change in Fe binding at the COOH-terminal site may be related to the greater stability of this site to acid denaturation (see Ref. 2).

Although the pertubations at residues 206 and 534 are consistent with the involvement of the adjacent histidines in iron binding, the basis of the effects in the second region of each domain is less obvious. A!though the sequences of resi- dues corresponding to 291-297 and 626-632 are strongly con- served in both domains of the chicken as well as human transferrin, no residues that are thought to function in iron binding are present in this region (i.e. histidines or tyrosines). However, given the uncertainties in the numbers of these residues that ligand with the ferric ion (see Ref. 2), there may be one or two as yet unidentified protein groups that contrib- ute to metal binding. Inspection of this region suggests that aspartates 291 and 297,627 and 632 are possible candidates as iron liganding residues, through the P-COZH groups. Studies are currently in progress to determine if any of these groups function in this way.

Acknowledgment-We wish to thank Vera Ondricek for her skilled technical assistance.

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2. Aisen, P., and Listowsky, I. (1980) Annu. Reu. Biochern. 49,357-

3. Williams, J . (1974) Biochem. J. 141, 745-752 4. Azari, P., and Feeney, R. E. (1958) J . Biol. Chem. 232, 293-302 5. MacGillivray, R. T. A,, Mendez, E., Sinha, S. K., Sutton, M. R.,

Lineback-Zins, J., and Brew, K. (1982) Proc. Natl. Acad. Sci.

6. Jeltsch, J . M., and Chambon, P. (1982) Eur. J. Biochem. 122, 291-295

7. Kaplan, H., Stevenson, K. .J., and Hartley, B. S. (1971) Biochem. J. 124, 289-299

8. MacGillivray, R. T. A., Mendez, E., and Brew, K. (1977) in Proteins oflron Metabolism (Brown, E. B., Aisen, P., Fielding, J., and Crichton, R. R., eds) pp. 133-141, Grune and Stratton, New York

9. Richardson, R. H., and Brew, K. (1980) J. B i d . Chem. 255,3377- 3385

10. Bosshard, H. R., Koch, G. L. E., and Hart,ley, B. S. (1978) J . Mol. Biol. 119, 377-389

11. Rieder, R., and Bosshard, H. R. (1978) J . Biol. Chem. 253, 6045- 6053

12. Zschocke, R. H., Chiao, M. T., and Bezkorovainy, A. (1972) Eur. J . Biochem. 27, 145-152

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15. Line, W. F., Grohlich, D., and Bezkorovainy. A. (1967) Biochem-

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17. Aasa, R., Malmstrom, B. G., Saltman, P., and Vanngard, T. (1963)

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20. Williams, J., Elleman, T. C., Kingston, B., Wilkins, A. G., and

21. Lineback-Zins, J., and Brew, K. (1980) J. Biol. Chem. 255, 708-

22. Hunkapiler, M. W., and Hood, L. E. (1978) Biochemistry 17,

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4,998-1005

1141

istry 6, 3393-3402

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Chem. 252,8108-8112

istry 17, 1105-1109

Kuhn, K. A. (1982) Eur. J. Biochem. 122, 297-303

713

2124-2133

Differential Kinetic Labeling of Transferrin

Differential Kinetic Labeling of Transferrin 9413

f"

: " 8 0 j

i I

i i i

100

8.8

7 2 0 7

4 0 F R A C T I O N N O

i

F R A C T I O N N O

c

- 2 0 4 0 60 8 0 100

FRACTION NO

-7 CN I

14 . 5 0 i., 75 1 60

" a y o ' O O L 0

4" 0 20 40

FRACTION N O

94 14 Differential Kinetic Labeling of Transferrin TABLE IY

sequencer a n a l y i ~ s a i F l f C N l C I C

B~ 200-

I 120

FRACTION NO

19.9 19.2

150i

1 0 r

i

FRACTION NO

ALA

VAL

1.03 1 1 1

2.08 1.56 I21 I 2 1

1.01 ".97 0 . 9 1 I I i ( I 1 1 1 1

0.88 1.11 I 1 1 I21

Differential Kinetic Labeling of Transferrin

FRACTION NO

9415