Metal-binding properties of a single-sited transferrin fragment

Metal-binding properties of a single-sited transferrin fragment

490 Btochtmtca etBiophystcaActa, 742 (1983) 490-495 ElsevlerBmmed~cal Press BBA31480 METAL-BINDING PROPERTIES OF A SINGLE-SITED TRANSFERRIN FRAGMEN...

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490

Btochtmtca etBiophystcaActa, 742 (1983) 490-495 ElsevlerBmmed~cal Press

BBA31480

METAL-BINDING PROPERTIES OF A SINGLE-SITED TRANSFERRIN FRAGMENT OLGA ZAK, ADELA LEIBMAN t and PHILIP AISEN *

Departments of Phystology and Btophyslcs, and Medwme, Albert Emstem College of Medwme, Bronx, NY 10461 (U S A ) (Recewed September 20th, 1982)

Key words Transferrm, Metal bmdmg~ Smgle-slte fragment

A single-sited iron-binding fragment of transferrin, prepared by proteolytic cleavage with thermolysin, has been characterized. The fragment, bearing no carbohydrate, must be derived from the N-terminal half of the protein's two homologous domains. The strength of iron-binding at pH 6.7 and 7.4, and the EPR spectroscopic features of its iron and copper complexes, establish it as carying the 'b' site of transferrin. Introduction The two sites of human serum transferrln differ in their chermcal and spectroscopic properties [1] However, these differences are not reflected in physiological differences between the sites when the protein functions as an iron donor in VlVO [2] Since extensive internal homology in the primary sequence of transfemn offers compelhng proof that the modern two-sited protein arose from duphcation and fusion of a pnmatlve gene specifying a single-sited protein [3], it is pertinent to ask whether the Iron-binding properties of a proteolytlcally generated single-sited transferrm fragment are unchanged from the properties of the corresponding site in the native molecule To study flus question we have turned to the procedure of Lineback-Zins and Brew [4] to Isolate a fragment containing only the site in the N-terminal domain of human transferrm.

Experimental procedures Protems Human serum transferrln was isolated from Cohn fraction IV-7, or from a 40-60% am1" Deceased * To whom correspondence should be addressed Abbrevmtmns Mops, 4-morphollnepropanesulfomc acid, Hepes, 4-(2-hydroxyethyl)- l-plperazaneethanesulfomcacid 0167-4838/83/0000-0000/$03 00 © 1983 Elsevter Bmmedtcal Press

monlum sulfate fraction of pooled bank plasma, by procedures previously described [5]. Chelateand iron-free preparations were also obtained according to standard methods in the hterature [5,6]. The single-sited N-ternunal fragment was prepared by thermolysln dlgesuon of iron-saturated transfernn largely following the techniques of Lineback-Zlns and Brew [4]. For some preparations, thermolysln (from Calblochem Corp.) was bound to cyanogen brormde-actlvated Sepharose according to the method of David and Reisfeld [7] No difference was observed in preparations obtained using free enzyme or tmmoblhzed enzyme Fragments were isolated from the digestion mixtures by gel permeation chromatography using 2.5 × 90 cm columns of Sephadex G-75 Superfine Since, as pointed out by Lmeback-Zms and Brew, the fragments consisted of several components revealed by SDS-polyacrylamlde gel electrophoresis, further purification was attempted by DEAE-Sepharose chromatography At least three, and sometimes four, fractions were obtained with a 1 5 × 90 cm column initially equilibrated with 0 05 M Tns-HC1 buffer at pH 8 0, and eluted with a 0 05 M-0.2 M linear gradient of the same buffer. Studies utilized the first peak elutlng from the DEAE column. Preparations were freed of iron by procedures similar to those used m obtainmg apotransferrm,

491

except that 0.1 M N a N O 3 was substituted for water following dialysis against citrate/acetate buffer. Exposure of fragment preparations to low lOmC strength often led to ~rreverslble preclp~tatmn. Equthbrmm dmlysts Procedures for this were similar to those used for characterizing the ironbinding properties of holotransferrln [10]. For studies near pH 6 7, 8-16 mM Mops was included m the preparations to stabdlze pH Thermodynamic constants for the binding of iron by slngles~ted fragments could be directly calculated at the p C O 2 of room air and the pH values of each cell, using the binding equatmns of Ref. 10. Effectwe stablhty constants at pH 6.7 and 7.4 were derived from these Other procedures Iron-binding capaoty was evaluated by spectrophotometnc titration at 470 nm using freshly prepared ferrous ammonium sulate as titrant. For spectroscopic stuches, preparations were loaded with iron as the 1:2 Fe(III)-mtrflotnacetate chelate [8], or with copper as 15 mM 65Cu in 0.1 N HC1 [9]. To ensure occupancy of the specific anion-binding site by bicarbonate a 50-100-fold excess of tlus anion was also present. Electrophoretic studies in 10% polyacrylamlde/ 1% SDS gels followed the techniques of Malzel [11], using molecular weight standards from Pharmacm Fine Chemicals. Optical spectra were recorded on a Cary model 14 recording spectrophotometer. EPR spectra were obtmned with a Vanan E-9 instrument, operating at 9 GHz, using a liquid nitrogen insert Dewar and preoslon sample tubes (Wdmad) Digital processing was accom-

phshed with a FabrlTek model 1070 signal averaging computer. Carbohydrate analyses were carried out by gas chromatography by the procedures of Blumenfeld and Adamany [12] using the PerlonElmer Model 910 gas chromatograph. Results and Discussion

Characterlzatton of thermolysm fragments In agreement w~th the original report by LlnebackZms and Brew [4], the fragment separated from thermolysln-cleaved transferrin by gel permeation chromatography typically contained one dormnant speoes with a molecular weight near 35 000. Further fractlonatlon by DEAE-Sepharose chromatography gave three or four pink fractions. The first of these, peak I, consisted of a major component of M r 34900 + 1 000 (n = 6), accounting for over 85% of the total protein, and manor components of slightly lower molecular weight No bands corresponding m moblhty to free thermolysln were seen m any preparations. A 1% solution of peak I, as estimated by the method of Lowry et al. [24] using apotransferrln for a standard, was found to have A ~ cm2s0= 10.2. Smaller components, presumably representing more highly digested fragments, tended to predomanate m other peaks so subsequent stu&es were carried out on peak I Determlnatmn of the equivalent weight for Iron binding of peak I, using freshly prepared ferrous ammonium sulfate as tltrant, yielded sharp end point values of 32900 and 33 200, respectwely Use of ferric nltrflotnacetate as tltrant generally

TABLE I E Q U I L I B R I U M B I N D I N G OF Fe 3+ TO T R A N S F E R R I N F R A G M E N T NEAR pH 7 4 The concentration of fragment was near 3 5 10 -5 M, and that of citrate was 0 03 M, m all cells Experiment

A B C

Total [Fe 3+ ] at eqmhbrlum (/~M) Protein

Buffer

15 5 595 127

11 3 439 107

pH

[Fe 3+ ]/[fragment]

-log[Fe)q ]

K

7 35 737 7 39

0 12 044 0 60

204 198 19 5

148 187 130 155±168 a

a Mean + S E

492 T A B L E II E Q U I L I B R I U M B I N D I N G O F Fe 3+ TO T R A N S F E R R I N F R A G M E N T pH 6 7 The concentratmn of citrate was near 1 5 m M in all experiments Experiments

A B C D E

[Fragment] (# M)

51 3 37 1 355 69 5 37 3

Total [Fe 3+ ] at e q m h b n u m ( # M) Fragment

Buffer

47 1 96 5 643 137 82 5

34 8 85 6 462 97 9 60 7

pH

[Fe3 + ] bound/[fragment]

6 69 6 63 679 6 72 6 85

0 24 0 30 051 0 56 0 58

-

log[Fea3q+ ]

17 7 17 3 17 7 17 2 17 6

r

32 0 248 41 4 30 1 26 2 309+29 a

a Mean 5: S E

failed to gtve sharp end points, presumably because of competition between chelator and transferrm for iron For studies of the iron-binding strength of the thermolysln fragment, 33 000 was taken for the equivalent iron-binding weight. Neither the components of DEAE peak I nor the cruder fractmn obtained by gel permeation chromatography contained detectable carbohydrate. From the studies of Evans and Wdhams [13] and of Brew and associates [3], demonstratlng that both carbohydrate chains are disposed in the C-terminal half of transferrln, the thermolysln fragments must be derived from the N-terrmnal domain of the transferrm molecule, confirnung results of Lmeback-Zms and Brew [4]. The strength of tron binding. The thermodynamic association constants for the reaction Fe 3+ + fragment" + HCO 3 ---) (Fe 3+ - f r a g m e n t - H C O 3 ) " - i

+

3H+,

evaluated near pH 6.7 and near pH 7.4 by the method of equihbnum dialysis using citrate as a competing complexmg agent [10], are presented in Tables I and II. From these constants, conditional or effective constants for the binding of iron to the fragment at each p H and at the p C O 2 of air, were calculated [10]. Results of these calculations are presented In Table III, along with corresponding constants for the binding of iron to each site of

native transferrm (from Ref. 10). At pH 7.4 the effective binding constant for the fragment corresponds closely to the constant for the 'b' site of holotransfernn and is nearly an order magnitude less than that of the 'a' site. At pH 6.7, the effective constant for the fragment satisfies the limiting condition for the 'b' site, but is two orders of magnitude less than that of 'a' site. On this basis, therefore, the 'b' site may be assigned to the N-terminal fragment of transferrin, in agreement with previous inferences based on the work of Evans and Wdhams [13] and Fneden and Aisen [14]. In their studies of the thermolysin fragment, Lineback-Zms and Brew suggested that 1t represented the acid-stable site of transferrm because of ItS capacity to bind iron at pH 6 or below [4]. The aod-lablle site, however, is charactenzed by loss of Iron at low pH, particularly when a competing iron-binding agent is present [15,16]. In the abT A B L E III EFFECTIVE STABILITY C O N S T A N T S F O R T H E BINDI N G O F I R O N IN A I R Data on t r a n s f e m n sites are from Ref 10

Fragment Transfernn 'a' site Transfernn ' b ' site

pH 6 7 (M - I )

pH 7 4 (M - t )

1 8 1017 2 9 1019 < 1 4 1018

5 7 1019 4 0 1020 6 8 1019

493 05 04 Z03

~

02

-~

.

01 350

.~. 400

450 500 550 WAVELENGTH (am)

600

650

Fig 1 Optical spectrum of dlfemc transferrm ( ) and thermolysm-cleaved N-ternunal fragment ( - - - - - - ) , each at an iron concentratmn of 9 7 10 -5 M

sence of such an agent, we have observed both sites of dlfemc transferrin to retam iron m acetate buffer at pH 5.7 at least for several hours, and at p H 6.0 for several days (unpublished data), findrags m reasonable accord with the data of Lestas [16]. Thus, the stability of the ferric complex of the thermolysm fragment at pH 6 does not necessarily ~mply that it is the acid-stable site. As m bovane serum transferrln [17], the optical

Fe2 T/'ans/'errtn

I

I

I

]

I

I

I

1300

1400

1500

1600

1700

1800

1901

GAUSS

Fig 2 EPR spectra of (A) dffernc transfernn at a total ~ron concentration of 8 5 l0 -4 M (4 25 l0 -4 M m protein) and (B) thermolysm-cleaved fragment at an iron concentration of 4 25 10 - 4 M Spectrum C, representing the 'a' site m the C-terminal domain, is obtaaned by &gltally subtracting spectrum B from spectrum A Buffer, 0 1 M H e p e s / 0 05 M KCI, pH 7 4 Microwave power, 25 mW, macrowave frequency, 9 155 GHz, modulation, l0 G at 100 KHz, temperature, 77 K

properties of natwe human dlfernc transferrm and the iron-loaded fragment, each at the same concentrauon of iron, are similar but not idenUcal (Fig. 1) More stnlong &fferences are apparent between the EPR spectra of the fragment and the &ferric native molecule (Figs 2A, B). The spectrum of the 'a' site (F~g. 2C) was obtained by &gltal subtraction of the spectrum of the fragment from the spectrum of &fernc transferrln at the same molar concentration of protein, or twice the concentration of iron. Although tins procedure rests on the assumption that the s~te m the fragment is structurally intact, the assumption seems reasonable m the hght of the eqmhbrium dialysis measurements and the optical stu&es. In agreement w~th stu&es with native transfern n in wluch, as judged by Makey-Seal electrophoresis [18] one or the other site is preferentially occupied [10], the sphttmg of 27 gauss between the two low-field peaks of the g' = 4.3 signal originating from the 'b' site complex, Is narrower than the corresponding sphttlng of 41 gauss m the 'a' site spectrum (Fig. 2). More strikingly, the amsotropy is appreoably greater m the 'b' site spectrum so that the signal amphtudes are only about half of those in the 'a' site spectrum. Thus, prewous analyses pointing to a difference between the m&wdual site EPR spectra of human serum transfern n are corroborated [10,19]. In related studies, differences between the EPR spectra of the two sites of ctucken ovotransferrm have also been established [20] Addmonal evidence that the fragment contains the 'b' or acid-labile site emanates from the EPR spectrum of its complex with 6SCu2+. The spectrum of a complex of the fragment with 6SCu, and oxalate as the obhgate anion (Fig. 3A), closely resembles that of a monocuprlc complex of holotransfemn with oxalate as the reqmred amon [9]. In a 'hybrid' 65Cu-complex of the fragment, in the presence of both oxalates, the low field, M ~ - ~3 hyperfine hne shows a sphttmg into five components in the presence of oxalate (Fig. 3B). These arise from the superposxtlon of spectra from the ternary complex of Cu 2+ , fragment and oxalate, and the ternary complex of Cu 2÷ , fragment and carbonate [9]. The lughest filed component of the oxalate complex almost exactly coincides with the lowest field component of the carbonate complex,

494 r - - - T - - - q carbonate oxalate[

L 2600

2800

, 3000 GAUSS

f 3200

B

I

I

I

I

2560

2580 GAUSS

l 2600

65Cu, thermolysm fragment and oxalate, prepared as described m Ref 9 The concentraUonof fragment is 2 3 l0 -4 M, that of copper is 1 7 10-4 M, and that of oxalate 7 8 l0 -3 M Buffer, 0 l Hepes, pH 8 8 Microwave power, 20 mW, microwave frequency, 9 139 GHz, modulauon, 5 G at 100 KHz, temperature, 77 K B The low-field hyperfme hne m the EPR spectrum of the Cu complex of the thermolysm fragment with oxalate and carbonate present In&cated superhyperfme sphttmgs are calculated from the data m Ref 9 The concentration of fragment Is 3 4 l0 - 4 M, that of copper ts 2 6 l0 -4 M, and that oxalate Is 7 6 10-4 M Bicarbonate is present at ambient concentration Buffer, 0 05 M Hepes/0 1 M KCI, pH 8 4 Microwavepower, 10 mW, rmcrowave frequency, 9 142 GHz, modulauon, 4 G, at 100 KHz, temperature 77 K Fig 3A The spectrum of a ternary complex of

to produce a five-hne p a t t e r n where the rmddle c o m p o n e n t represents the superimposition of two hnes As only the ' b ' site wdl accept oxalate as the obligated a n i o n when Cu 2÷ ~s the specifically b o u n d metal ion, the assignment of the ' b ' site to the N - t e r m i n a l d o m a i n of the fragment is verified On the two-sztedness of transferrm. The present studies l n & c a t e that the i r o n - b i n d i n g properties of the ' b ' site in holotransferrln are well preserved m the ' h a l f - t r a n s f e r r m ' fragment c o n t a i n i n g only the ' b ' sxte. Thus, ~t is unlikely that the overall p r o t e i n structure exerts great influence o n the local b i n d m g sites It m a y be, therefore, that the sole biolog~cal advantage conferred b y d o u b h n g the size a n d n u m b e r of b i n d i n g sites of transferrm d u n n g the course of biochemical e v o l u u o n [3] is to achieve a sufficient size to avoid glomerular filtration a n d u n n a r y loss of the o r c u l a t m g protein [21]. However, the two sites of t r a n s f e r n n are mequlvalent, i n d e p e n d e n t , a n d u n e q u a l l y occupied by tron in the o r c u l a U o n [10,22,23]. The posslbdlty still remains, therefore, that the f u n c t i o n of transferrm tn regulating the flow of iron a m o n g sites of storage, utlhzaUon, a n d a b s o r p t i o n exploits the differences wtuch exast between the sites

Acknowledgements This work was supported, in part, by G r a n t A M 15056 from the N a t i o n a l Institutes of Health. W e are grateful to Dr. A.M. A d a m a n y for carrying out the c a r b o h y d r a t e analyses.

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495 10 Alsen, P, Lelbman, A and Zweler, J (1978) J Blol Chem 253, 1930-1937 11 Maazel, J V , Jr (1971) Methods Vlrol 5, 179-246 12 Blumenfeld, O O and Adamany, A M (1978) Proc Natl Acad Scl U S A 75, 2727-2731 13 Evans, R W and Wllhams, J (1978) Blochem J 173, 543-552 14 Fneden, E and Alsen, P (1980) Trends Blochem Scl 49, XI 15 Prmclotto, J V and Zapolskl, E J (1975) Nature 255, 87-88 16 Lestas, A N (1976)Br J Haematol 32, 341-350 17 Brock, J H and Arzabe, F R (1976) FEBS Lett 69, 63-66

18 Makey, D G and Seal, U S (1976) Bloclum Blophys Acta 453, 250-256 19 Aasa, R (1972)Blochem Biophys Res Commun 49, 806-812 20 Keung, W - M , Azan, P and Plulhps, J L (1982) J Blol Chem 257, 1177-1183 21 Wdhams, J , Grace, S A and Wllhams, J F (1982) Blochem J 201,417-419 22 Lelbman, A and Alsen, P (1979) Blood 53, 1058-1065 23 Wdhams, J and Moreton, K (1980) Biochem J 185, 483-488 24 Lowry, O H , Rosebrough, N J , Farr, A L and Randall, R J (1951) J Blol Chem 193, 265-275