Circular dichroism of iron, copper, and zinc complexes of transferrin

ARCHIVES

OF

B[OCHEMISTRY

Circular

AND

148, 27-36 (1972)

BIOPHYSICS

Dichroism

of iron,

Copper,

and

Zinc

Complexes

of Transferrin’ B. NAGY2 Department

of Muscle

S. S. LEHRER

AND

Research, Boston Biomedical Research Institute; and Department Harvard Medical School, Boston, Massachusetts3 Received

July

15, 1971; accepted

of Neurology,

Oct,ober 7, 1971

The absorption bands of copper-transferrin are optically active with well-defined positive dichroic bands in the visible range having ellipticities of [81620= 3,620 deg cm2 dmole-1 and [el,,6 = 6,700 deg cm2 dmole-1 in terms of bound copper. In irontransferrin there are also two bands; a negative ellipticity band with [8]ass = -16,000 deg cm2 dmole-1 and a positive ellipticity band with [&so = 6,000 deg cm2 dmole-1 in terms of bound iron. Additional copper binding to iron-transferrin produces an absorption band at 675 nm with an associated negative ellipticity band. Zinc binding to apotransferrin, 2 moles per mole, affects 1 mole of tyrosine per mole of Znz-’ bound, and the generated tyrosinate spectrum is optically active. There is optical activity in apotransferrin in the 280 nm region attributable to tryptophan, and optical activities in the 270 and 230 nm regions suggest possible disulfide contributions. Neither the tryptophan optical activity nor the optical activities in the region of 270 nm and 230 nm are affected by metal binding. Apotransferrin has an estimated a-helical content of 17-I@$, and there is no observable change in this value when 2 moles of Cue+ or ZP are bound per mole of protein.

Human serum transferrin possesses a single polypeptide chain (2) and two identical carbohydrate side chains (3). It has been suggested that the single polypeptide chain of transferrin from different sources (4-6) is made up of two duplicate polypeptide segments (7). This could very well account for there being two equivalent metal-binding sites which under physiological conditions are occupied by iron. The equivalence of the binding sites is supported by linear changes with metal binding in the absorption spectrum (a), magnetic resonance (S), optical rotatory dispersion (9),

and intrinsic fluorescence (10). Optical rotatory dispersion studies (ORD) (11) showed a Cotton effect associated with the visible absorption band produced by Fe3+ or iVn2+ binding but not with the absorption produced by Cu2+ binding, suggesting that copper, owing to its coordination properties, is in a symmetric complex. This study deals with the visible and ultraviolet circular dichroism (CD) and ORD spectra of copper- and iron-transferrin. Our results indicate that, copper binds asymmetrically to transferrin. The spectral changes caused by the addit#ional binding of Cu2+ to copper- and iron-transferrin were also studied by CD and absorption measurement in the 350-700 nm spectral range.4 CD spectra in the 280 nm region show optical activity contributions from trypto-

1 This work was supported by grants from the National Institutes of Health (AM 11235 and AM 11677). A preliminary report has been presented (1). *Recipient of NIH Career Development Award # l-K3-GM-13-497. 8 Reprint request cards should be sent to the Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA, 02114.

4 This additional binding is called nonspecific in contrast to the specific binding of two moles of Cua+ or Fe3+ to apotransferrin. 27

Copyright

0

1972 by Actrdemic

Press,

Inc.

28

NAGY AND LEHRER

phan and, possibly, tgrosine residues in the apoprotein. In view of the greater stability to denaturation and proteolysis of iron-transferrin than apotransferrin (12) it was also of interest to use the CD and ORD measurements to obtain further information regarding possible conformational changes of the protein moiety produced by metal binding. Due to the large absorption of Fe3+ and CW+ complexes in the ultraviolet, studies were mainly performed with Zn2+ because it forms colorless complexes (13). It is assumed to bind to the protein at the same site as Fe3+ and Cu2+ (13). From these studies it appears that the binding of Zn2+ or Cu2+ affects only tyrosyl side chains and causes little or no change in peptide backbone conformation. MATERIALS

AND METHODS

Human apotransferrin (Behringwerke) from Hoechst Pharmaceuticals, Inc. was used. The molar ratio of iron to transferrin was less than 0.03 as estimated from the visible spectra. Solutions of apotransferrin in 0.001 M NaHC03 , 0.1 M NaCl and 0.01 M HEPES buffer, pH 8 were used. The same solvent was used for each experiment. The protein concentration was estimated spectrophotometrically using 6278= 9.23 X lo4 ~-1 cm-1 (14). Spectrophotometric titrations with Cu*+ and with Fe3+ were performed as outlined previously (10) using 10 cm cells for the visible spectral region and 1 cm cells for the ultraviolet region. The same samples were used also for the CD and ORD measurements with 10 cm cells in the visible and 1 cm or 0.1 cm cells in the ultraviolet region. Spectrophotometric titrations with Zn2+ were carried out in double compartment mixing cells (15). One compartment contained apotransferrin and the other ZnClz in the same solvent. The baseline was then determined and the difference spectrum was recorded after mixing the sample cell. This procedure was repreated with each concentration of ZnC& . Since mixing resulted in twofold dilution of both protein and ZnCl, , we checked the effect of dilution on the protein spectrum. Dilution of the protein with the salt-free solution did not cause a spectral change. Visible and ultraviolet spectra were obtained either with a Cary 15 or a Beckman DK2 spectrophotometer. For ORD and CD spectral measurements a JASCO Durrum ORD/UVB spectropolarimeter wit,h a ROUSSEL-UCLAF JOUAN type CD attachment was used. All spectral measurements were made on filtered solutions (Millipore, Inc., HAWP, 0.45 p).

CaZcuZations. The molecular weight value of 81,000 was used for transferrin. Molar extinction coefficients of the bound metal were calculated from the protein concentration and the titration results. The optical rotation is expressed as the reduced mean residue rotation corrected for the Lorenta solvent dispersion [m’]h = o1x[MRW/100] [3/(nah + 2)], where CYis the measured specific rotation, MRW the mean residue weight taken as 115, and 72is the refractive index. The subscript X refers to the wavelength of measurement. The ratio of the refractive index correction factor, 3/(%*x + 2), of 0.1 M NaCl to that of water at 589 nm was found to be essentially unity (0.998). On the assumption that this ratio is constant at all wavelengths, only the water correction was applied. Ellipticities [e] are given either per mole of bound metal or per protein mean residue weight according to the relation [e] = 2.303 (4500/r) (Et-Ea) where q. and ea are the molar or mean residue weight extinction coefficients of the left and right circularly polarized light. Rotational strengths, R (erg cm3), of the circular dichroic bands were calculated from the CD spectra according to Moscowitz by assuming Gaussian type bands for the transitions (16). The calculated values are given as the reduced rotational strength [RI, which has a more convenient magnitude and is a dimensionless number defined by [R] = lOOR/ pnps , where pr, is the Debye unit and PB is the Bohr magneton. The numerical value of [R] is 1.08 X 10” times that of R. RESULTS

Specific binding of Cuz+ to apotransferrin. The ORD spectra of apotransferrin, Cu2+transferrin, and Fe3+-transferrin in the spectral range of 360-700 nm are shown in Fig. 1, There is an appreciable Cotton effect associated with an absorption band centered at 460 nm for iron-t,ransferrin as previously observed (9, 11). However, in contrast with previous reports (9, 11) the ORD spectrum of Cu2+-transferrin consistently differed from the ORD spectrum of apotransferrin, although the anomaly was less prominent than that in the ORD spectrum of Fe3+transferrin. The presence of optical activity for Cu2+-transferrin is more clearly indicated in the CD spectrum (Fig. 2). The absorption spectrum of Cu2+-transferrin (Fig. 2) has two well-defined absorption bands in the visible spectral range (2, 10). The one at 675 nm is a broad nonsymmetrical peak with ecu = 260 M-I cm-‘. The second peak is at 440 nm with ecu = 2,220

29

CD OF METAL-TRANSFERRINS

IN-~cm-*. There is also a broad band in t#he near ultraviolet, rising to a maximum around 300 nm. The CD spectrum shows two distinct dichroic bands, one of them at 620 nm with a value of [0] = 3,260 deg cm2 dmole-l

OF-50 n d

-100

,

I 400

600

500 WAVELENGTH,

nm

FIG. 1. Optical rotatory dispersion of apotransferrin, iron-transferrin, and copper-transferrin. Ordinate: ORD values given as reduced mean residue weight rotation, deg cmz dmole+. -, apotransferrin; -.-, iron-transferrin; -, copper transferrin. Conditions : protein concentration 7.5 mg/ml, pH 8.0, 22”, light path 10 cm. The metal to protein molar ratio in each case was 2.

400

600

500 WAVELENGTH,

FIG. 2. Absorption and circular sorption in terms of protein-bound lengths :> 500 nm. Left ordinate: bound copper in deg cm2 dmole- I. 7.5 mg/ml, CuCL added in a molar

in terms of bound copper. The position of this CD band indicates that the absorption band at 675 nm is composed of at least two transitions with an optically active transition on the short wavelength shoulder. The second positive dichroic band with [e] = 6,700 deg cm2 dmole-’ is at 426 nm and is also slightly shifted from the band maximum at 440 nm. Another positive dichroic band is indicated by the CD spectrum below 350 nm, but its position could not be ascertained owing to a very large absorption of coppertransferrin at shorter wavelength. The titration of apotransferrin with copper was followed by monitoring the absorption peaks and the circular dichroic bands. Upon addition of CuC12,as can be seen in Fig. 3, the related absorption and circular dichroic changes at 440 and 426 nm, respectively, show no further increase above about 2 moles of Cu2+ added per mole of transferrin. At the long wavelength bands (As75and CDs20), however, simple saturation of the absorption or CD peaks is not observed. The increase in absorption at 675 nm at a molar ratio >2 indicates that further Cu2+ is bound at other sites. The related circular dichroism at 620 nm shows a maximum with further decrease of circular dichroism at copper: protein molar ratios > 2.

700

nm

dichroism spectra of copper-transfer&. - -, molar abten-fold increased absorption for wavecopper; -.-, absorption; right ordinate: molar ellipticities of protein -, circular dichroism. Conditions : protein concentration ratio of copper to protein of 2, pH 8,22’, light path 10 cm.

NAGY AND LEHRER

30

-16 I

400 I

MOLAR

2

RATIO OF CR

3

4

TO TRAWFERR,,,

FIG. 3. Spectrophotometric (A) and circular dichroic (B) titration of apotransferrin with copper. A. Left ordinate: molar absorption of transferrin at 440 nm, 0; right ordinate: molar absorption of transferrin at 680 nm, A. B. Mean residue weight ellipticities of transferrin, in deg cm2 dmole-1 at 430 nm, 0, and at 626 nm, A. The vertical broken line indicates the approximate saturation of binding sites of transferrin by copper. Conditions : protein concentration 7.5 mg/ml at the beginning of the titration; corrections were made for the dilution upon additions of CuC12 solution, pH 8.0, 22”, light path 10 cm. Both spectral and circular dichroic measurements were made on the same sample.

NonspeciJic binding of copper to iron-transfer&n. The nonspecific binding of copper was studied by adding Cu2+ to iron-transferrin. Since the affinity of iron to the specific-binding sites in apotransferrin is much higher than that of copper (2, 13, 17), copper cannot replace the bound iron even when it is added in about tenfold excess. Figure 4 shows the CD spectra of irontransferrin and of iron-transferrin with 3 moles of Cu2+ added per mole of protein, Iron-transferrin has a negative CD band at 455 nm with [0] = - 16,000 deg cm2 dmole-’ in terms of bound iron, associated with its absorption band at 470 nm. There is another positive CD band at about 360 nm, with [0] = 6,000 deg cm2 dmole-’ in terms of bound iron. The addition of threefold excess of copper does not affect the CD bands in

500 WAVELENGTH,

I

600

,

700

nm

FIG. 4. Circular dichroic spectrum of irontransferrin and of an iron-transferrin-copper complex. Ordinate: -, ellipticity per mole of bound iron before addition of copper; --, after addition of three moles of copper to 1 mole of Fe*+-transferrin. Conditions: protein concentration 5.3 mg/ml, Fe3+-transferrin contains 2 moles of Fe3f per mole of protein, pH 8.0, 22”, light, path 10 cm.

the 350-520 nm region confirming that copper does not replace the bound iron. However, a new negative CD band centered at about 600 nm is produced by the addition of Cu2+. Changes in absorption at about 600 nm are also associated with the additional binding of copper. Both the absorption and CD changes caused by increasing concentrations of added Cu2+ to iron-transferrin are shown in Fig. 5. These data can explain both the lack of saturation of the absorption band at 675 nm and the decreasing positive CD at 620 nm noted earlier. In the case of the CD titration, since the negative CD band associated with additional binding of copper overlaps the positive CD band of copper-transferrin at 620 nm, a gradual decrease in the magnitude of the ellipticity band occurs as the nonspecific binding increases. Speci$c binding of zinc to apotransferrin. The binding of Zn2+ was followed by measuring the uv difference spectrum by a method similar to that used in the case of conalbumin (18). The difference spectrum

CD OF METAL-TRANSFERRINS

500

600

700

WAKLENGTH.

so0 nm

FIG. 5. Changes in absorbance (A) and circular dichroism (B) of iron-transferrin in the 500-700 nm region on addition of copper. A. Ordinate: Ae =

(eFePI-trllnsferrin+nCu2t)

Ordinate:

A[6’]

=

-

(EF&-transferrin).

([e]Fel+-tranaferrin+nCu”

B. -

A, n = 2; 0, 72 = 3; where n = m.oles of copper added per mole of transferrin. Conditions: protein concentration 5.3 mg/ml, iron-transferring contained 2 moles of Fe3+ per mole of protein, pH 8.0, 22’, light, path 10 cm. ([e]Fe’+-transferril,).

0,

?z =

31

1;

240

260

2so

WAVELENGTH,

300

nm

FIG. 6. Difference absorption spectrum of einctransferrin vs. apotransferrin. Conditions: protein concentration 1.0 mg/ml (1.2 X KP rd), ZnCll concentration 5 X 10-r M, pH 8.0, 22’, light path 0.88 cm.

OS

produced by the addition to apotransferrin of Zn2+ in a 4: 1 molar ratio is characterized by a maximum at 295 nm, a minimum at 275 nm and another maximum at 245 nm (Fig. 6). Upon titration of apotransferrin with increasing amounts of Zn2+ isosbestic points appear at 265 and at 280 nm indicating that only two distinct states with different optical properties are involved in the spectral change. As seen from the titration curves (Fig. 7), the absorption changes at the three peaks do have a well-defined break at a Zn:protein molar ratio of about 2. At higher ratios there is a slight but monotonic change in the absorption indicating further binding to sites other than the specific-binding sites. The difference spectrum arising on titration with Zn2f is very similar to that produced by the ionization of tyrosine (19, 20). The absorbance changes at the three peaks occurring upon titration are given in Table I. Assuming that these changes of transferrin with Zn2f (see Fig. 7) are due to ionization of tyrosine, the use of the molar differ-

04

B 2 4

0

1

I 2

4

6

I s

MOLAR RATIO OF Zn*+TO TRINSFERR~N

7. Spectrophotometric titration of apotransferrin with zinc. Left positive ordinate: change of absorbance at 245 nm, 0. Left negative ordinate: change of absorbance at 274 nm, q . Right ordinate: change of absorbance at 295 nm, A. Conditions: protein concentration 1 mg/ml, varying the concentration of added ZnClz , pH 8.0, 22”, light path 0.88 cm. Fra.

ence spectral values for tyrosyl dissociation at the two characteristic wavelengths, 295 and 245 nm (20, 21), shows that about two tyrosyl residues are affected by binding of 2 moles of Zn2+ to 1 mole of apotransferrin. ORD and CD spectra of copper and xinc-

32

NAGY AND LEHRER TABLE

COMP-LRI~ON

OF ABSORPTION FERRIN

CHANQFS WITH THE

I

BT 295, 274, AND 245 nm ON BINDING OF Znzt BY HUMAN MOLAR ABSORPTION VALUES OF TYROSINE Wavelengths (nm) 274

AEtMi8a Aetryosine Moles of tyrosine/mole of transferriP Molar ratio of Znz+/transferrinf Moles of tyrosine/mole of Zn boundg

5,290 i 80 2,330b 2.27 f 0.04 2.30 f

0.08

0.99

-1,260 f 100 - 550d (2.3) 2.0 f (1.15)

0.1

TRANS-

245

25,860 f 510 11,100” 2.33 f 0.05 2.58 f

0.05

0.90

a Values corresponding to the intersection of the two slopes of the titration curve (Fig. 7) ; average of six determinations. The values are given aa differences in the molar absorption coefficient of transferrin. b Value taken from Beaven and Holiday (21) is differences between molar absorption coefficient of tyrosine in 0.1 N NaOH and that at neutral pH. c Value from Donovan (20), acetyltyrosine at pH 11 ‘us. acetyltyrosine at pH 7.5. d Value calculated by assuming that 2.3 moles of tyrosine per mole of transferrin are affected by Zn2+ binding based on values obtained at 245 and 295 nm. BValues calculated as A~trsnsferrio/A6try~~i~~. f Values determined from spectrophotometric titrations (Fig. 7). g Values obtained by dividing the values of moles of tyrosine/mole of transferrin by the values found for moles of Zn*+/mole of transferrin. Value in parenthesis is less precise than those at wavelengths 95 and 245 nm due to the small spectral changes at 274 nm.

transferrin in the 260-320 nm wavelength range. ORD and CD measurements in the 260-320 nm region show that there is optical activity associated with the aromatic amino acid side chains in apotransferrin, Zn2+transferrin, and Cu2+-transferrin (Fig. 8). There are positive CD bands centered at 291.5 nm, 283 nm, and a somewhat less distinguishable peak at about 278 nm superimposed on a broad negative peak. The difference CD spectra of apotransferrin vs. Cu2+- or Zn2+-transferrin showed a new negative band centered at about 292 nm. This agrees well w&h t,he absorption maximum of the ionized tyrosyls (Fig. 6). There is also a Cotton effect anomaly with the right sign and magnitude in the ORD spectrum after addition of Cu2+ or Zn2+ as required by the correspondence of CD and ORD. This Cotton effect is characterized by a trough at about 304 nm and a peak at about 278 nm with a crossover point at about 290 nm (see Fig. SC). All the aromatic side chain dependent CD peaks completely disappear in 6 M guanidine hydrochloride. ORD and CD spectra of apo- and Zn-transferrin in the 200-240 nm wavelength range.

The features of the far ultraviolet ORD spectrum of apotransferrin (Fig. 9) include a trough at 235 nm, a crossover at 220 nm, a shoulder on the positive side at about 216 nm, and a peak at about 203 nm. The interesting feature of the ORD is that both the trough and the peak are red-shifted from the position usually found in a helical model peptide, viz., from 233 nm to 235 nm and from 199 nm to 203 nm, respectively. A rough estimate of structure can be derived from the ORD trough having a value of [m’]235 = -3,800 deg cm2 dmole-‘. Taking [m’]233for a random coil as -1,800 and for a 100 % a-helix as -13,000, the relatively low value of 16-17 % a-helix content is obtained. The CD spectrum of apotransferrin shows two overlapping negative bands in the 200240 nm wavelength range with peaks at 221-222 nm and a somewhat larger one at 212 nm. The CD spectrum crosses over to positive values at 203 nm indicating a positive peak below 200 nm. There is also a noticeable shoulder at 230-234 nm on the longer wavelength side of the CD band centered at 221-222 nm. Comparing the

CD OF METAL-TRANSFERRTNS

9 -400

--so0 &

.

-

2

BQQQ

0

‘-./.I 8

‘0

I 200

FIG. 8. Circular dichrosim and optical rotatory dispersion of apotransferrin, zinc-transferrin and copper-transferrin in t’he absorption region of the aromatic amino acid side chains. A. Ordinate: circular dichroism given as the mean residue weight ellipticity in deg cm3 dmole-1. -, apotranaferrin; Zn*+-transferrin. 0 cu2+A-A, transferrin; -- -, the differenci betxkeen the ellipticities of Zna+-transferrin and apotransferrin. B. Ordinate: optical rotatory dispersion given as reduced mean residue weight rotation in deg cm% dmole-I. -, apotransferrin; A, Zn**-transferrin; 0, Cu2+-transferrin. C. Ordinate: the difference between the optical rotatory dispersion spectrum of Cua+- or Zn2+-transferrin and apotransferrin, - -. Conditions: protein concentration 1.4 mg/ml, zinc or copper to protein ratio 2, pH 8.0, ZZ”, light path 1 cm.

negative CD peak value of apotransferrin at 221-222 nm [e] = -6,800 deg cm2 dmolel with that found for 100% a-helical polypeptide, - 38,000 deg cm2 dmole- l (22)) gives again an a-helix content of about 18 % for apotransferrin. Comparison of these protein-conformation dependent Cotton effects of apotransferrin with metal-transferrin was feasible in the case of Zn-transferrin, only partially in the case of Cu-transferrin, but not in the case of Fe-transferrin, owing to the high optical density of the Cu- and especially Fe-transferrins in the far ultraviolet range.

210 220 WAVELENGTH, nm

230

240

FIG. 9. Optical rotatory dispersion and circular dichroic spectra of apotransferrin in the 195-240 nm wavelength region. Left ordinate: optical rotation a.qthe reduced mean residue weight rotation in deg cm*dmole-1, -. Right ordinate: CD aa the mean residue weight ellipticity in deg cm2 dmole-I, - -. Conditions: protein concentration 1.85 mg/mI, pH 8.0, 22O,light path 0.01 cm.

Such a comparison is given in Fig. 10. Neither the ORD nor the CD spectrum appears to be affected in the 216-250 nm wavelength range by binding 2 moles of Zn2+ or Cu2+ by apotransferrin. The ORD and CD spectra of Cu2+-transferrin are essentially the same as those of Zn2+- and apotransferrin at wavelengths greater than 226 nm. At shorter wavelengths the comparison becomes difficult because the signalto-noise ratio decreases owing to the absorbance of the Cu2+-complex. DISCUSSION

Asymmetric binding of copper and iron to human transferrin. The observation of CD bands associated with the visible absorption bands of Cu2+-transferrin indicates asymmetric binding, in contrast with the previous conclusion of Ulmer and Vallee (9, 11) deduced from optical rotatory dispersion alone. A close scrutiny of the ORD spectrum (Fig. 1) indicates only a slight

34

NAGY

-2

-2

,” -4 ‘0

-4

-6

-6

AND LEHRER

B

220

230

9 x ‘2 P

240

WAVELENGTH,rm

FIG. 10. Optical rotatory dispersion and circular dichroic spectra of apotransferrin, Zna+-transferrin and Cu*+-transferrin in the 216-250 nm wavelength region. Left ordinate: CD (filled symbols) as the mean residue weight ellipticity in deg cm2 dmole-I. Right ordinate: ORD (empty symbols) aa reduced mean residue weight rotation in deg cm* dmole-I. A, A, Zn*+-transferrin; 0, 0, Cue+-transferrin. The solid lines were obtained with apotransferrin. Conditions: protein concentration 0.84 mg/ml, pH 8.0, 22”. Metal to protein ratio, 2, light path 0.1 cm.

suggestion of Cotton effects associated with copper transferrin. The overlapping Cotton effects associated with the two positive CD bands explain why only the slight effect, occurs in the ORD spectrum. These CD bands of approximate reduced rotational strengths of [R] 620= 2.8 and [RlaO = 8.3, in terms of bound Cu2+, have about the same width and are separated by about one width. The Cotton effect trough associated with the 620 nm CD band and the maximum of the Cotton effect associated with the 430 nm CD band partially cancel each other. The CD spectrum of copper-transferrin with its two positive ellipticity bands apparently differs from the reported CD spectra of a copper-conalbumin complex exhibiting a single negative peak at 540 nm (23). This may indicate that the copperbinding site has a different ligand environment in conalbumin than in transferrin or that the conalbumin used still contained some bound iron. Reinvestigation of these questions is in order.

Titrations of transferrin with Cu2+ using the 440 nm absorption peak (2), electron spin resonance (2, 24, 25), optical rotatory dispersion (II), and int.rinsic tryptophyl fluorescence (10) have previously produced information regarding t’he stoichiometry, the environment of the sites and the equivalence of the sites. This study supports the previous conclusions and further suggests that the two sites are equivalent since changes in optical activit’y were linear up to 2 moles of Cu2+ per mole of transferrin. Further weaker binding of Cu2+ was observed to occur by measurement of the absorption and CD changes produced by the addition of l-3 moles of Cu2+ per mole of Fe3+-transferrin. This nonspecific binding that did not affect the spectral properties attributable to Fe3+ binding has an absorption peak between 620 and 650 nm and a negative CD peak between 580 and 610 nm which shifted t,o the red as more Cu2+ was added. The furt,her increase in absorption and decrease in CD in this spect’ral region, which were observed during t#itration of apotransferrin with Cu2+, were most probably due to the spectral contribution of the nonspecifically bound Cu2+. Participation of tyrosine in complexing Zn2+ and Cu2f to transjerrin. In addition to ligands involving nitrogen, tyrosyl side chains have been proposed as ligands both in transferrin and conalbumin, viz., three tyrosyls for iron and two for copper (25,SS). According to a more recent study on conalbumin two tyrosyl residues appear to be coordinated to each trivalent metal atom and one tyrosyl residue to each divalent met,al atom (12). From the present study it appears that one tyrosyl residue is involved in the binding of 1 mole of Zn2+ to human serum transferrin. The tyrosine dissociation spectrum produced by Zn2+ binding is identical with that found with Z$+-conalbumin; the ratio of the absorbancies at the two maxima, 245 nm and 295 nm, is also very similar; 4.8:1 was found in this study for transferrin while 4.7: 1 was reported for conalbumin (18). The tyrosine: metal ratio and spectral similarities suggest great similarities of the binding sites in both proteins. Optical activity of aromatic amino acid side chains. The near ultraviolet ORD, CD and

35

CD OF METAL-TRANSFERRINS

absorpt,ion spectra have provided information regarding t’he aromatic side chains. In view of the studies on small peptides and proteins (27-30) the sharp positive CD peak at 291-292 nm is most probably due to tryptophan. The two small peaks (253 nm and 275 nm) which were also seen in polyL-tryp CD spectra (31) are probably also due to t’ryptophan. The intensities of all of these peaks do not change on binding Zn2f or Cu2+, suggesting that the environmental symmet,ry of tryptophan does not change on binding metal. The previously reported small change in absorption upon binding of Cu2+ and Fe3+ (10) attributable to perturbation of tryptophan is not inconsistent with these results, for certain effect,s such as charge changes may influence the absorption spectrum wiithout significantly altering the optical activity, or the absorption of tryptophans not contributing to the optical activity may be perturbed. The binding of either Zn2+ or Cu2+ produces a negative CD band centering on 292 nm which is in the wavelength region where tyrosinate spectra generated by metal binding appear. This suggests that one or more t,yrosine(s) interacting with the metal are involved in an opt,ically active transition. Peptide baclcbone cunformatian. Transferrin appears to have a low content of optically active peptide backbone conformation which, expressed as a-helix, amount’s to about 17-B%. The lack of change in the far uv, ORD and CD spectra upon binding of Zn2+ or Cu2+ is evidence for t,he lack of conformational changes involving the peptide backbone upon specific binding of metal ions. Reasons for the increased stability of metal t,ransfmerrin against heat denaturation (12) need not involve metal induced peptide backbone conformational changes, but rather a stabilization of an existing conformation. There are 33-35 half cysteine residues present per mole of transferrin (5, 6), and on the basis of the hydrodynamic behavior of the molecule it appears that it contains a large number of disulfide bonds (6). There is also evidence for optically active disulfide transitions (32)- a shoulder at 230 nm OII the negative CD band at 222 nm and a broad negative band in the region of 270-280 nm. This shoulder at 230 nm on

the 222 nm negative CD band is present in apotransferrin and apparently is unchanged in metal transferrin. The optical activities of the disulfides might explain the shift of the Cotton effect trough to 235 nm. Further experimental evidence is necessary, however, to substantiate the implication of disulfide bonds, especially in view of the difficulties in interpret,ing similar bands in RNase (33, 34). The possibility of contributions by the approximately 5 % carbohydrate content to the CD at wavelengths higher than 220 nm (35) can be excluded since optically active absorpt,ion bands in sugars are usually below 200 nm. However, for amino sugars (approximately f$ of the carbohydrate content in transferrin (3)), the first band is around 209 nm (36). On the basis of the reported optical activity of N-acetylglucosamine (36) and assuming simple additivity of contributions, its effect would be less than 2 % at 220 nm. ACKNOWLEDGMENTS We are grateful to Miss Karen Luebbers and Mrs. Grace Kerwar for their expert technical assistance. REFERENCES 1. NAGY, B., AND LEHRER, S. S., Third InterAbstract national Biophysics Congress, IAS, p. 11. Cambridge, MA, 1969. 2. Aass, R., MALMSTROM, B. G., SALTMAN, P., AND VANNGARD, T., Biochim. Biophys. Acta 76, 203 (1963). 3. JAMIESON, G. A., J. Biol. Chem. 240, 2914 (1965). A., AND GROHLICH, D., 4. BEZEOROVAINY, Biochim. Biophys. Ada 147, 497 (1967). 5. GREENE, F. C., AND FEENEY, R. E., Biochemistry 7, 1366 (1968). 6. MANN, K. G., FISH, W. F., CHADWICK-C• X, A., AND TANFORD, C., Biochemistry 9, 1348 (1970). 7. PHILLIPS, J. L., AND AZARI, P., Biochemistry 10, 1160 (1971). 8. AISEN, P., LIEBMAN, A., AND REICH, H. A., J. Biol. Chem. 241, 1666 (1966). 9. ULMER, D. D., AND VALLEE, B. L., Biochemistry 2, 1335 (1963). 10. LEHRER, S. S., J. Biol. Chem. 244,3613 (1969). 11. VALLEE, B. L., AND ULMER, D. D., Biochem. Biophys. Res. Commun. 8, 331 (1962). 12. AZARI, P. R., AND FEENEY, R. E., J. Biol. Chem. 232, 293 (1958).

36

NAGY

AND

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