Biophysical Chemistry 99(2002) 181–188

0301-4622/02/$ - see front matter� 2002 Elsevier Science B.V. All rights reserved.PII: S0301-4622Ž02.00182-5

EPR study of non-covalent spin labeled serum albumin andhemoglobin

S. Cavalu *, G. Damian , M. Dansoreanua, b cˆ ¸

University of Oradea, Faculty of Medicine and Pharmacy, Department of Biophysics, RO-3700 Oradea, Romaniaa

‘‘Babes-Bolyai’’ University, Department of Physics, RO-3400 Cluj-Napoca, Romaniab

University of Medicine and Pharmacy ‘‘Iuliu Hatieganu’’, Department of Biophysics RO-3400 Cluj-Napoca, Romaniac

Received 5 February 2002; received in revised form 31 May 2002; accepted 5 June 2002

Abstract

Electron Paramagnetic Resonance(EPR) was used to investigate the Tempyo spin label(3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) as a report group for the interactions and the conformational changes of lyophilizedbovine serum albumin(BSA) and bovine hemoglobin(BH), as function of pH values in the range 2.5–11. The EPRspectra are similar with those of other non-covalently spin label porphyrins in frozen solution at very low temperatures.This behavior indicated a possible spin–spin interaction between the hemic iron and the nitroxide group. The changesin the EPR spectra as function of the pH are discussed in terms of conformational changes of the proteins. Spectralsimulations and magnetic EPR parameters reveal the following:(i) one single paramagnetic species, with Gaussianline shape, was used for the best fits of experimental spectra in the case of serum albumin samples; and(ii) aweighted sum of Lorentzian and Gaussian line shape in the case of hemoglobin samples. The representation ofcorrelation time vs. pH, reveals a dependence of degree of immobilization of spin label on the conformational changesof proteins in acidic and basic environment.� 2002 Elsevier Science B.V. All rights reserved.

Keywords: EPR; Spin label; Serum albumin; Haemoglobin

1. Introduction

Proteins are polymeric chains that are built frommonomers called amino acids. All structural andfunctional properties of proteins derive from thechemical properties of the polypeptide side-chain.The ionizable groups of the side-chains of chargedamino acids are often involved in biochemical

*Corresponding author. Fax:q40-59-41-8266.E-mail address: scavalu@xnet.ro(S. Cavalu).

transactions(binding, catalysis). Therefore, pHusually has rather dramatic effects on the functionof proteins. Polar residues are both buried as wellas on the surface of the protein. They either formhydrogen bonds with other polar residues in theprotein or with water. Non-polar residues do notinteract favorably with water. However, a signifi-cant number of non-polar residues are also foundon the surface of the protein. Therefore, it isimportant to study the interactions of proteins withwell-defined molecules that will confer the total

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Fig. 1. The chemical structure of Tempyo spin label.

molecules sufficient affinity for the binding site ofinterest on proteinw1,2xThe successful application of spin labeling to

protein structure investigations is limited by thepossibility to chemically change specific sidechains in proteins. However, useful information onprotein properties can be obtained by non-covalentspin labeling if the affinity of the protein for thelabel molecules is great enough to affect theirmotional freedomw3–6x. The rate of rotation(ortumbling) of the spin label influences the lineshapeof its EPR spectrum. Therefore, the EPR signal ofa spin label covalently or non-covalently bondedto a biomolecule can yield a range of informationabout its structural environment in conventionalESR and allows for full spectral coveragew7,8x.In the same time, EPR has been an invaluable

tool for probing microscopic molecular motions ina variety of systems, including isotropic solventsw9x, liquid crystals w10x, model membranes andbiomoleculesw11–13x.In the present work, non-covalent spin labeled

bovine serum albumin(BSA) and bovine hemo-globin (BH) with Tempyo spin label(3-carba-moyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) wereinvestigated both in liquid and lyophilized sam-ples, in the pH range 2.5–11, in order to obtainuseful information related to the interactionbetween the nitroxide group and the functional siteof the proteins.Interactions of spin label with hemic or non-

hemic proteins might affect the spin label spectraand in the same time it is well known that the pHstrongly influences the conformation of proteinsleading to significant changes in the type anddegree of these interactionsw14x. In this pH range,we followed the effect of protein conformationalchanges on the interactions between the nitroxideand the functional groups of proteins and also thepH influence on molecular motion emphasized bythe EPR spectra of the spin label.

2. Materials and methods

Powdered bovine serum albumin and hemoglo-bin ()95% methemoglobin) from Sigma Chemi-cals, were used without further purification.Proteins were hydrated in phosphate buffer physi-

ological saline at a final concentration of 10 M.y3

Tempyo spin-label(3-carbamoyl-2,2,5,5-tetrame-thyl-3-pyrrolin-1-yloxy), from Sigma Chemicals(Fig. 1), was added to the liquid samples of eachprotein in a final concentration of 10 M(pro-y3

teinyspin label molar ratio 1:1) and the pH valueswere adjusted to the desired value in the range2.5–11. An amount of 5 ml from each sample waslyophilized for 30 h aty5 8C and used for theEPR measurement, at room temperature.EPR spectra for both liquid and lyophilized

samples, were recorded at room temperature witha JEOL-JES-3B spectrometer, operating in X-band(9.5 GHz), equipped with a computer acquisitionsystem. Samples were placed in quartz capillarytubes. The spectrometer settings were: modulationfrequency 100 KHz, field modulation 1 G, micro-wave power 20 mW. The computer simulationanalysis of spectra, for obtaining the magneticcharacteristic parameters, was made by using aprogram that is available to the public through theInternet(http:yyalfred.niehs.nihyLMB).The lineshape of an EPR spectrum depends,

among other factors, on the orientation of theparamagnetic center with respect to the appliedmagnetic field. In a powder, or a frozen aqueoussolution, the paramagnetic centers will be fixedwith a random distribution of orientations, and inthe case of anisotropic g- and hyperfine interac-tions this will lead to a broadened EPR spectrum,since all orientations contribute equally. In theliquid state, however, the paramagnetic centers arenot fixed but undergo rotational fluctuation. In thecase of fast rotation, the anisotropic interactionsare thereby averaged to zero, giving rise to sharpEPR lines. If the velocity of the rotational motion

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decreases, the EPR spectrum will approach that ofthe powder spectrum. Therefore, a rotational cor-relation time for a paramagnetic molecule can alsobe determined by EPR using semiempirical for-mula w15x.For isotropic motion in the rapid tumbling limit,

the spectra will be isotropic with the averages ofthe principal components of the g-values andhyperfine splitting factor,a . The rate of theN

isotropic motion determines the relative widths ofresonances and the width,DH , of an individualm

(hyperfine) line, in the first approximation can bewritten as a function of thez component of thenitrogen nuclear spin number(msy1, 0, 1)w16,17x:

2DH sAqBØmqCØm (1)m

where theA coefficient includes other contribu-tions than motion. The termsB andC are functionsrelated to the rotational correlational time(t) andcan be defined as a function of peak-to-peak linewidth of the central line,DH wGx, and the ampli-0

tudes of themth line I w14x:m

B E1 I I0 0C FBs DH y0 y y2 I ID G1 y1

Nws0.103v DgDae

w z3N 2 2 y1x |xŽ .Ž . Ž .q3 dg da t 1q 1qv t (2)B e By ~4

B E1 I I0 0C FCs DH q y20 y y2 I ID G1 y1

w 36 N N2 2 xŽ . Ž .s1.181Ø10 Da q3 da t 1yw x cy 8

z12 2 2 2y1 y1|Ž . Ž .= 1qv t y 1qv t (3)c c e c~8

in which

1 1N N N N N N NŽ . Ž .Da sa y a qa , da a ya (4)zz xx yy xx yy2 2

1 1Ž . Ž .Dgsg y g yg , dgs g yg (5)zz xx yy xx yy2 2

and v s8.8=10 Na M, a is the isotropicy6 N NN

hyperfine splitting andv the ESR spectrometere

frequency in angular units.

In the range from 5=10 to 10 s(motiony11 y9

in the rapid tumbling limit) and magnetic fieldabove 3300 G,Dg and Da vanish, and theN

correlation timest andt are directly related toB C

the B andC coefficients by the following simplerelationsw5x:

t st sK ØB (6)B z 1

t st sK ØC (7)C x,y 2

whereK s1.27=10 andK s1.19=10 . They9 y91 2

average correlation times is:1y2Ž .ts t Øt (8)B c

The slow motion of spin probe, lead to abroadening of the EPR lines. In this case, therotational correlation time,t, is larger than 10y9

s, and thus, the relation(8) is not applicable.The isotropic nitrogen hyperfine splitting chang-

es to a powder like spectrum, with the peak-to-peak distance between the external peaks of thespectrumw2Øa9 (N)x depending on the magnitudezz

of the rotational correlation time,t w18,19x. Anoth-er lineshape theory for slow isotropic Brownianrotational diffusion of spin-labeled proteins hasbeen developed by Freedw9x. Thus, the correlationtime can be evaluated from the ratio of theobserved splitting between the derivate extremea9 and principal valuea , determined from rigidzz zz

matrix spectrumw5,7x:

bB Ea9zzC Ftsa 1y (9)aD Gzz

Thea andb parameters depend on the kind ofthe diffusion process, wherea andb are empiricalconstants, which are tabulated in e.g. Poole andFarach (1987) w15x. For small spin probe, theintermediate jump diffusion is preferablew15x.

3. Results and discussion

In the low concentration liquid state BSA andBH solution, the Tempyo spin label which is arelatively small molecule, gives rise to a spectrumwith narrow lines and constant hyperfine splitting,typical for fast isotropic rotational motion(Fig. 2)with a very low rate of migration between proteinmolecule and water. For this kind of rotation, the

184 S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188

Fig. 2. EPR spectrum of the Tempyo spin label at low pH.

Fig. 3. Experimental(——) and simulated(««) EPR spectraof Tempyo spin label in lyophilized bovine serum albumin atvarious pH values.

Table 1Magnetic parameters values of Tempyo spin label in lyophili-zed bovine serum albumin at various pH values

pH gxx gyy gzz A (G)xx A (G)yy A (G)zz

11 2.01028 2.00841 2.00441 4.72 8.53 35.489.5 2.00113 2.00762 2.00484 6.16 5.91 33.968.1 2.01128 2.00781 2.00461 6.41 5.69 32.986.7 2.00113 2.00742 2.00500 6.15 6.55 32.205.5 2.01147 2.00809 2.00505 6.03 7.83 35.454.5 2.01259 2.00672 2.00588 7.79 6.98 35.523.5 2.01243 2.00660 2.00538 8.28 5.34 35.462.5 2.01269 2.00662 2.00459 6.38 5.66 35.91

rotational correlation time can be estimated fromthe intensity ratio of the low-field and high-fieldN-lines using a semi-empirical formula(8). Forthe Tempyo spin label in BSA and BH aqueoussolution, the calculated rotational correlation timeswas 2=10 s and 2.5=10 , respectively,y10 y10

which is consistent with fast rotation as expectedfor a small molecule.No detectable changes were observed in EPR

spectra of aqueous solution of Tempyo spin labelwithout proteins, at different pH values.The characteristic EPR spectra of a Tempyo spin

label in lyophilized BSA is due primarily toanisotropy in the nitrogen hyperfine couplingwhich is typical for slow rotation. For slow rota-tions, the EPR spectrum of spin labels depends, ina much more complicated fashion, on the com-bined influences of molecular motion and magneticinteractions. Fig. 3 displays the experimental andsimulated spectra for Tempyo spin label in lyo-philized BSA at different pH values. In order tofind the magnetic parameters, the experimentalEPR spectra were simulated. The best fit of exper-imental EPR spectra of Tempyo spin label inlyophilized BSA, was obtained assuming a singleparamagnetic species with magnetic parameterslisted in Table 1, and a Gaussian lineshape corre-sponding to static dipolar interactions.The main feature of the EPR spectra of Tempyo

spin label in lyophilized haemoglobin(Fig. 4),like the Tempyo spin label in lyophilized BSA,exhibit the characteristics to slow motion of spinlabel but with more broadening of the lineshape.The best fit of the simulated experimental EPRspectra can be obtained by assuming the presenceof two functional groups in hemoglobin associated

with two non-equivalent paramagnetic species.Computer simulations indicate a weighted sum ofmixed Gaussian lineshapes(static dipolar interac-tions) and Lorentzian lineshapes(spin–spin inter-actions). Generally, the broadening of the Gaussianlineshape are due to static dipolar interactions of

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Fig. 4. Experimental(——) and simulated(««) EPR spectraof Tempyo spin label in lyophilized bovine hemoglobin at var-ious pH values.

Fig. 5. The experimental EPR spectrum and its subspectra ofTempyo spin label in lyophilized hemoglobin at pH 2.5

the spin label molecules whereas the broadeningof the Lorentzian lineshape is due to to spin–spininteractionsw20x. Figs. 5 and 6 present the EPRspectra of Tempyo spin label in the lyophilizedBH at low and high pH, in which the contributionof each species is displayed. At low pH(pH 2.5),the main contributions are due to species withGaussian line shape(;80%). This contributiondecreases to;50% at high pH(pH 11). The firstspecies with Gaussian lineshape and poor resolvedhyperfine splitting is not influenced by the pres-ence of the hemic iron and, therefore, we assumeto be located far from the hemic group. The secondspecies with Lorentzian lineshape and a wellresolved hyperfine structure is located, probably,near the hem group, giving rise to a spin–spininteraction between the nitroxide radical and theparamagnetic iron of the hem group. Our resultsare in accordance with covalently labeled methe-

moglobin and other porphyrins in frozen samplesunder 50 K w21,22x. In these previous studies,spectra of covalently labeled methemoglobin wereanalyzed by using perturbation calculations inorder to estimate the iron to nitroxyl distances andit was suggested that plausible distances are in therange of 14.5–17.5 A. Theg tensor andA tensor˚components used in the simulation for the best fitvalues of the simulation of the effective powderspectrum, are presented in Table 2.Due to increased spatial restrictions of protein

structure in the vicinity of the label, by lyophili-zation, the mobility of spin label is slow on theEPR time scale(;5=10 s ), leading to a6 y1

broadening of the EPR lines, with the peak-to-peak distance between the external peaks of thespectrumw2Øa9 (N)x depending on the magnitudezz

of the rotational correlation time,t. Generally,broadening of the peaks in an EPR spectrum is

186 S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188

Fig. 6. The experimental EPR spectrum and its subspectra ofTempyo spin label in lyophilized hemoglobin at pH 11.

Table 2Magnetic parameters values of Tempyo spin label in lyophili-zed bovine hemoglobin at various pH values

pH gxx gyy gzz A (G)xx A (G)yy A (G)zz

11 2.01242 2.00771 2.00372 4.81 8.70 34.01L( )

2.01117 2.00497 2.00610 6.42 7.76 33.16G( )

9.5 2.00935 2.00839 2.00478 6.19 13.89 34.82L( )

2.01079 2.00695 2.00465 6.11 7.29 33.06G( )

8.1 2.01133 2.00702 2.00464 8.38 6.68 33.87L( )

2.01500 2.00428 2.00592 8.64 6.50 32.60G( )

6.7 2.00115 2.00773 2.00425 3.27 8.20 33.73L( )

2.01213 2.00476 2.00556 7.43 8.88 35.84G( )

5.5 2.012442 2.00821 2.00471 9.52 7.32 34.62L( )

2.001050 2.00592 2.00541 5.86 6.12 35.45G( )

4.5 2.01609 2.00768 2.00522 9.20 7.66 36.03L( )

2.01198 2.00416 2.00523 7.43 8.88 35.15G( )

3.5 2.01265 2.00816 2.00413 4.14 5.39 36.59L( )

2.01169 2.00287 2.00506 7.06 9.36 35.46G( )

2.5 2.01344 2.00844 2.00506 9.83 10.38 36.26L( )

2.01270 2.00471 2.00548 4.67 5.80 35.78G( )

(L), Lorentzian lineshape;(G), Gaussian lineshape.

Fig. 7. Correlation times(t) as a function of pH for Tempyospin label in lyophilized bovine serum albumin(j) and bovinehemoglobin(m).

indicative of immobilization of the spin label,whereas sharpening of the peaks is indicative ofan increase in label mobility. By comparing theapparent nitrogen hyperfine splittingwtermeda9zz

(N)x with the nitrogen hyperfine splitting obtainedfrom their rigid limit valueswa (N)x, the rotation-zz

al correlation times can be calculated using for-mula 9. The values ofa andb coefficients dependon the motional model. The study of the influenceof different diffusional models on the spectral lineshape in the regime of slow motional spin labelby high EPR fields has shown that jump diffusionmainly affects the line widths at the same motionalrates w23x. In our calculations, the intermediatejump diffusion model was considerate, with coef-ficients values ofas5.4=10 s andbsy1.36y10

w15x. The average correlation time for differentvalues of pH are plotted in Fig. 7. As shown infigure, the pH influences the rotational correlation

time. In the acidic pH range the NH groups of2

the label molecule as well as those of the aminoacids residues are protonated. The fact thatt showsgreater values in this range followed by a signifi-cant decrease in the basic pH range, indicate alow mobility of spin label in acidic environmentwhile an increasing of mobility can be noticed inthe basic pH range. A minimum mobility can beobserved around the isoelectric point of both pro-teins (for BSA pHs4.8, and for BH, pHs6.8).i i

From the pH dependence of correlation time(involving the mobility of the label as well), weassume that in an acidic environment the mobilityof spin label molecules are reduced due to the

187S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188

formation of hydrogen bonds between the NH2

group of spin label and side chains of neighboringamino acids. One can correlate this observationwith the fact that serum albumin undergoes revers-ible isomerization in the pH range 2.7–7 from theexpanded form characterized by 35%a-helix con-tent, to normal form characterized by 55%a-helixcontent accompanied by a decrease inb-sheetw24,25x. It is well known thatb-sheet conformationfavors the formation of hydrogen bonding.By comparing with the BSA case, we notice

that the mobility of Tempyo is greater with respectto hemoglobin, which is not surprising if we takeinto account that hydrogen bonding opportunitiesdepends on theb-sheet content: in hemoglobin theb-sheets represent 50% while in BSA the percent-age varies from 70 to 45%, depending on the pH.As shown in Fig. 7 the mobility of this species

increases in an acidic environment, reaching amaximum value corresponding to pH 6.8 followedi

by a slow decrease. We suggest that in basic pHrange, where the label is not subject to strongelectrostatic interactions, dipolar and spin–spininteractions are manifested almost with the samecontribution in the broadening of the spectrum.

4. Conclusions

EPR spectroscopy is very useful to study themobility of nitroxide radicals with respect to hemicor non-hemic proteins in different environmentalconditions. Non-covalent labeling of proteins cangive valuable information on the magnetic inter-actions between the label molecule and the para-magnetic center of the proteins. The relevance ofthis interaction can be obtained from lineshapeanalysis: computer simulations for non-hemic pro-tein assume a Gaussian lineshape, while for hemicprotein a weighted sum of Lorentzian and Gaussiancomponents is assumed. The contribution of theeach line shape to experimental spectrum dependson the pH.

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