Interactions of the Lanthanide- and Hapten-Binding Sites in the Fv

Biochem. J. (1976) 155, 37-53 37Printed in Great Britain

Interactions ofthe Lanthanide- and Hapten-Binding Sites in the FvFragment from the Myeloma ProteinMOPC 315

By RAYMOND A. DWEK,* DAVID GIVOL,t ROBERT JONES,* ALAN C. McLAUGHLIN,*SIMON WAIN-HOBSON,* ALASTAIR I. WHITE* and CAROLYN WRIGHT*

*Departments of Biochemistry and Molecular Biophysics, University of Oxford,South Parks Road, Oxford OX1 3QU, U.K.,

and tDepartment of Chemical Immunology, Weizmann Institute for Science, Rehovoth, Israel

(Received 27 August 1975)

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with theFv fragment of the mouse myeloma protein MOPC 315 were investigated by the tech-niques offluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclearmagnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenchedby the haptens (fluorescence enhancement, e = 0.15) and enhanced by Gd(III)(E = 1.14) and other lanthanides. The binding of the haptens studied here is insensitive topH in the range 5.5-7.0 (dissociation constant KH = 0.3-1.0#UM) and shows 1 :1 stoicheio-metry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent;the binding constant (KM) varies from 10puM at pH7.0 to 700pM at pH4.8. La(III) bindingis less sensitive to pH. The pH-dependences of the metal-binding constants imply that agroup in the protein with pKa>6.2 is involved in the binding, and probably also othergroups with lower pKa values. 3. The apparent binding of the haptens is weakened about20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complexwith an interaction between the two binding sites is derived in Appendix I to explain theexperimental results at two pH values. 4. Time-dependent fluorescence changes areobserved in the presence ofGd(III) at pH5.5. A two-state kinetic scheme involving a 'slow'conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour.5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on thesubsequent addition of lanthanides are consistent with the binding scheme for haptensand lanthanides proposed from the fluorescence studies. A difference between the limitingquenching ofthe e.s.r. signal from the bound haptens in the presence ofsaturating concen-trations of Gd(l) and La(m) is attributed to dipolar interactions between boundGd(111) and the nitroxide moiety of the bound hapten. The residual quenching withGd(IH) allows an estimate of 1.2nm to be made for the distance between the two para-magnetic centres. 6. The 270MHz proton difference spectrum of the Fv fragment resultingfrom the addition of La(III) suggests that any metal-induced conformational changes aresmall and involve relatively few amino acid residues on the Fv fragment. 7. The titrationcurves ofchemical shift versus pH for the three histidine residues in the Fv fragment showthat one histidine residue has its pKa lowered from 6.9 to 6.4 in the presence of La(III).The decrease in pKa does not arise from direct competition with a proton for a metal-binding site on the histidine residue. The change in pKa and chemical shift of this residuetogether with the observation that a small number of residues are perturbed on La(III)binding suggest that the histidine residue is in the vicinity of the metal ion. 8. The additionof Pr(11l) results in chemical shifts of the aliphatic proton resonances of the Fv fragmentand also broadening of some aromatic resonances. This broadening is unexpected andpossible reasons for it are discussed.

Much of our initial programme to determine in globulin A) myeloma protein MOPC 315 has in-solution the anino acids and their orientations in and volved finding suitable paramagnetic centres thatnear to the combining site of the Fv fragment$ from would bind to the protein (Dwek et al., 1975a). Suchthe Dnp (dinitrophenyl)-binding IgA (immuno- centres provide reference points for high-resolution

t Abbreviation: Fv fragment, variable region of heavy (n.m.r.) (nuclear-magnetic-resonance) mappingand light chain. studies (Dwek, 1973). In a previous paper (DwekVol. 155

R. A. DWEK AND OTHERS

et al., 1975a) we showed how a Dnp-spin-labelledhapten could be used to perturb the proton high-resolution n.m.r. spectrum of the Fv fragment, andby using difference spectroscopy the n.m.r. spectrumof the amino acid residues in and around the combin-ing site was obtained. We assigned resonances in thisspectrum to histidine-97 in the light chain andhistidine-102 in the heavy chain. However, the factthat a particular nuclear resonance is perturbed bythe spin-labelled hapten and consequently 'appears'in the difference spectrum can only place that nucleusin a sphere of given radius from the spin-label group-ing. The actual orientation of a nucleus can beobtained from the lanthanide perturbations of thenuclear resonances (Dwek, 1973), and the finding ofone specific lanthanide-binding site on the Fv frag-ment was thus most fortunate (Dwek et al., 1975a).Our preliminary data (Dweket al., 1975b) suggested

that there was an antagonistic effect between haptenand metal binding, each weakening the other's bind-ing about 20-fold. It is, however, a prerequisite forhigh-resolution n.m.r. mapping studies to have a fullknowledge of all the equilibria existing in solution,and this gave the impetus to the present work. Inthis paper we consider a number of aspects of thebinding of haptens and lanthanides to the Fv frag-ment ofMOPC 315 myeloma protein with particularreference to possible conformational changes inducedby binding, and the apparent interaction between thetwo binding sites.

Materials and Methods

Materials

Fv fragment from the mouse myeloma protein,MOPC 315, was prepared as described previously(Hochman et at., 1973) and then freeze-dried andstored at -20°C. No decrease in hapten-bindingcapacity or any other changes in properties weredetectable after 3 months. Frozen solutions in 0.15M-NaCl (approx. 200.uM-Fv fragnent, based on amol.wt. of 25000) were stable for at least 2 weeks.Protein concentrations were determined by weighingand by absorbance (el"mI'l -1.5 at 280nm). Thetwomethods agreed within ±5%. Unless otherwise stated,all fluorescence and e.s.r. (electron-spin-resonance)experiments were carried out in 0.15M-NaCI/0.05M-Pipes [piperazine-NN"-bis.(2-ethanesulphonic acid)]buffer (analytical grade) adjusted to a pH value in therange 5.0-7.0 by addition of 5M-NaOH.

Oxides of the lanthanides (Koch-Light Ltd.,Colnbrook, Bucks., U.K.) were dissolved in 31OM-HCI and the resulting chlorides evaporated to drynessby gentle heating. The residues were dissolved inwater to give 100mM stock solutions (unbuffered;pH approx. 6.0), which were stored frozen for severalweeks. Precipitation of lanthanides was negligible at

-NO2 CH3

02N Q NH NO

CHACH3

(1)NO2

CH302N Q NH- CH2 CR

NO

CH3

(T1)

pH6. Metal concentrations were checked by titrationwith EDTA, by uWing Eriochrome Black T as anindicator, and agreed with weighing to ±5 %.The spin-label haptens studied were N-(1-oxyl-

2,2,6,6-tetramethyl-4-piperidinyl)-2,4-dinitrobenzene(I) and N-(1-oxyl-2,2,5,5-tetramethyl-3-methylamino-pyrrolidinyl)-2,4-dinitrobenzene (II): They weresynthesized as described previously (Dwek et al.,1975a). Stock solutions (2mM) were prepared bydissolving the desired amount in ethanol and werestable for 2 months at -200C and showed no loss ofthe nitroxide e.s.r. signal. e-Dnp-lysine (BDHChemicals Ltd., Poole, Dorset, U.K.) was chro-matographically pure. Stock solutions were preparedin water.

Methods

Fluorescence measurements. These were made on aPerkin-Elmer/Hitachi MPF-2A spectrofluorimeterwith- the cell compartment thermnostatically main-tained at 25°C. Typical protein concentrations were0.5,M. Protein fluorescence was excited at 295nmand observed at 330nm. A correction for attenuationof incident and emitted light by haptens free in solu-tion was made from a calibration curve obtained atthe same excitation and emission-wavelengths from atryptophan blank of similar fluorescence intensity tothe protein solution. The correction factor at eachhapten concentration was almost independent of thehapten used or the intrinsic tryptophan fluorescence.No corrections were necessary for metals up to a con-centration of 3mm or for the ethanol in which haptenswere added.

In the titration of Tb(III) fluorescence by monitor-ing the 540nm emission band, a filter with a cut-offat 430nm was placed in the emission beam to avoidsecond-order detection of the 280nm radiation fromthe exciting radiation. Also, a correction was applied

1976

38

LANTHANIDE AND HAPTEN BINDING TO FRAGMENT Fv FROM MOPC 315

for the large baseline change in the presence of pro-tein, caused by scatter and tryptophan fluorescence.

Electron-spin-resonance measurements. These weremade on a Jeol JES-PE e.s.r. spectrometer operatingat X-band (9.25GHz). Titrations were performed at20°C on samples ofFv fragment (10-40uM) ofvolume0.3-0.4ml contained in a flat aqueous sample cellwith a syringe attachment for mixing. This arrange-ment obviated disturbing the instrumental settingsduring a titration. All measurements were correctedfor dilution (maximum 10-20%). It was usually alsonecessary to apply a small baseline correction tosignals from bound haptens owing to the signals fromfree haptens and free Gd(III).

Nuclear-magnetic-resonance studies. High-resolu-tion n.m.r. spectra were recorded at 270MHz byusing a Bruker spectrometer with an OxfordInstrument Co. (Oxford, Oxon., U.K.) superconduct-ing magnet. The methods used have been describedpreviously (Dwek et al., 1975a). Measurements weremade at 30±+1C. Chemical shift values are reportedas p.p.m. downfield from the sodium salt of 3-(tri-methylsilyl)propanesulphonic acid, used as an exter-nal standard.

Ultracentrifuge studies. These were carried out at20°C in a Beckman model E analytical ultracentrifugeby using the schlieren optics with a standard cell andAnD rotor operating at 52000-56000rev./min.

Results and Discussion

Fluorescence

Binding of haptens and lanthanides to Fv fragmentstudied by using protein fluorescence. Protein fluor-escence quenching is an established method for deter-mining the binding of Dnp haptens to antibodies(Velick etal., 1960) and to the Fv fragment (Hochmanet al., 1973; Haselkorn et al., 1974). The same tech-nique has also been used here to detect the binding oflanthanides to the Fv fragment, although the relative

changes are much smaller. In the following sectionsthe terminology is as defined in Appendix I. All re-sults refer to Gd(III) and spin-label hapten (I) unlessotherwise indicated.

(a) Haptens. The binding of hapten (I) to Fv frag-ment was studied in the pH range 5.5-7.0 by usingprotein concentrations ([A]) of about 0.5M (basedon mol.wt. 25000) and hapten concentrations ([H])of 0-30,uM. The fluorescence intensity was correctedfor Dnp absorption as indicated in the Materials andMethods section. In the presence of saturating haptenthe fluorescence is strongly quenched (enhancement,8AH = fAH/fA = 0.15), suggesting that a large pertur-bation occurs in one ormore tryptophan residues. Thebinding curve could be readily analysed on the as-sumption of 1:1 stoicheiometry. Initial estimates ofthe dissociation constant, KH, were therefore obtainedby using the formula, KH- (1 -f)2[HhT/f, where f isthe ratio of the change in the fluorescence at theequivalence point to the total change in fluorescenceat saturation. Estimates ofKH and 6AH were then usedin a curve-fitting program, which gave the final valuesSAH= 0.15, KH = 0.3-0.6AM. Both 6AH and KH arealmost pH-independent in the range 5.5-7.0.The behaviour with hapten (II) and with Dnp-

lysine was very similar to the above, and their dis-sociation constants, KH, are given in Table 1.The low solubility of haptens (I) and (II) in water

means that it is necessary to add them to aqueoussolutions in a suitable solvent, in this instanceethanol. However, the apparent binding constant ofDnp-nitroxide (I) to Fv fragment at pH6.8 (KH,.app.)is very sensitive to small concentrations of ethanol inthe solution, although the limiting fluorescence(fAH) is not significantly altered. Ethanol alone causesa small enhancement in protein fluorescence. Theeffects of ethanol appear to approximate to those of acompetitive inhibitor, since they are reversed by asuitably high hapten concentration. They mightsimply arise from non-specific binding of ethanol inthe hydrophobic hapten-binding pocket, but the

Table 1. Binding constants (uAM) ofhaptens and lanthanides to Fvfragment in 0.15 M-NaC/10.05 M-Pipes buffer

Fluorescence experiments and e.s.r. experiments were performed at 25°C and 20°C respectively. The binding constants aredefined according to the scheme of Appendix I.

Binding constantKM for Gd(III)

pH5.5140

pH6.811

TechniqueProtein fluorescence

KH

KHM for Gd(III)

KMHI for Gd(II1)

KMH for La(III)Vol. 155

Hapten (I) Hapten (I)0.3 0.3

380040006.5

500400-800

4.05-104.0

Hapten (11) Dnp-Lys0.55 0.6 Protein fluorescence

0.3-1.0 Hapten e.s.r.280 Protein fluorescence

600-1200 Hapten e.s.r.5.0 10 Protein fluorescence5-10 Hapten e.s.r.5.5 Protein fluorescence

39


possibility of reversible denaturation cannot be ruledout. A 10% (v/v) ethanol solution raises KH,app. from0.3/iM to 6pM, but below 2% ethanol the effects onhapten binding are minimal. This restriction is easilymet for fluorescence experiments (total volume3.Oml), butmay inconvenience hapten titrations usinge.s.r. or n.m.r. where the total volume is muchsmaller, if artifacts are to be avoided.

(b) Lanthanides. Protein fluorescence was moni-tored as for hapten binding. The fluorescence changeson adding lanthanides were slow (see below) and2-4min was required to reach a steady value.

(i) Gd(HI). The fluorescence enhancement is fairlyconstant (EAM = fAM/fA = 1.14±0.03) over the pHrange 5.0-7.0, although there are moderate changes(approx. 20%) in the intrinsic protein fluorescence inthis pH range. Since the lanthanides precipitateabove pH7.0, and the fluorescence enhancement onthe addition of Gd(III) declines rapidly below pH5.0,the investigation of the pH variation of Gd(III) bind-

2000

1000

500

200

100

50

20

pH

Fig. 1. pH-dependence ofthe binding ofGd(III) andLa(III)to Fv fragment

The binding was monitored by the enhancement ofproteinfluorescence of Fv fragment (1-2,UM) at 340nm, in 0.05M-Pipes buffer/0.15M-NaC1, at 25°C. 0, Gd(Il); 0, La(HI).

ing is limited to the range shown in Fig. 1 (pH4.8-7.0).The Gd(III)-binding constant, KM, was estimated

from the concentration for half-maximal change influorescence. KM is very sensitive to pH (varying from700pM at pH4.8 to 10pM at pH7.0), implicating anionizable group on the protein in the lanthanide bind-ing. The data for Gd(III) in Fig. 1 (lower curve) implythat at least one group with pKa > 6.2 is involved inthe lanthanide binding. Since the observed slope isgreater than 1, the data would also be consistent withother groups having PKa values between 5.0 and 6.0being involved. The data do not, however, distinguishbetween two possibilities, i.e. direct competition ofthe protons and the Gd(III) for the same site(s) in theprotein or an indirect effect of an ionizable group inthe Gd(III)-binding site (i.e. transmitted through aconformational change in the protein).At pH6.8, KM is invariant (±10%) over the protein-

concentration range 0.26-4.2jM. Thus any protein-protein interactions would appear to be unimportantas regards lanthanide binding. This is supported byultracentrifuge studies in the same buffer at 80M(2.0mg/ml) protein concentration. Exposure to9mM-Gd(III) for 12h had no significant effect on thesedimentation coefficient at 20°C (s = 2.3-2.6S) andno heterogeneity in the sample could be detected.At pH7.0, the use of Fv-fragment concentrationscomparable with KM (7.5,UM and 15M) has permittedthe stoicheiometry of binding to be determined bymeans of Scatchard plots. The fractional change inprotein fluorescence was used as a measure of freeand bound protein, which thus assumes that Gd(III)-binding sites not producing a fluorescence changecan be neglected. With this limitation, the analysisindicates that the fluorescence enhancement is dueto Gd(H) binding at a single site [KM = 8-12,uM,n (number of binding sites) = 1.0±0.2/mol].

(ii) Other metals. Tb(III) produces similar changesin protein fluorescence to Gd(II) at pH5.6. La(III)also produces comparable enhancements at pH6-7,but the limiting enhancement falls to an unsuitablevalue (approx. 5%) for carrying out titrations belowpH5.1. With Gd(III) this degree of enhancement(approx. 5%) occurs at pH <4.6. The upper curve ofFig. 1 was obtained by direct measurement of thefluorescence enhancement on titrating with La(MI),which varied from 1.05 at pH5.1 to 1.18 at pH6.8.La(III), normally considered a suitable diamagneticcontrol for experiments with Gd(MH), apparentlybinds to Fv fragment with a similar affinity to Gd(III)at pH5.1 (KM; 500,pM), but binds an order ofmagni-tude more weakly than Gd(III) at pH6.8(KMT 100pM). This implies some slightly differentmechanism of binding for the two metals. From theslope of the Gd(III)-binding data it seems likely thattwo or more groups with overlapping ionizations areinvolved in the binding. To explain the discrepancy

1976

40


with La(II), a possibility is that some parameter such characteristic fluorescence change. This is inconsis-as ionic radius of the lanthanide is critical for binding tent with simple displacement ofhapten by metal andat the multiply ionized site (i.e. at high pH) but less suggests that a ternary complex is formed. The datacritical for binding in the partly ionized site (i.e. at may be analysed on this basis according to the theorypH5.1). Nevertheless, a group of pK> 6.2 seems to developed in Appendix I to give the dissociationbe implicated in the binding of both lanthanides. constants of hapten and Gd(III) to Fv fragment in

Eu(III) gave no detectable fluorescence change on the absence (KH and KM) and the presence (KMH andbinding at pH5.9, which was therefore studied by KHM) of the other ligand. In the analysis the assump-measuring the apparent binding of Gd(III) in the tions [H]free. [H]totaj and (M]free; [Mitotat havepresence of Eu(III) or by reversing the Gd(III) en- been made, which are valid since for most of thehancements by Eu(JII). By assuming purely competi- data [M]total, [H]total .7[A]total = 0.37juM.tive behaviour, the binding constant was obtained Appendix I predicts that the decrease in proteinfrom the formula: fluorescence (AF) as a function of hapten concentra-

KM,app = Km(l + [I]/K1) tion for different (constant) Gd(III) concentrationswill give a set of hyperbolae (Appendix I, eqn. 7).

where [I] and K1 are the concentration and dis- This is verified by the double-reciprocal plots derivedsociation constant of the competing lanthanide. At from Fig. 2 (Fig. 3). The apparent binding constantspH5.9, Eu(III) was found to have a very similar bind- [KD(M)] for each different Gd(III) concentration areing constant (50-80flM) to that of Gd(III) (40pM). replotted in the inset as a function of the Gd(II)

Ca(II), which is an important factor in complement concentration. The inset also shows a plot of totalfixation (Lepow et al., 1963), does not apparently fluorescence at infinite hapten concentration [F'(M)]compete for the lanthanide-binding site. At pH6.8, (obtained from the extrapolated values of AFO(M)2.OmM-Ca(II) has no effect on protein fluorescence in the double-reciprocal plots] as a function ofGd(III)and does not alter the Gd(III)-binding constant concentration. This secondary plot of F'(M) may be(KM = 20M). analysed according to eqn. (15) ofAppendix I to give

(c) Binding studies in the presence of both hapten KHM. The secondary plot of KD(M) may be analysedand lanthanide. At both pH5.5 and 6.8 the binding of according to eqn. (11) of Appendix I to give LH,hapten (I) is substantially affected by the presence of KMH and KHM. The values of KHM determined fromGd(III), and vice versa. Fig. 2 shows that the presence the two methods, i.e. from the Gd(III) concentrationof Gd(III) both weakens the apparent binding con- for half-maximal change in KD(M) (0.50mM) and forstant of hapten and decreases the apparent fluor- half-maximal change in F'(M) (0.80mM), agreeescence change on hapten binding. At the highest fairly well and give some indication of the errors inmetal concentrations, a limit is approached with a the methods. The value for the fluorescence enhance-characteristic apparent binding constant and a ment in the ternary complex may also be obtained

from the replot ofF'(M) and is eAMH = fAMH/fA = 0.71.The calculated values of the binding constants aresummarized in Table 1.

80 , X , r I T Two points are apparent from the data in Table 1.First, since the model predicts that the four bindingconstants should be related according to the equation

60 KMKMH = KHKHM, and all four binding constants*44= > § A may be independently determined, the data may beCd 4.2

40 4 *, = = = _shown to be reasonably consistent with the assumed0e _ MX>,,0.531.06 model, noting that the experimental determinations

55 0CS50.27 of the binding constants may show as much as 50%10! 20 Y°°046 00'133 1 scatter. (This is particularly true for values ofKM and

KHM, since the observed changes in fluorescence onaddition of metal are small.) Second, the presence of

0 4 8 12 16 20 24 hapten is seen to weaken the binding of Gd(III) by afactor of 10-20-fold and vice versa. The analysis con-[Hapten] (pM) firms that this antagonism is an indirect effect, i.e. not

Fig. 2. Antagonism of hapten (I) and Gd(III) binding to a direct competition of hapten and Gd(1II) for theFv fragment at pH6.8 same site. This implies that the hapten and the Gd(III)

The apparent binding of hapten (I) to Fv fragment must have distinct binding sites on the Fv fragment.(0.38juM) was monitored by the protein fluorescence quen- This is consistent with previous water proton-ching at 340nm at several fixed Gd(III) concentrations relaxation-rate studies on the effect of Dnp-aspartate[value (mM) beside each line], in 0.05M-Pipes buffer/ on the binding of Gd(III) to Fv fragment (DwekD.15M-NaCI at 25°C. et al., 1974). The interaction between the two distinct

Vol. 155

41

I

41

41

I

I

1

7

c

I


4.0

3.0- ~~~~~~~1.0

0 2 3 4

[Gd(TIr)] (mM)

4.2

I

Ce,.

0.0440.016

-I.0-0.8-0.6-0.4-0.2 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

1/[HaptenJ (#M-1)

Fig. 3. Graphical treatment ofdata for the antagonism ofhapten (I) and Gd(III) binding to Fv fragment at pH6.8

Double-reciprocal plots ofthe curves in Fig. 2 for the apparent binding ofhapten (I) to Fv fragment (0.38pM) at several fixedGd(III) concentrations [value (mM) beside each linej. The inset shows replots from the intercepts ofthe apparent dissociationconstant for hapten (I) (o, K'D(M)] and the total fluorescence change [A, '(M)] as a function of Gd(If) concentration.

1-%'4

5 lo 15

[Gd(III)] (mM)

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1.0* l~~~~~~~~~~/[Hapten](,llM-Fig. 4. Antagonism ofhapten (I) and Gd(III) binding to Fv fragment at pH5.5

Double-reciprocal plots of the apparent binding of hapten (I) to Fv fragment (0.57pM) were determined by protein-fluorescence quenching at 340nm at several-fixed Gd(III) concentrations [value (mn) beside each line] in 0.05M-Pipesbuffer/O.15M-NaCl at 25°C. The inset shows a replot of the apparent dissociation constant for hapten (I) [K'D(M)] as a func-tion of Gd(TII) concentration.

1976

42


binding sites could be a direct interaction between twoadjacent sites (i.e. partial overlap of the sites) or anindirect interaction (mediated by the protein)between two widely separated sites.The analysis was repeated at pH5.45 (see Fig. 4).

A comparison of the calculated binding constants atthe two pH values (see Table 1) shows two interestingresults. The binding constant of hapten feither in thepresence or in the absence of Gd(III)] is insensitiveto pH in the range 5-7. However, the binding con-stant ofmetal (in both the presence and the absence ofhapten) is quite sensitive to pH. This is consistentwith the results of the previous sections.

La(III) gives similar effects to those of Gd(JII) atpH6.9 (see Table 1), except that a higher concentra-tion of La(III) is necsary, because of the intrinsic-ally weaker binding constant of La(III) to Fv frag-ment. The behaviour of hapten (II) is also similar tothat of hapten (I) (see Table 1).

(d) Time-dependent changes on binding lantha-nides and haptens to Fv fragment. In the absence ofGd(III) the fluorescence quenching on addition ofhaptens occurs rapidly on the time-scale requiredfor a measurement (approx. 10s). However, in thepresence of Gd(III), the fluorescence quenching onthe addition of hapten is markedly slower and re-

quires a period of minutes to reach a new equilibrium.Similarly, the fluorescence change on the addition ofGd(III) requires minutes to equilibrate. This is shownin Fig. 5 for the consecutive addition of Gd(III)(different concentrations) and hapten (3.3.uM) atpH 5.5. In each case, there is a rapid fall of thefluorescence on the addition of hapten, followed by afurther slow decrease. The slow decrease is alwayscharacterized by a single exponential half-life (z) asindicated by the semi-log plots shown in the inset toFig. 5. The addition of EDTA completely reversesthe Gd(III)-induced changes (in both the presenceand the absence of hapten), indicating that the reac-tions are reversible. This is further substantiated bythe observation that r is a function only of the finalmetal and hapten concentration and is not affected byadding metal and hapten in the reverse order (seeFig. 6).

Since the binding equilibria involving the haptenand the metal would be expected to be rapid (Eigen& Hammes, 1963; Hammes, 1968), the slow changessuggest that in the presence of metal a slow equili-brium between two or more different conformationsof the protein is displaced. The rapid changes in thepresence of hapten [but not Gd(III)] suggest that thehapten binds preferentially to a conformational

0

Cu._

.'

Cu

0D

._$-

CZ

.aucoV)u

0 I00 200 0 100 200 300 4

Time (s)400

Fig. 5. Relaxation ofFv-proteinfluorescence in thepresence ofGd(ll) atpH5.5The time-dependence is shown of the fluorescence intensity at 340nm on a.ddition of Gd(II) and the further change onaddition of a fixed concentration of hapten (I) at several Gd(III) concentrations in 0.05M-Pipes buffer/O.15M-NaCl at 25°C.Fv fragment (Fv; 0.5 M) was added at the time indicated by the arrow. Gd(III) was added at the following concentrations:(1), O.13mm; (2), 0.27mM; (3), 0.53mM; (4), 1.06mM; (5), 2.10mM. Hapten (I) (3.3gM) was added at t-O for part (b). Theinset shows semi-log plots of the second change (AF) after addition of hapten.

Vol, 155

31

-


0.04

0.02

(a)

-

0 2

[Gd(II)] (mM)0.04

0.02

(b)

IR

-I - - @

0 10 20 30

[Hapten] (#uM)Fig. 6. Dependence of the relaxation of Fv-fragmentfluorescence on (a) Gd(III) and (b) hapten (I) concentra-

tions atpH5.5

The reciprocal of the half-time for the fluorescence change(r) is plotted against Gd(III) concentration in the presence(----) and absence ( ) of 3.3puM-hapten (I) (a) andagainst hapten concentration in the presence of 1.06mM-Gd(HI) (b). The curves are computed fits (see the text).Protein concentrations were 0.3-0.5pM (@) or 1-3pM (O).Relaxation times in the presence of both ligands weredetermined with the hapten added last, except for onemeasurement where the addition was reversed (A).

must, however, be pH-dependent, since r is muchsmaller at pH7 than at pH5.5.Appendix II shows a method for the quantitative

analysis of this slow relaxation based on a two-statemodel. The analysis confirms that, for a given haptenand metal concentration, the relaxation processescan always be described by a single T value (AppendixII, eqn. 8). In principle, a detailed treatment of thefluorescence changes and their half-times as a func-tion of metal and hapten concentrations could giveestimates of the intrinsic dissociation constants Kmand Km, for binding to the two states of the proteinseparately, for which the constants derived in Appen-dix I (KM etc.) are weighted averages. The forward andbackward velocity constants of the isomerizationk+1 and k-L could also be obtained. Thus the kinetictreatment can potentially give more informationthan the equilibrium treatment. Here we consideronly a limited amount of data, sufficient to establishthe validity of the two-state treatment as a firstapproximation; to determine the constants of thesystem uniquely rapid reaction techniques, such astemperature jump, will probably be required inaddition.

Fig. 6(a) shows the dependence of r-' (see Appen-dix II) on Gd(III) concentration in the presence andabsence of hapten. Fig. 6(b) shows the dependence ofzr- on hapten concentration in the presence ofGd(III). Eqn. (5) of Appendix II predicts that thechange in r-1 (relative to the value at zero metal con-centration) should be hyperbolic. For the case withno hapten present, eqn. (5) simplifies to:

A(ir1)=['c(M) ~71(k)=+1( - 1) [M]Km+ [M]

state that predominates in the solution. The mixtureof slow and fast changes observed in the presence ofGd(III) after the addition of hapten (see Fig. 5) canthen be qualitatively understood in terms of the hap-ten binding rapidly to one conformation and thendisplacing the equilibrium between the two conforma-tions. Adding metal to a solution containing haptenand Fv fragment gives only the slow change, as mightbe expected, since the starting equilibrium then lieswell to one side.

T is independent (±10%) of protein concentrationin the range 0.3-3.0M both with Gd(III) alone andwith Gd(III) plus hapten, indicating that polymeriza-tion is unlikely to be an important factor in the equi-libria. In addition, ultracentrifuge studies with 80pM-Fv fragment indicate that there is negligible poly-merization in the presence of Gd(III), hapten, or

both together. Hence the changes probably representan isomerization of the protein. This isomerization

The metal concentration for half-maximal changegives Km directly, and the starting and final values ofr-I give (k+1+k-L) and k-1+k+1 (Kn/K,) respect-ively. Eqn. (10) enables us to relate these constantsto the already determined statistical dissociationconstant KM. Putting k+l/k-l = L, the isomerizationconstant, and KmIKm' = c, the ratio of intrinsic dis-sociation constants for the two states, it can readilybe seen that:

x (M00) 1 + LcT (M=0) 1+L

From the data of Fig. 6, it is difficult to obtainaccurate estimates of T-N(0) and Km. The continuousline is computed for Km= 80juM, -r1(O) = 0.1 5s-,r-'(M = oo) = 0.015s-1, but it is possible to varyKm and T-1(0) over a twofold range and still obtain afit within the experimental limits of the data. Itshould be noted that the errors are particularly largeat low metal concentrations both because T is short

1976

44


and also because the fluorescence enhancement(maximum 15 Y.) is considerably decreased. L and ccannot be determined independently from Fig. 6,but by using the estimate (1+Lc)/(1+L) 0.1, thelimits can be placed as 0 < c < 0. 1, with the correspond-ing limits for L of 9 <L < oo. Physically, this suggeststhat in the absence of hapten the protein ispredominantly (>90%) in one form (A'), and thatGd(III) then binds almost exclusively (at least a factorof 10 more strongly) to the other form (A). The lackof time-dependence in the absence ofGd(III) suggeststhat the hapten preferentially binds to the form thatpredominates in free solution (A').The conclusions are obviously consistent with the

conclusions from the equilibrium-binding studies,but provide much more insight into the apparentantagonism between the two binding sites. In terms ofthe above model, the Fv fragment exists free in solu-tion predominantly in the form that binds the haptentightly and the metal ion weakly. The addition ofmetal ion then shifts the equilibrium towards theconformation that binds the metal ion tightly andthe hapten weakly, producing an apparent weakeningof the hapten binding constant. The addition of hap-ten would have a reciprocal effect. Thus, whereas thephysiological importance of the lanthanide bindingto Fv fragment remains to be determined, the bindingproperties of the lanthanide may be used as a monitorof subtle conformational changes in the Fv fragmentthat occur on hapten binding.

Binding ofTb(III) to Fvfragment by Tb(III) studiedby fluorescence enhancement. The addition of Fvfragment to Tb(III) solutions causes little change inthe fluorescence-excitation spectrum (emission at540nm) except for a large increase in a band centredon 280nm. Titrations of the 540nm emission band(excitation 280nm) with Tb(III) (0-500,pM) werecarried out in the presence and absence of protein(2AM) at pH5.6. The results suggest that Tb(III)binding (KM 300,pM) is substantially weaker thanGd(III) binding and that the fluorescence of thebound Tb(III) is enhanced by a factor of 100-200(assuming a 1:1 stoicheiometry). The excitationspectrum suggests that the mechanism of enhance-ment is energy transfer from tryptophan to the metal.

Electron spin resonance

Binding of spin-label haptens, and the binding oflanthanides in the presence of spin-label haptens.(a) Haptens. The e.s.r. signals of haptens (I) and (II)change on binding to Fv fragment from the narrowlines characteristic of a mobile label to broader linescharacteristic of a less mobile label, particularly forhapten (II). The use of e.s.r. spectra of a series ofhaptens to probe the geometry, rigidity and polarityof the combining site has been described (Dweket al., 1975a,b). Because of the very high intensity of

Vol. 155

the free-label signal in comparison with the bound-label signal it is impossible to observe the latterdirectly except at the extremes of magnetic field. Forthis reason label II, which gives a greater peak-to-peakseparation, is more convenient to monitor than labelI. The intensity of the narrow high-field (right-hand)line can, on the other hand, be used directly as ameasure of the free hapten concentration in the pre-sence of bound hapten, since the contribution ofimmobilized labels to this region of the spectrum isnegligible. Fig. 7 illustrates the method for simul-taneously monitoring the free and bound hapten in atitration of a fixed total concentration of hapten (II)(20,uM) with an increasing concentration of Fv frag-ment (0-60,pM) in 0.1M-Pipes buffer/O.15M-NaCI,pH6.9. The inset shows the decrease in signal fromthe free hapten as the protein concentration is in-creased. The data are consistent with 1:1 stoicheio-metry and a dissociation constant (KH) of 0.3-1.O0UM,in good agreement with fluorescence titrations (Table1). In this titration the concentration of ethanol wasconstant and as low as possible for addition of thehapten (approx. 1%).

If the titration is performed in reverse (increasingthe hapten concentration at fixed protein concentra-tion) or the signal from bound hapten is monitored,small but distinct apparent deviations from the ex-pected stoicheiometry occur. They may partly beconnected with the need to add relatively large con-centrations of ethanol (see above) or, for hapten (I)only, with the limited solubility in aqueous solutions(approx. 30,4M), but could also indicate heterogeneityofthe bound hapten with regard to spectral character-istics. This might arise if the hapten bound weakly ata second site or could bind to two conformationalstates of the protein and displace the equilibriumbetween them. These complications stress thatchanges in the signal of the bound hapten must beinterpreted with caution.

(b) Lanthanides. (i) Gd(III). When Gd(III) is addedto solutions ofFv fragment (Q0-30,uM) atpH 6.9 in thepresence of a moderate concentration of hapten (I)or (IL) (25AuM) changes in intensity of the signal fromboth bound and free hapten occur. The signal ofbound hapten decreases hyperbolically as a functionof Gd(IH) concentration with an apparent KM of600-800,UM and a limiting intensity of <0.2 of thestarting value. The signal of free hapten increasesover the same range of metal concentrations, thenfalls steadily at higher concentrations of metal(>2mM). We interpret the initial rise as being due todisplacement of hapten from the protein as the metalbinds. The subsequent fall is probably due to dipolarinteractions between the two paramagnetic species infree solution, since a control containing no proteingives a very similar decrease. The decrease in signalfrom the bound hapten may be due to other causesthan displacement, and this is discussed below.

45


CUC

Cd200('to)

100

0 20 40 60

Total [Fv fragment] (#M)

Fig. 7. Changes in the e.s.r. spectrum ofhapten (II) binding to Fvfragment

The e.s.r. spectrum [(a) bound-hapten signals, (b) free-hapten signals] of a fixed total concentration of hapten (II) (20pM)is recorded at increasing concentrations ofFv fragment (0-60AM) in 0.05M-Pipes buffer (pH6.9)/0.15M-NaCl at 20°C. Theinset shows the binding of hapten (II) to Fv fragment as monitored by the e.s.r. signal of the free hapten. The bar representsa magnetic yield of t mT.

The displacement can be explained in terms of theabove fluorescence analysis. Monitoring the high-field line provides estimates of the concentrations offree hapten and (by difference from the total haptenadded) of bound hapten. The amount of hapten dis-placed from the protein by a saturating concentrationof Gd(III) can similarly be estimated. The protein isthen in only two forms (AM and.AMH) and so thedissociation constant, KMH= [AM][H]/[AMH], canbe estiated. The values for K, for haptens (I) and(II) are 5-10pM, in agreement with the results fromfluorescence measurements (Table 1).With hapten (I) it is impossible to ensure 100%

saturating conditions with metal present because ofthe limited solubility (3OuM), but this difficulty can beavoided with the five-membered-ring hapten (1I),which is soluble in aqueous solutions to greater than100pM. Under these conditions there is no detectabledisplacement ofhapten fromthe protein when Gd(JII)binds, but there is still a decrease in signal from thebound hapten, which- is hyperbolic and character-ized by limiting relative amplitude of 0.4 andKM,,m. =1100pM at pH6.9. This must reflect anintrinsic change in the AH complex on bindingGd(III). The value I00p,m is of the same order as thedissociation constant KHiM = [AH][M]/[AHM] deter-mined by fluorescence (Table 1). The e.s.r. results

thus appear to support the binding scheme proposedabove.

Effect ofpH on Gd(1II) titrations. A series of fourtitrations ofFv fragment with Gd(III) were performedin the pH range 5.9-7.0 in the presence of concentra-tions of free hapten (I) near to its solubility limit(approx. 30#m). The bound and free hapten wereboth monitored as above. The changes in free haptenwere used to estimate the decrease expected in thesignal ofbound hapten at infinite metal concentrationdue purely to displacement. In every case the dis-placement of hapten from the bound site was nomore than 30% of the total bound hapten, whereasthe observed signal decrease was much larger(50-80%). After correction for this displacement,the relative amplitude of the e.s.r. spectrum for thebound site (with metal present) was found to varyfrom 0.3-0.4 at pH7.0 to 0.7 at pH5.9. Thus theremust be a pH-dependent change affecting the signalfrom the immobilized hapten in the bound site in thepresence of metal. KM. p. for Gd(IM) increases from0.8mm at pH7.0 to 4.0 M at pH5.9, which corre-sponds satisfactorily to the variation in KHM detectedby fluorescence.

(ii) Other lanthanides. The above results indicatethat there is a real decrease in the broad e.s.r. signalfrom bound haptens on the formation of the ternary

1976

46

LANTHANIDE AND-HAPTEN BINDING TO FRAGMENT Fv FROM MOPC 315

complex with Gd(III). This could occur because ofmagnetic dipolar interactions (Leigh, 1970; Dwek,1973) between the bound Gd(III) and the boundnitroxide. However, in view of the earlier studies asecond possibility, that the metal may induce a changein mobility of the hapten, which is reflected in theshape of the e.s.r. spectrum, must be considered.The effects of titrating Fv fragment (354uM) with

La(III), Eu(III) and Gd(III) on the e.s.r. spectrum ofbound hapten (II) were compared at pH6.9 underconditions where no displacement occurs (80100-lOM-hapten). With the diamagnetic La(III) there is a de-crease in signal on binding, though much smallerthan with Gd(III) (limiting relative amplitude = 0.65)with KM,app.t4mm. Also, the binding of Eu(III)causes an apparent quenching (limiting amplitude= 0.5, KM,app. = 2.0mM). However, diamagneticLa(III) cannot cause paramagnetic quenching andEu(1II) would be expected to produce a significantlysmaller paramagnetic quenching than Gd(III), be-cause of the large difference in their electron-spin-relaxation times (Dwek, 1973).

It would then appear that a significant amount ofthe apparent quenching of the e.s.r. spectrum fromthe bound spin-label signal is due to a mechanismdistinct from the paramagnetic quenching effect.This could be due to a further immobilization of thenitroxide moiety on binding of the lanthanides,causing a further broadening of the e.s.r. signal ofthe bound spin label. Because of the partial overlapof the e.s.r. spectra from the bound and free spin-labelled haptens, it is difficult to ascertain directly ifthe width of the bound nitroxide spectrum altersunder these conditions. If, however, the effects of thediamagnetic La(III) are taken as a control for the'conformational' effect of lanthanide binding on thee.s.r. spectrum of the bound spin label, the extraquenching effect (0.4/0.65 0.6) of the paramagneticGd(III) may be used as a first approximation to theparamagnetic contribution to the quenching. If theestimate for the eectron-spin-relaxation time ofGd(lll) of 0.l5ns (Jones et al., 1974) is assumed, anestimate of 1 .2nmfor the distance between the Gd(II)and the nitroxide can be made (Dwek, 1973).

This distance estimate must be considered verytentative because of the small difference in quenchingbetween Gd(III) and the La(III) control and becauseof the anomalous behaviour with Eu(III). Thetheory of paramagnetic quenching of the boundnitroxide e.s.r. spectrum by paramagnetic ions(Leigh, 1970) suggests that Eu(III) should producenegligible quenching relative to the La(III) control.That this is not so may mean either that the effects areentirely 'conformational' or that there is some othermechanism of quenching with paramagnetic ionswith very short electron-spin-relaxation times, suchas Eu(IIl), that may have to be considered. Such amechanism may be unimportant for Gd(1II), which

Vol. 155

has a long electron-spin-relaxation time, but untilsufficient model systems have been studied with avariety of lanthanides it is not really possible toevaluate this.The fluorescence and the e.s.r. results, taken to-

gether, confirm the existence of a specific lanthanide-binding site on Fv fragment. This site interacts, eitherdirectly or indirectly, with the hapten-combining site.The magnitude ofthe lanthanide-binding constants inthe absence of haptens, and their pH-dependence,suggests that at least two ionizable groups on theprotein are involved in binding the metal. The knownproperties of lanthanides in other systems (Phillips& Williams, 1966; Williams, 1974) further suggestthat these groups are probably carboxylate ions.

High-resolution n.m.r. studies

The 270MHz proton spectra of the Fv fragment(1.2mM) at pH4.33 in the presence and absence ofLa(III) (19mM) are shown in Fig. 8. The differencespectrum resulting from the presence of La(III) hasan intensity less than 5% of that of the Fv fragmentspectrum and is also shown in Fig. 8. This suggeststhat any conformational changes resulting from theaddition of metal are relatively small and probablyinvolve few residues. The advantage therefore ofn.m.r. studies over the fluorescence studies is clearlyseen; n.m.r. gives direct structural information. Al-though the fluorescence studies allow the time-dependence of the conformational changes in thepresence ofmetal to be monitored, they give no infor-mation on the extent of any such changes, which arenow shown to be relatively small.

Fig. 9 shows the titration curves of the chemicalshift of the C-2 and C4 protons versus pH for thethree histidine residues in the Fv fragment. Thetitration curves in the absence of La(1II) are in goodagreement with those previously published (Dweket al., 1975a,b). The addition of 19mm-La(III)results in both chemical shifts and a slight decrease inPKa value (from 6.9 to 6.4) of one of these histidineresidues. The high concentration of La(111) used inthese experiments is necessary because of the weakmetal-binding constants, particularly at low pH.At pH values ;6.5 the binding of La(III) is aboutfive times stronger than at pH5.0 and a limited titra-tion using 21m-La(III) between pH values 6.5 and8.5 gave results in excellent agreement with thoseobtained at the higher La(lII) concentrations. Thisshows that any effects caused by a change in ionicstrength from the introduction of a relatively highconcentration of La(III) are small.The slight decrease in the pKa of the histidine

residue (0.5 unit) shows that La(III) does not competewith a proton for the nitrogen-binding site on thehistidine ring. If that were the case, the addition ofLa(1II) would be expected to decrease the binding

47


(b)

.vf1vv.

I I10 5 0

p.p.m.

Fig. 8. 270MHzproton spectrum ofFvfragmentfrom protein MOPC 315

The concentration of the Fv fragment was 1.2mM and the sample contained 0.15M-NaCI. The spectra were recorded at30°C and pH4.33. (a), No addition; (b), in the presence of l9mM-La(II1); (c) (a-b), difference spectrum at normal intensity;(d) (a-b) x 4 enlarged difference spectrum.

constant of the proton by a factor of {I + [La(III)]/KLa(III)}, where KLa(II) is the binding constant ofLa(III) to the Fv fragment in the high-pH regionabove the pKa of 6.2 as determined by fluorescence.Since this binding constant has been determined to be100pM, the addition of l9mM-La(III) would lead to a191-fold decrease in the proton-binding constant or adecrease in the pKa value by 2.3 units. This is not con-sistent with the observed decrease in the histidine pKavalue of only 0.5 unit. This result is not really sur-prising, since the chemistry of the lanthanides is suchthat they would not be expected to bind to nitrogenligands such as histidine (Phillips & Williams, 1966).However, the change in chemical shift and pKa of thishistidine residue and also the small number of resi-dues in the Fv fragment perturbed on adding La(III)would suggest that this histidine residue is in thevicinity of the metal ion. It is also worth noting thatthe presence of La(III) broadens the C-2(H) reson-ance of this histidine residue and to a lesser extent theC-4(H) proton. This could result from either a metal-induced conformational change bringing one ormore protons near to this histidine residue with con-

sequent dipolar broadening, or from chemical ex-change phenomena (Dwek, 1973). In the latter casethe addition of La(III) would have to result in thehistidine residue existing in two environments withthe time-scale for exchange between them being inthe slow to intermediate region on the n.m.r. time-scale (Dwek, 1973). This could happen, for instance,if one of the La(III)-chelating groups moves backand forth in a 'breathing' movement with consequentalterations in the environment of the histidine.A general broadening ofthe proton n.m.r. spectrum

of Fv fragment also results from the presence ofPr(III), which is much more pronounced than withLa(III). Titrating Pr(III) at pHI4.8 into Fv fragment(1.1 mM) results initially in shifts in the aliphaticregion and 6-CH2 region of the proton n.m.r.spectrum of the Fv fragment. The fraction of reson-ances shifted increases as the Pr(III) concentration isincreased to 0.92mM, consistent with slow exchange(Dwek, 1973). Throughout this range, however,there appear to be few shifts in the aromatic region.As the Pr(III) concentration is further increased thereare still detectable shifts in the aliphatic region, but a

1976

48

(c)

(


8.0 IP

--4._

g._

CO

'a

7.5

7.0

4 5 6pH

Fig. 9. Titration of the histidine chenfragmentfrom protein MOPC 315 in j

the presence (O) of saturatFor details see the text. The C4 proresidue with pKa 8.2 were not resolvLa(III).

general broadening of the wholeapparent. This broadening increicontinues up to l9mM-Pr(III), anvery difficult to observe any chanresidue resonances. In general Prcause shifts in nuclear resonancesThe observed broadenings, l

aromatic region, are more chaiexpected for paramagnetic metaltron-spin relaxation times. One p4which may account for this broadeical exchange phenomenon involbetween Pr(III) and the aromatiiferent environments of the aromience different pseudo-contact swould, of course, be in additiomerely from the presence of th4La(III) should provide the apprcblank.] Again if the relative motthis would lead to broadeningAlternatively or additionally, therinteraction between the Pr(II1) alwhich would result in broadeninmuch the same way as has been otlabel hapten (Dwek et al., 197RVol. 155

the broadenings with Pr(III) are unexpected, andsuch effects may present a limitation on the use oflanthanide ions to obtain geometrical information insolution in some cases. The mechanism of broadeningis unclear and thus further investigations to test thegenerality of the effects, to find out in which systemsthey are most likely to be observable, and to test thevalidity of theoretical models such as the one pro-posed by Gueron (1975), are merited.

Conclusion

The magnitude of the binding constant of metal tothe Fv fragment would, by comparison with modelcompounds, suggest that the metal is co-ordinatedto at least two carboxyl groupings on the Fv frag-ment. The group with the PKa value of about 6.2(determined by fluorescence) associated with metalbinding is not a histidine residue and is therefore mostprobably a carboxyl group of one residue actingas part of a 'dibasic' acid. This situation has beenfound in lysozyme for the lanthanide-binding site,which is between aspartate-52 and glutamate-35

7 8 9 (Blake & Rabstein, personal communications). Thehistidine residue which has pKa. 6.9 in the absence

nical shifts for the Fv of lanthanide is most probably in the vicinity ofthe absence (o) and in the lanthanide-binding site. The conformationalring La(III) changes on metal binding as detected by fluorescence)tons for the histidine studies have been shown by high-resolution n.m.r.

(ed in the presence of studies to involve only a few residues, suggesting thatthey are reasonably localized. The antagonistic effectsof hapten and metal binding suggest that the twobinding sites are close. Confirmation of this comes

n.m.r. spectrum is from the e.s.r. studies with lanthanides in the presenceases as the titration of a spin-labelled hapten, which give a distance ofId consequently it is 1.2nm between the lanthanide-binding site and theges in the histidine- nitroxide moiety. The Fv fragment exists in at leastr(III) is expected to two different conformers, each with different affinities(Dwek, 1973). for hapten and metal. In the absence of either haptenparticularly in the or metal, the Fv fragment exists predominantlyracteristic of those (>90%) in one conformation. The hapten bindsions with long elec- preferentially to this form, whereas the metal bindsossible explanation, preferentially to the other form. Since the lantha-Dning, is some chem- nides and the haptens both bind tightly and specific-ving relative motion ally, this suggests that the myeloma protein mayc residues. The dif- exist in a multiplicity offorms, each form displaying aatic residues experi- specificity for a different class of ligands.shifts. [Such shifts'n to any resultinge metal, for which)priate diamagneticLion is slow enoughof the resonances.e could be a dipolarnd nearby residues,kg of resonances, inbserved for the spin-5a,b). Nevertheless,

We thank Professor R. R. Porter, F.R.S., ProfessorD. C. Phillips, F.R.S., Professor R. J. P. Williams, F.R.S.,and Miss Elizabeth M. Press for their encouragement andinterest in this work. We also thank the Medical ResearchCouncil and Science Research Council for their support.R. A. D. thanks the Royal Society for the award ofa LockeFellowship. A. C. M. is a recipient of a post-doctoralfellowship from the Canadian Medical Research Council.We thank Dr. I. D. Campbell for many helpful discussionson high-resolution n.m.r. and J.E.O.L. Co. Ltd. for theloan of an e.s.r. spectrometer.

49

50 R. A. DWEK AND OTHERS

Refeces

Dwek, R. A. (1973)Nackear Magneti Resonne io-,.-chemistry: Application to En.zyme Systems, ClarendonPress, Oxford

Dwek, R. A., Knott, J. A., McLaughlin, A. C., Myatt,R. W., Press, E. M., IPice, N. C., Richards, R. E. &White, A. I. (1974) Proc. Rare Earth Res. Conf. 1Ith 1,184-193

Dwek, R. A., Knott, J. C. A., Marsh, D., McLaughlin,A. C., Press, E. M., Price, N. C. & White, A. I. (1975a)Euir. J. Biochlem. 53, 25-39

Dwek, R. A., Jones, R., Marsh, D., McLaughlin, A. C.,Press, E. M., Price, N. C. & White, A. I. (1975b)Philos.Trans. R, Soc. London Ser. B 372, 53-74

Eigen, M. & Hanunes, G. G. (1963) Adv, Enzymol. Relat.Areas Mol. Biol. 25, 1-38

Gueron, M. (1975) J. Magn. Reson. 19, 58-66Hammes, G. G. (1968) Acc. Chem. Res 11,321-329Haselkorn, D., Friedman, S., Givol, D. & Pecht, I. (1974)

Biochemistry 13, 2210-2222Hochman, J., Inbar, D. & Givol, D. (1973) Biochemistry

12, 1130-1135Jones, R., Dwek, R. A. & Fors6n, S. (1974) Eur. J. Bio-

chlem. 47, 271-283Leigh, J. S., Jr. (1970) J. Chem. Phys. 52, 2608-2612Lepow, I. H., Naff, G. B., Todd, E. W., Pensky, J. &

Hinz, C. F. (1963) J. Exp. Med. 117, 938-1008Phillips, C. S. G. & Williams, R. J. P. (1966) Inorganic

Chemistry, vols. 1 and 2, Clarendon Press, OxfordVelick, S. F., Parker, C. W. & Eisen, H. N. (1960) Proc.

Natl. Acad. Sci. U.S.A. 46, 1470-1482Williams, R. J. P. (1974) Recent Res. Cancer Results48,1-11

APPENDIX I

Denivation of the Equations for the Fluorescence Titrations of FvFragment with Haptens and Metal Ions

In the thermodynamic scheme (Scheme 1), Arepresents the myeloma protein fragment (Fv), H thehapten and M the lanthanide metal. The appropriatedissociation constants are defined as shown. As usual,we have the condition KHKHM = KMKMH.From -the conservation equation for A and by

using eqns. (l)-(4) we obtain

[A]= [AlI1 + ] + [M] + [H][M]

KH KM KHKHMwhere [A]- is the total amount of antibody present.The total fluorescence, F, in a solution containing

a fixed amount of antibody, (AlT, and arbitraryamounts ofhapten and metal may be given byF=fA[AI+fAM[AM]+fAH[AH]+fAMH[AMH] (5)

wherefA is the fluorescence per unit concentration ofthe antibody and fAM, fAn and fAMH are similarlydefined. We can therefore write

(M] (H] ~ [H1 M)~]T(fA +fAM f-+AHLHK+t H][n [A]TVA +f~k- +fAny +./'MH KHKHM

(H] [M] [H][M]H KM KH HM

(6)

As in any titrati9n, what is usually measured is thedifference between the initial fluorescence and thefluorescence after the addition of an arbitraryamount of ligand (either metal or hapten). For ouranalyses in this paper we consider titrations in which

Scheme 1. Bindingschenmeformetal, haptenand theantibodyfragmentH [H][A]

A AH KH ~~~~~~~[AH]kh

Km - MJ[A] (2)M M [AM]

km khm

[HJ[AM] 31KMH- [AMH

AM H AMH KHM - [M][AH] (4)

H [AMH]1976


the metal concentration is held constant and thehapten concentration is varied, and define AF as

AF = F(H, M)- F(0, M)

[H]=eAFO(M) -KDM (7)

('+K'(M)}where

We note that a double-reciprocal plot of AK, versusthe metal concentration will give KIM from theextrapolated value on the abscissa and a value of(KMH-KH) on the ordinate. Thus all four bindingconstants in Scheme 1 can be unequivocally deter-mined. The fluorescence yields for each of the fourcomplexes may also be easily obtained.fA is obtainedfrom the fluorescence of an arbitrary concentrationof the myeloma fragment (Fv). fAH and fAM are ob-tained either directly from the fluorescence of an

fAH fAMH[M]\ I [M]\( [M]( 1 +- [Ml IH +HK jF+ _ tA AKM, K KHKH"AFo(M) = [A]T HMH+KK) i +KM fA+ M _ -H H HM/

( KM /\K K Kk[MlAM- AKH KHKI,m

and

[M]1+KM

KL(M)= 1 [M] (9)

KH KMKMH

If the free metal concentration remains constantduring the titration with hapten, then eqn. (7) is a

normal saturation (Michaelis-Menten) curve withan apparent Michaelis constant KD(M), given byeqn. (9), and an apparent total change in fluorescence,AF,,(M), given by eqn. (8). Both KD(M) and AFo(M)depend on the total metal concentration, and in thelimit when M -O- 0, KD = KH. KHM and KMH may be

found by a simple replot of the apparent Michaelisconstant Kj, since

AK, = KD(M) - KD(0)

[M]KHM

= (KMH -KH) [MI

1 +KmKHM

(10)

(11)

Fv-fragment solution in the presence of saturatinghapten and metal respectively or from the standardextrapolation procedures for binding complexes.fAMH is obtained either in a similai direct way or by analternative way as follows. We define F'(M) as

F'(M) = AFo(M) + F(O,M) (12)F'(M) is the total fluorescence intensity at infinitehapten concentration for an arbitrary metal ionconcentration. Then it can be shown that

F'(M) = fAHKHM +fAMH[M] (13)KHM + [Ml

Eqn. (13) may be rearranged into a Michaelis-Menten form by analogy with eqns. (9) and (10), i.e.

AF'(M) = F'(M) - F'(O) (14)

/[M

= (fAMH -fAH) KH[MIKHM

(15)

The usual double-reciprocal plot will then give KHMand (fAMH-fA,l) from the intercepts.

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APPENDIX II

Derivation of the Relaxation Times for the 'Two-State'Model ofFv Fragment

We assume that the Fv fragment exists in two con-formations, designated A and A'. Both forms of Fvfragment are assumed to bind hapten (H) and metalions (M). The reactions in Scheme 1 are considered.The ratio [A']/[A] is given by L. The kinetic equationsmay be written

d[A'] - k+ [A] - k-1[A'] - k+h[H][A']+ k' h[A'H] - k+m[M][A'] + k'n,[A'M] (1)

dA'M]-= k+2[AM]- k-2[A'M]- kLm[A'M]dt+ k4m[A'][M] - k4mh[A'M][H] + kuimh[A'MH] (2)

In principle, we have eight coupled differentialequations. Since the observed relaxation times arelong (of the order of seconds), we assume that thehapten- and the metal-ion-binding processes occurmuch more rapidly than the relaxation between thetwo different forms of Fv fragment. Then the twobinding processes will be essentially at equilibriumduring the relaxation process. Eqns. (1) and (2) canthus be rewritten

d[ = k+ [A] -k-[A']dt

=['M k 2[AM] - k+2AA'M]dt

(3)

(4)

The conservation equation for the Fv fragment maybe written as

[A] + [A'] + [AM] + [A'M] + [AH]+ [A'H] + [AMH] + [A'MH] = [A]T

Using the following equilibrium conditions

K = [A][H][AH]

[A][M][AM]

[AH][M]Khm [AMH]

kt [A'][H]h [A'H]

K' [A'][M][A'M]

[A'H][M][A'MH]

eqn. (3) may be rearranged to give

d[A'] = -'1[A'] +dt :T

k+J[A]T[Ml [H]I [M]1+- +- 1+hK+m K+h K+hm/

where hl-I is defined below.Therefore the relaxation rate for the equilibrationbetween [A] and [A'] is

[MI [H] I []\{l + Km Kh K+ m/) (5)T1 _ k_ +1 M] [H](+ [M]))

+-m+ Kh1 +hml

A'k+m

AA+km1

k+m

AH+M I

I

k-m

k+h

AM+H I

Ik+hm

k-hm

k+mhAM+H

k-.h

AM

AH

AHM

AHM

k+2AM I

k-2

k'+m

A'+ Mk'-m

k'+lxA'+ H I

k'-h

k'+hmA'H + M

k'-hm

k'+mhA'M + H

k'-mh

Scheme 1

1976

52

A/M

A'M

A'H

A'HM

A'HM


It is important to note that these are not the samebinding constants as defined in Appendix I. The bind-ing constants in Appendix I are average values of thebinding constants for the two forms of the Fv frag-ment.The same procedures can be followed to determine

the relaxation rate, T2-', for the equilibration be-tween [AM] and [A'M].

-l (1 ~~+[M,] + [H,] 1 + [,]Km' Km + K4Km(+[]+[H]1 ))

Km Kh Khm,1

What is the relationship of T2 to rT? By using theequilibrium conditions for the metal-binding reac-tions and eqn. (4), we can write

d[A'] Km' d[A'M] KdtdA]= [Km]* d[A M] =L_k-2[A'] + k+2K [A] (7)

Comparing eqn. (7) with eqn. (3) implies that

k-2 =k-K'Km = kK.

Inserting these conditions into eqn. (8) givesT2 = Tj = 'r (8)

This means that the antibody and the antibody-metal complexes will relax with the same time-constant, r, given by eqn. (5). Likewise, a similaranalysis shows that the other antibody complexes,i.e. the antibody-hapten complexes and the anti-body-hapten-metal complexes, will relax with anidentical time-constant. This means that the observedrelaxation should follow a single exponential decay.

Also, we note that the binding constants in Appen-dix I may be related to the intrinsic binding constantsto the two forms of the antibody fragments. Forinstance

1 L1 Kh Th (9)KH 1 +L

1 L1-+1 KmKm

KM 1+LThus the reciprocal of the apparent binding constantin the fluorescence titrations is simply the weightedaverage value of the reciprocals of the binding con-stants to the two different forms of the antibody.

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Documents

Interactions of the Lanthanide- and Hapten-Binding Sites in the Fv