Resonance energy transfer in pepsin conjugates

J. Mol. Biol. (1969) 44, 71-88

Resonance Energy Transfer in Pepsin Conjugates R. A. BADLEY AND F. W. J. TEALE

Department of Biochemietry University of Bir&n&m Ed&a&m, Birmingham, England (Received 30 Decqmber 1968, and in rev&d

form 14 iKay

1969)

A series of pepsin derivetivez wt~ prepared in which a substrate rmalogue containing a chromophore selected to be a resonance energy acceptor from tryptophan wan covalently attached to the active site of the enzyme. The decreasesin pepsin fluorescence intensity and lifetime produced by chromophoree differing in absorption speotrum and intensity could be attributed to resonance energy transfer, and showed that the tryptophan groups emitted independently and had different probabilities of tranzfer to the surface chromophore. The e5Iciency of sensitization and the depolarization of the of fluorescent chromophores by tryptophan, chromophore fiuorezcence zensitized by tryptophan absorption, indicated similar quantum yields and random orientation for these residues. The observed efflcienciez of energy transfer were very much higher than those calculated for model systemz having a randomly diztributed and oriented array of either 5xed or rapidly rotating dipoles transferring energy to one acceptor placed in the surface of the volume containing the dipoles. The reason for this discrepancy in pepsin and two other proteins haa been considered in termz of non-uniform distributions, intertryptophan energy transfer and the low polar&ability of the macromolecular interior.

1. Introduction The general adequacy of the F6rster equation (FGrster, 1969) to describe quantitatively resonance energy transfer of excited states has been confirmed by recent experiments with donor-acceptor paira of fixed separation and random orientation (Latt, Cheung & Blout 1965; Stryer & Haugland, 1967; Gabor, 1968). The transfer rate is a function of the geometry and spectral properties of the componenta, and the appropriate refractive index of the intervening medium ; and since in principle all the parameters can be determined independently, any one can be calculated if those remaining are known. In practice, the mutual orientation of donor-acceptor pairs is the quantity most difficult to determine, unless rapid rotation about flexible linkage effectively randomizes the system during the lifetime of the donor excited state, or transfer occurs from a fixed array of randomly oriented donors to an acceptor group. In proteins, energy transfer measurements may indicate the distribution of fluorescent tryptophan and tyrosine groups within the macromolecule. Generally one is dealing with an array of donors, in which each has fluorescence parameters determined by its particular environment. Since the separation between residues is relatively small, the possibility of energy migration between similar donors mu& also be considered, especially in the case of tyrosine, in addition to the possibility of transfer between tyrosine and tryptophan or between tryptophans. Many aromatic residues are deeply 71

72

R. A. BADLEY

AND

F. W.

J. TEALE

embedded in the polypeptide framework and constitute an array of fixed dipoles, but the rotational freedom of surface groups must be considered. Many examples are known of energy transfer from protein tryptophan to chromophores located in the macromolecular surface, but quantitative interpretation of many of these observations is complicated by multiple acceptors or heterogeneity (Stryer, 1959,1966; Green, 1964; Steiner, Roth & Robbins, 1966; Weber & Daniel, 1966). Very high transfer efficiencies such as are found in haeme proteins (Weber & Teale, 1959) are also disadvantageous, since quantitative information about the donor-acceptor separation is only provided when donor emission and transfer rates are comparable. In order to make useful energy transfer measurements, it is desirable to attach, preferably covalently, a single acceptor group of selected spectral properties to a detlned portion of the macromolecular surface. This can be done conveniently by using the specific substrate-binding properties of an enzyme catalytic site (Haugland & Stryer, 1967). Proteases are especially convenient for this purpose, as they have only one active site, high tryptophan content and bind substrates ranging in size from amino acids to large macromolecules. In this communication, the fluorescence parameters and other properties of pepsin, afEnity-labelled with substrate analogues designed to be tryptophan energy-transfer acceptors, are reported and the observed transfer efficiencies are compared with those calculated for various model systems.

2. Materials and Methods Twice or thrice crystallized swine pepsin from several sources (Sigma, Mann and Worthington) was used without further purification, tests having shown that the material was homogeneous for the purpose of the experimental techniques used. Denatured haemoglobin substrate powder for pepsin assay was a Worthington product. D- and L-j3-phenylalanine and DNSt-L-/3-phenylalanine were obtained from Koch-Light. Other reagents were Analar grade from British Drug Houses or Koch-Light. All solvents were of spectroscopic or Analar grades. Sephadex G25 (coarse grade) was obtained from Pharmacia Ltd. Preparation

of the substrate anulogue diazoketoner!

The diazoketones shown in Fig. 1 were prepared by treating the acid chlorides of Nsubstituted phenylalanine with diazomethane in diethylether. The appearance of the characteristic infrared absorption band at approximately 4.7 pm was used to monitor the reaction,

cl?+ RNH-CH(CH2 CsHs)-COCHN2 + HCl RNH-CH(CHsCsHs)-COCl + CH,Nz _t where R = amino blocking group. N-substituted D-, L- or DL- phenylalanines were prepared by elimination of HX between an amino group and a halide-containing reagent : RI-X + HsN-R2 ---+ RINH-Rp + HX RI = DNP, DNS, PS, TOS, or benzylcarboxymethyl (the residue of phenylalanine). Rs = benzylcarboxymethyl or MNP. BDAP was formed similarly from 2,3-diamino-nL-propionic acid and 2 equivalents of FDNB. Dimsmethane was prepared in ethereal solution (about 0.4 M) from p-tolylsulphonylmethylnitrosamide by the procedure of de Boer & Backer (1903). N-TOS-L-Phe-COCHN2 was prepared by the method of Delpierre BEFruton (1966). N-DNP-Phe (L or D) was prepared by the method of Sanger (1946). N-DNP-Phe-COG? (L or D). Thionyl chloride (2 ml.) was added to N-DNP-Phe (L or D) t Abbreviations used: DNS-,(dansyl) l-dimethylaminonaphthalene-5snlphonyl;HX, hydrogen halide; DNP, 2,4-dinitrophenyl; PS, pyrene-3-aulphonyl; TOS, (tosyl) p-toluene-sulphonyl; MN%‘, m-nitrophenyl; BDAP, 2,3-bis (2,4-dinitrophenylamino) m-proprionic acid; FDNB, l-fluoro2,4-dinitrobenzene; DMF, dimethylformsmide; NBS, N-bromo succinimide.

ENERGY

TRANSFER

IN PEPSIN

Reagent formula

CONJUGATES

Abbreviation for reagent N-TOS-Phe-COCHN2

0

NH-FH-COCHN,

No2

CH2-0

P-

W,

CH3’

N 0 8 0

73

(L or D)

N-MNP-DL-Phe-COCHNz

$H243

N-DNS-L-Phe-COCHN2

S02-NH-CH-COCHN,

N-DNP-Phe-COCHN,

~0,

(L orD)

EDAP-COCHN,

NO2

S02-NH -CH - COCHN,

N-PS-L-Phe-COCHNz

cH2*

FIU. 1. Struotural formulae of the pepsin substrata analogus cl&ok&ones. (3 g) in diethyl ether (40 ml.) at 0°C end stood for 2 hr at 20°C. The product isolated by solvent removal was recrystallized from ether/cyclohexene. Yield = 2-8 g (80%); m.p. = 182 to 184OC. N-DNP-Phe-COCHN, (L or D). N-DNP-Phe-COCl (L or D) (0.6 g) was dissolved in ethereal diazomethane (20 ml,) at 0°C end left overnight at 20%. The solid product obtained by solvent removal was recrystallized from ether/cyclohexane. Yield = 02 g (40%); m.p. = 9OW(d). N-DNS-L-Phe-COCHN, was prepared from N-DNS-L-Phe in a manner similar to that desoribed shove. The acid chloride precipitated from the cold ethereal solution and was obtained by filtration. N-PS-L-P~COCHN~. Pyrene-3.sulphonylchloride was prepared by the method of Vollmann, Beaker, Corell BEStreeok (1937). L-Phenylahmine (O-6 g) and pyrene-3-eulphonylahloride (1 g) were dissolved in acetone (60 ml.) end w&er (40 ml.). Excess solid sodium bicarbonate was added and the mixture shaken for 12 br at 20°C. Acetone was removed on B rotary evaporator after filtration. Aoidiflcation to pH 2 precipitated a yellow solid, isolated by flltretion. After washing with hot chloroform, the product was recrystallized by aoidiflcation of pH 8 aqueous solution. The dried produot (0.6 g) was added to thionyl chloride (0.3 ml.) and DMF (1 ml.) at 4O’C. The produat was isolated by the addition of diethylether at 0°C. The dried solid in DMF (1 ml.) was added to ethereal diazomethane (10 ml.) at 0°C and then stood for 12 hr at room tempemture. After solvent removal and recrystallization from DMF/ether mixture, the semi-crystalline product WBB obtained. N-MNP-DL-P?M-COCHN~. a-Bromo-hydrociie acid was prepared according to Marvel (1966). m-Nitro-aneline (0.8 g) was refluxed in sodium-dried dioxan (30 ml.) together with a 60% sodium hydrid-il dispersion (1 g) for 1 hr under a nitrogen atmosphere. a-Bromohydrocinnamic acid (2.8 g) in dry dioxan (8 ml.) was added and refluxing continued a further hour under the nitrogen atmosphere. After cooling to 2O”C, water

74

R. A. BADLEY

AND

I?. W. J. TEALE

(70 ml.) ~8s added and the solution extrmted twice with petroleum ether (2 x 50 ml.). The aqueous portion we8 further extracted with diethylether (2 x 50 ml.) and then acidified with hydrochloric 8cid. After extraction with methylethylketone (2 x 50 ml.), the combined extracts were dried down 8nd redissolved in diethylether (50 ml.), treated with activated charcoal, filtered and dried with anhydrous magnesium sulphate. Filtration and solvent removal yielded 8 yellow solid which ~8s identified 8s N-MNP-nn-Phe by scidbase behaviour, ultrrwiolet, visible and infrared spectroscopy. The diazoketone ~8s prepared 8s indicated previously. BDAP-COCHN2. nn-2,3-Diaminopropionic acid was prepared sccording to Fischer (1907). The BDAP wa6 formed by the action of FDNB (2.2 equivctlents) on disminopropionic acid in 60% aqueous ethanol saturated with sodium bicarbonate. The product ~8s isolated similarly to N-DNP-L-Phe above. It ~8s identified by acid-base behaviour, ultraviolet, visible and inf&red spectroscopy. Chrometography on Whetman no. 1 paper (LeggettBailey 1962) with sever81 solvent systems indicrsted one component only (m.p. 265”d). The diazoketone was prepared ss indicated previously. Preparation

of the pepsin conjugates

Pepsin oonjugates were prepared by coupling diazoketone to the enzyme in 10% ethanol, acetone or DMF solutions in pH 5.5, lO-%r-8cetate buffer in the presence of cupric ions. The concentrations of pepsin rend cupric ion were kept below 10m4M and 10e3M, respectively, to prevent protein precipitation. In 8 typic81 preparation, pepsin and cupric chloride were dissolved in buffer to which wss edded at room temperature sufficient diazoketone, in the organic solvent, to saturate the solution with reagent. The mixture W&B stood at 4°C Etnd the time for complete reaction, measured by peptic ctctivity and fluorescence quenching, varied from 2 to 24 hr according to the n&rue of the amino blocking group and the solubility of the reegent. Precipitated excess diezoketone wss removed by centrifugation (100,000 g) for 30 mm. 1.5-ml. portions of the clettr supernstant frsction were further puri6ed by passing through 8 pH 5.5 buffer equilibrated Sephadex G25 (cosrse) column (10 cm x 1 cm; void vol. ebout 5 ml.). The conjugate emerged in the 2 ml. 8fter the void volume. The insolubility of some of the diazoketones in aqueous media meant that the dilute conjugates after column fractionation needed concentrating. This ~8s done by lyophilis8tion or v8cuum dialysis.

Methods Ultraviolet absorption spectra were obteined with 8 Gary 14 recording spectrophotometer. Fluorescence excitation ctnd emission spectrs were obtained with a double monochrometor instrument of convention81 design, using 8 stablized D.C. xenon arc (250 W, A.E.I.) 8 Bausch & Lomb 500 mm 1200 lines/mm grating monochromator for excitation, and 8 Beusch t Lomb 260 mm 600 lines/mm monochrometor with 8 selected photomultiplier (E.M.I. 6266A) for detection. Photocurrents were measured with 8 Pye Scalamp gelvanometer or with a combination of phase-sensitive detector, digit81 voltmeter (Weir Electrical Co.) and pen recorder (Control Instruments). Fully corrected spectra were obtained 8s previously described (Teale & Weber, 1957). Absolute quantum yields were determined by the method of Weber & Teale (1957), taking c8re that the glycogen standard gave little forw8rd scattering. Fluorescence decay times were measured by two methods; with a nanosecond deuterium fl8sh lemp and sampling oscilloscope (M8ckey, Pollsck & White, 1965) and also with 8 20-MHz modulation and phase spectrofluorometer, to be described elsewhere. Crtlculations showed th& the error in average lifetime measurements of heterogeneous systems, due to the modul&ion-weighting of each component, ~8s negligible when the longest lifetime present wa8 6 nsec. Excitation and emission polarization spectra were obtained with 8 direct-reading modification of the instrument of Weber (1966), in which anelysis of the fluorescence spectrum could be carried out. Polarized excitation with bsndwidths sm8ller than 1 nm ~8s selected with 8 Bausch BELomb 500 mm 1200 lines/mm monochromator. The cuvette holder could

ENERGY

TRANSFER

IN

PEPSIN

CONJUGATES

75

be controlled by thermoatet between 0 and 60%. Reletive quantum yields were measured by preparing solutions of equal over-all absorption at the exoitation wavelengths; the measured intensity was corrected for solvent absorption and light-scattering. The wave-

length cslibratious of the Cery spectrophotometer end the various grating monochromators were made identical by using murow lines of a mercury arc. Melting of pepsin

pointe (uncorrected) were taken with a Kofler hotbench. The proteolytio was myed by digestion of denatured haemoglobin using a method

activity baaed on

that of Delpierre & Fruton (1966). The tryptophau fluorescence of human plesme albumin was ebolished by treatment with NBS (Teale, 1961). DNS conjugates of this modified albumin and of ovomucoid were prepared with dansyl chloride by the method of Weber (1962). Unless otherwise stated, all pepsin solutions were examined at pH 6.6 in 0.01 n-acetate buffer at 20°C.

3. Results The number of emitting components in pepsin was determined by the matrix method of Weber (1961). The average excitation, and emission values, &ken every 10 nm between 266 to 296 nm and 306 to 366 nm, respectively, with band-widths of 6 nm are shown in Table 1, together with the percentage differences of the 2 x 2 minors. Inspection of the matrix showed that the maximum contribution of tyrosine was 3% of the total emission. Comparison with the absolute yield of pepsin fluorescence, measured as O-22fO-06, indicated a direct tyrosine absolute yield of about O-01. The choice between the assumption of non-fluorescent tyrosine or efficient transfer to tryptophan was made by measuring the absolute quantum yield of TABLE 1 Excitation

and eh98~

Exaitation wavelength (nm)

306

266

7

mu&ix for pepsin in aquecnur solution (pH 5.5) showing the percentage difference8 of the 2 x 2 rninm8 Emission wavelengths (run) 326 336

316 12-b

19

-2 276

0

14.6

286 296

1

I

I

II 270

I

I,,

260

I 280

I

Excitation over-all

70 0

46

,

44 -1 62 0

47

42

at various excitation

wavelengths.

,

,-

04-

2 @20

FIQ. 2. Relative

49

68

38

I

21.6 0

-1

$2

21

g

47

66 +4

9

24

0

36

+12

365

+1

38

$2

19

23.6 0

26 +3

345

quantum

11’1 290

I!_ 300

310

wavelength (nml

yield of pepsin fluorescence

78

R. A. BADLEY

AND

Fluorescence

F. W. J. TEALE

lifetime

FIG. 3. Absolute quantum yield ageinst fluorescence exoited at 280 nm. 1. Native pepsin (pH 5*6), 0.01 M-aoetate buffer. la. Correated for fractional absorption of tryptophan in pepsin. 2. Denatured pepsin (pH 7), 0.01 M-phospbate buffer. 2a. Corrected for fractional absorption of tryptophan in denatured pepsin. 3. Glyoyl-tryptophan; this and all following (except 8) pH 7, 0.01 M-phosphate buffer.

hsec)

lifetime 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

for indole derivatives

and pepsin,

Tryptophan methyl ester. 2-Methyl-indole, L-Tryptophan. Indole. L-Tryptophan (pH 10.2). Skatole. Indole-3-butyric acid. Indole-N-acetic acid. N-Methyl indole. Indole-3-acetic acid.

pepsin every 5 nm between 260 to 300 nm, observing the emission at 345 nm. The variation in yield with excitation wavelength is shown in Figure 2. Native pepsin at 20°C showed a single decay time, using both single-flash, phase-shift and demodulation methods, with r=4-6 fO-1 nsec. In Figure 3 the absolute yields and lifetimes of several indole compounds, and native and denatured pepsin are shown. The pepsin lifetime was independent of excitation wavelength between 260 to 300 nm. The catalytic activity of pepsin was completely abolished by the attachment of one mole of substrate analogue per mole of enzyme. The stoichiometry of the reaction was determined by absorption spectroscopy, using a value of 50,900 cma/m-mole for the pepsin ebsorbancy maximum at 278 nm (Perlmrtnn, 1966). The pyrene derivative showed some non-specific attachments in addition to active site l&belling. Generally, the characteristic absorption of the attached chromophore differed slightly from that of the chromophore in aqueous solution, suggesting a relatively lower polarizability of environment in the protein. The n-phenylalsnine reagents reacted much more slowly than the corresponding L-isomers, although the ratio of the initial rates decreased with increasing chromophore bulk. Successive reactions with different reagents showed that the same single catalytic site was concerned in all the reactions. The absorption spectra of five pepsin-bound chromophores and the fluorescence spectrum of pepsin are shown in Figure 4. The fluorescence spectra of the conjugates showed little shift in the tryptophan band as the transfer efficiency increased ; but the tyrosine component, the quantum yield of which hardly changed, became more easily detectable as the tryptophan contribution decreased. In Table 2 the fluorescence intensities and lifetimes of pepsin conjugates excited at,

ENERGY

TRANSFER

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77

Wavelength (nml FIQ. 4. Absorption spectra of the chromophores the pepsin relative fluorescenoe spectrum. TABLE

lkzctiondfhorescence

ettaohed

to pepsin. The dashed line represents

2

intensities and lifetimes of the pep&n conjugates, together with the overlup integral.9 (J)

Pepsin conjugate Pepsin N-TOS-L-Phe-Pepsin N-MNP-Phe-Pepsin N-DNS-L-Phe-Pepsin N-DNP-L-Phe-Pepsin BDAP-Pepsin

J x 101%mam-mole-l 0 1.2 3.6 20.0 41.0

Obs F/F0 1.0 1.0 0.73 0.365 0.19 0.13

Obs r/z0 1.0 1.0 0.86 0.64 0.38 0.33

290 nm are compared with the values in the native enzyme, together with the ctppropriate overlap integral (J) calculated from equation (1) by numerical integration of linear wavelength values of absorption intensity end of fluorescence measured at constant wavelength bandwidth.

(1)

In equation (l), IA is the pepsin Huoreeoence intensity at wavelength h and eAis the molar rtbsorbsncy of the chromophore at the same wavelengfh. The reciprocal of the

R. A. BADLEY

78

AND

F. IV.

J. TEALE

tryptophan excitation polarization values for the conjugates in pH 5.5 buffer at O”C, plotted against the fractional lifetime for several excitation wavelengths are shown in Figure 5. The fluorescence was observed at 345 nm. The increased polarization values in the conjugates corresponded well with their decreased average lifetimes. The polarizations at constant excitation wavelength, extrapolated to zero lifetime, are compared with corresponding values measured with tryptophan in pure glycerol in

303 nm ,A---‘4-e.

+-+---+-------

t 0

I

I 0.2

I

I

I 0.4

Fractional

310nm

I O-6

I

fluorescence

FIG. 5. Reciprocal polarization egainst fractional jugates, excited at 310, 303 and 270 nm.

I 0.8

lifetime

I

I I.0

k/To)

fluorescence

lifetime

for the five pepsin con-

Figure 6. The fractional fluorescence intensity of each conjugate compared with native pepsin was almost independent of the exciting wavelength, although a slight increase was noted when exciting the absorption edge of pepsin at 300 nm. The DNS-conjugate showed a fluorescence maximum at 517 nm sensitized by transfer from pepsin tryptophan groups excited at 290 nm. Comparison with the positions of the band maximum of DNS-amide in dioxanlwater mixtures indicated a dielectric 0.4 F-----F

0.1 0.05 I I 260 270

I

1 280

1

1 290

Wavelength

I

I 300

I

I 310

(nm)

FIG. 6. Fluorescence polarization spectra of tryptophan pepsin conjugates, extrclpOl8t0d to zero lifetime (2).

at Smite

viscosity

in glycerol

(1) and in

environment of about 30 at the pepsin-active site. The fully corrected excitation spectrum of the DNS fluorescence is shown in Figure 7, together with that of DNSphenylalanine. The sensitization of DNS fluorescence by pepsin amounted to 17,500

ENERGY

TRANSFER

IN

PEPSIN

CONJUGATES

79

cm2/m-mole s.t 230 nm. The resolution of the spectral detail at 292 nm characteristic of tryptophan compared with the over-all pepsin absorption spectrum stressed the selective transfer from tryptophan rather than tyrosine. The excitation spectra of the

260

320

300

280

Wavelength

340

360

(nm)

FIG. 7. Fluoreecenoe excitation spectra of (1) tryptophan fluorescenoe in pepsin, (2) 617 nm fluorescenoe of DNS-Phe-pepsin and (3) dansyl-L-phenylalaninnine in 65% dioxan-water(pH 56).

residual tryptophan emission at 345 nm and of sensitized emission of DNS at 517 nm were identical. Except for the tosyl derivative, the conjugates showed a selective decrease in the tryptophan emission, permitting the tyrosine component at 304 nm to be resolved. In Figure 8 the excitation polarization of DNS-Phe-pepsin in the spectral regions of sensitized and direct DNS fluorescence excitation is shown, together with those of

0

280

I

11.1 300

‘If( 320 Wavelength

340

360

1’

380

bun)

FIQ. 8. Densyl fluoreeoenoe polar&&ion speotra of daueylamide in glycerol (l), dansyl (3). coid and modified dansyl human plaame albumin (2) and DNS-Phe-pepsin

ovomu-

DNS-labelled ovomuooid and modified human plasma albumin. The polarization spectrum of DNS-amide is also included. To measure the DNS polarization spectrum in a conjugate in the absence of tryptophan sensitization, observations were made of DNS conjugates of ovomucoid and NBS-modified plasma albumin. These contained no fluorescent tryptophan and gave no sensitization in the region of tryptophan absorption. The polarization spectra, after correction for rotational effects, were nearly coincident with that of the pepsin conjugate at wavelengths longer than 310 nm. At shorter wavelengths the pepsin conjugate showed marked depolarization, relative to the comparison systems, evidently produced by tryptophan sensitization. 6

80

R.

A. BADLEY

AND

F. W. J. TEALE

4. Discussion The fluorescence of pepsin is characteristic of tryptophan in a relatively polar environment, so that the emitting residues are of the exposed type (Fig. 4). Inspection of Figure 2 shows that at 275 nm there is a fraction 0.32 of absorption which does not lead to tryptophan emission. This value is minimal, as the absorption at 300 nm cannot be attributed solely to tryptophan. Cystine bridges, of which there are three in the molecule, certainly contribute, and probably tyrosine also. To interpret Figure 2 we require the fractional absorption of tyrosine and tryptophan, calculated from the molar ratio of these residues in pepsin. The number of tryptophans present is estimated as five or six by spectrophotometric assay (Arnon & Perlman, 1963; Edelhoch, 1967), whereas smaller values are given by chemical or solvent perturbation methods (Dopheide & Jones, 1968 ; Herskovits & Sorensen, 1968). There is some evidence that high values are given by the spectrophotometric assay (Davidson, Sajgb, Noller & Harris, 1967), so that in the present work five tryptophans per mole have been assumed. On this basis, taking 16 tyrosine and 14 phenylalanine residues, the molar absorbance maximum (50,900 cm2/m-mole) and band-shape of pepsin can be matched reasonably well. The fractional absorption of tyrosine at 276 nm is

16 x 1340 16x1340+5x5550

=0*43. This figure, taken together with the

inactive absorption fraction indicated in Figure 2, shows that at least three quarters of the tyrosines do not transfer to tryptophan. The virtual absence of tyrosine emission and sensitization suggests effective direct deactivation of the singlet state, probably through interaction with some of the 41 carboxylate groups known to be present in pepsin (Clement, 1967). If no tyrosine sensitization occurs, the absolute quantum yield of tryptophan in pepsin can be calculated as 0~22/1--0~43=0~385. This value agrees well with the measured lifetime, plotted together with corresponding values for various indole compounds in Figure 3. The approximate proportionality between q and 7 values shows that the processes which reduce the natural lifetime and maximum quantum yield in the various compounds all take place during the singlet excited state. That the relationship also holds for native and denatured pepsin confirms the absence of tyrosine transfer and suggests that the factors determining the quantum yield of tryptophan in pepsin also act at the singlet level. Possible mechanisms are collisional quenching, charge-transfer in the excited state, resonance energy transfer or exciplex formation. The tryptophan groups must, moreover, have comparable yields if they are independent, but the possibility of energy transfer from residues differing widely in quantum yield from the ultimate emitter must not be overlooked. The stereospecificity of the reaction of the diazoketones with pepsin, the complete inhibition of the enzyme and the absorption spectra of the conjugates show that in each case one residue of the substrate analogue is attached at the single active site, probably through esterification of a carboxyl group (Zeffren & Kaiser, 1967 ; Erlanger, Vratsanos, Wasserman & Cooper, 1967). That the conjugates represent a set of resonance energy transfer donor-acceptor systems differing mainly in the overlap integral term is suggested by the fluorescence intensity and lifetime measurements presented in Table 2. Native pepsin and its tosyl derivative have identical emission parameters, so that quenching by bound copper ions remaining from the preparative method can be discountled (Lundblad &

ENERGY

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PEPSIN

CONJUGATES

81

Stein, 1969). This conclusion was supported by the absence of effect of concentrated solutions of EDTA on the fluorescence of the conjugates. Furthermore, it is probable that large conformation changes in the macromolecule, which might modify the tryptophtm environments, do not acoompany conjugation. In those conjugates having little chromophore absorption relative to pepsin (TOS, MNP and DNS conjugates), no perturbation of the tryptophan absorption spectrum compared with the native enzyme was discernible. Thus, although the possibility of direct quenching of some tryptophan residues through complex formation with the added chromophore cannot be entirely discounted, the effects illustrated by Table 2 can be largely attributed to resonance energy transfer from the array of tryptophan residues to the N-linked chromophore in the substrate analogue. Before considering the over-all efiloiency of transfer, other properties of some of the conjugates must be discussed. In the DNS and pyrene conjugates, the chromophore fluorescence sensitized by tryptophan absorption is readily observed. The correspondence between the degree of sensitization of DNS %uorescence (66% of total tryptophan) and the decrease in tryptophan yield (35%) support the conclusion that all the tryptophans have similar quantum yields. In general, the sensitization will be smaller or greater than the degree of donor quenching when those donors favourably situated for resontmce transfer have quantum yields greater or smaller than the average value, since the fractional absorption of ertch residue is almost independent of its quantum yield (Van Duuren, 1961). In addition to the foregoing considerations, some idea of the tryptophan orientation is provided by observations of the direct and sensitized DNS fluorescence polarization. For a fixed donor-acceptor pair, the sensitized fluorescence polarization is given by the isotropic depolarization equation of Perrin (1929). The depolarization produced by a randomly oriented array of donors can be calculated by combining the Perrin equation with that of Weber (1954), corrected by taking into account the Ka term of Fijrster (1959). The %nal result is :

where P,, is the donor-limiting fluorescence polarization in the absence of rotations at the absorption wavelength, PA is the acceptor polarization directly excited at the wavelength of the overlap maximum and PDAis the observed sensitized polarization. Thus for small values of PD and PA, the sensitized polarization is approximately equal to the product of the donor and acceptor polarizations, and is not zero for fixed randomly+riented systems. After correction for the small amount of direct DNS excitation, PDA was calculated as 0940 &0902 (Fig. 8; 290 nm). Substituting into equation (2) the measured values of PA = O-25, P, = O-135, we obtain P,, = 0.035. This agreement is probably to some extent fortuitous, but suggests that the tryptophans concerned in energy transfer approximate to a quasi-random array. Good evidence that the tryptophan residues emit almost independently is the observation that the lifetimes of the series of conjugates listed in Table 2 decrease more slowly than the corresponding intensities. Since for each donor F/P, = T/T~, this indicates that we are observing an turay of tryptophan donors differing in transfer probability as a consequence of their specific orientation and position in the macromolecule. The consequent weighting of the lifetime by intensity gives an average value greater than

a2

R. A. BADLEY

AND

F. W. J. TEALE

the average intensity. The dependence of the transfer efficiency on the overlap integral also leads to the same conclusion. For each donor, F,/F = 1 + J/J,,, where J1,a is the overlap integral which produces 50% transfer efficiency. A linear relationship between F,/F and J characterises a system with a single J1,a value. Inspection of Table 1 shows that this simple relationship does not hold, because here the donors have different J1,a values. The e&cienoy of transfer decreases more slowly with decreasing overlap integral than in simple systems, an effect which has also been observed in a protein by Green (1964). As the donor lifetime of each conjugate is shortened by transfer, the tryptophan fluorescence polarization in aqueous solution at constant temperature increases. The linear plot of reciprocal polarization against average lifetime (Fig. 5), when extrapolated to zero lifetime, gives the limiting polarization in the absence of all timedependent depolarizing processes such as group rotation or energy migration. In contrast, depolarization by torsional oscillations or overlapping transitions would be unaffected. The values obtained by extrapolation at various wavelengths are considerably smaller than those for tryptophan in rigid media. The origin of this depolarization has still to be investigated, but probably does not lie in any appreciable amount of intertryptophan transfer. The small value of R, calculated for intertryptophan transfer (R, = 1-O nm) compared with the average residue separation (E 2-O nm) in pepsin would suggest a low efficiency for this process. Until the positions and orientations of the tryptophan groups in the macromolecule have been determined by X-ray methods, one can only compare the observed residual fluorescence intensities and lifetimes with those calculated for randomised model systems (see Appendix). Arrays of randomly oriented donors transferring to a single acceptor were considered in which all the dipoles were either rapidly rotating during the donor lifetime, or rigidly fixed in direction. The characteristic distance R, for each conjugate was calculated by using equation 8, setting q = 0*385,n = 1.5, the appropriate value of J taken from Table 2 and for the rapidly rotating dipole case setting Ka = 213. Pepsin has a molecular weight of 34,163 (Rajagopalan, Moore & Stein, 1966) and density 1.35 g/ml., so that a diameter of 43 A can be assigned to the equivalent anhydrous sphere. In Figure 9 the theoretical fractional intensities and lifetimes as functions of the parameter R,/D are shown for donors uniformly distri-

:: Lk

0

I

1

I

I

0.2

0.4

0.6

0.8 R,/D

FIG. 9. Computed curves for a spherical model, cence lifetime, rotating dipoles (l), fractional cence, rotating dipoles (3). The experimentally fluorescence; 0, frectional lifetime.

I.0

I.2

I.4

with one surface acceptor; fractional fluoresfluorescence, fixed dipoles, (2) and fractional fluoresdetermined values arc indicated: 0, fractional

ENERGY

TRANSFER

IN

PEPSIN

83

CONJUGATES

buted within a sphere of diameter D with one acceptor group in the surface, together with the experimental values for the pepsin conjugates. It will be seen that for a given acceptor the transfer e%kiency is higher for rapidly rotating dipoles than for the rigid randomly oriented system. While the experimental points lie on curves similar in form to the theoretical lines, supporting the assumption of a distribution of donor transfer probabilities, in each conjugate the observed transfer e&iency is higher than either of the theoretical values. This high eficiency may indicate a favourable orientation of the acceptor transition moments, although this could hardly be expeoted in each of the conjugates, and almost certainly arises from the distribution of the tryptophan residues. Since the geometry of the donor array is a critical faotor, over-all shapes other than spherical must be considered. From crystallographic rtnd X-ray diffusion data (Vazina, Lednev & Lemazhikhim, 1966), pepsin approximates to a prolate ellipsoid of revolution with axial ratio approximately 2. The theoretical curves computed for one acceptor placed either at a pole or on the equator of such an ellipsoid are shown in Figure 11, together with the experimental points. It can be seen that while the polar case gives e&iencies lower than those for a sphere of equal volume, the equatorial case, through compensating effects, hardly differs from the spherical model. The observed high transfer eiikkncy certainly does not stipport the ellipsoidal shape with a polar acceptor position. In addition to pepsin, tryptophan fluorescence quenching produced by a single acceptor moiety per macromolecule has been reported for chymotrypsin (Hauglad & Stryer, 1967), carbonic anhydrase (Chen, 1967) and avidin (Green, 1964). The transfer parameters for these proteins and pepsin a,re included in Table 3. In every case the TABLET Theoretical and observedjluorescence intensities, equivalent anhydrous sphere diameter and energy transfer parameter R, for pepsin, chymotypsin, curbonic anhydrwe and avidin, with one energy transfer acceptor group per mole of protein

Protein

pepsin Pepsin Pepsin Carbonic tUlhydW3 Avidill Avidin Chymotrypsin

Acceptor

MNP DNS DNP BDAP DNS DNP Lipoate Anthraniloyl

t A is the F/F, value calculated $ B is the F/F,, value calculated

Diameter of equivalent sphere (4

Bo(A)

Theoretical F/F0 At BZ

ObsemedF/Fo

43 43 43 43

19.5 22.9 31-l 35-o

o-71 0.60 0.32 O-23

0.77 0.67 0.44 0.35

0.73 o-35 o-19 o-13

41 58 68 38

22.9 30.3 12.8 20.0

0.55 0.62 o-93 0.61

0.64 0.70 0.95 0.68

0.27 o-1 04-0.56 0.35

for rapidly rotating dipoles. for randomly oriented fixed dipoles.

observed quenching is much greater than that predicted for either of the spherical model systems. To obtain agreement, the tryptophan groups in these proteins would need to be uniformly distributed in spherical volumes of average diameter 33 A for

84

R. A. BADLEY

AND

F. W. J. TEALE

pepsin, 29 A for carbonic anhydrase, 26.5 A for chymotrypsin and 27 A for avidin. For pepsin, this sphere approximates in diameter to the short axis of the ellipsoidal molecule. An arrangement approaching this can be seen in the structure of papain, where the active site is situated in a cleft closely surrounded by the tryptophan residues (Drenth, Jansonius, Koekoek, Swen & Wolthers, 1968). In addition to such non-uniform distributions, other factors must be considered. Inter-tryptophan transfer has been suggested to account for features of the polarization spectrum of proteins (Konev, Bobrovich & Chernitskii, 1965). The pathway of energy migration would be determined by several factors, including the proximity, mutual orientation, quantum yields and differences in absorption and fluorescence spectra between residues. Provided migration does not occur specifically away from the acceptor position, inter-tryptophan transfer always increases the over-all fluorescence quenching produced by an acceptor, since indirect transfer from remote donors can occur. Calculations based on the relatively low probability of inter-tryptophan migration show that the whole of the increase cannot be accounted for by this mechanism. Finally, the refractive index assigned to the internal milieu of the macromolecule must be considered. The experimental value (Heller, 1945) is not strictly appropriate, as it represents an average of the internal and surface domains. The average polarizability of the material between each donor-acceptor pair should strictly be considered; and in some cases indirect routes for transfer may occur. To reconcile the observed and theoretical transfer efficiencies, the refractive index of the pepsin interior domain must be decreased substantially. It is very probable that the high transfer eficiency observed in pepsin has its origin in more than one of the possibilities outlined above. APPENDIX

Energy Transfer from an Array of Rapidly Rotating or Stationary Dipoles to One Acceptor Dipole The rate of transfer nD+A from a donor dipole situated at distance R from an acceptor is given by (Fiirster, 1959): 9000.ln 10.K2+J nD+A = 128 rr6n4 ,rOR6 N

(3)

where J is the donor-acceptor overlap integral (equation (l)), T,, is the donor natural lifetime, n the refractive index of the intervening medium at the wavelength of spectral overlap, N is Avogadro’s number and K2 is an orientation factor. If the fluorescence intensity F, is reduced to F by energy transfer to the acceptor then :

F -F,=

nF nF

-k

%

+

nR +

1

EZ

nD+A

nD4A

(4)

1+nF

i-

nR

where n.F and nR are the rates of donor emission and radiationless deactivation, respectively.

The natural lifetime r0 = L , where 7 is the observed mean lifetime of 4

the donor having quantum yield q, and since 7 = nF

1 +

we have : nR

ENERGY

F E=

TRANSFER

IN

PEPSIN

CONJUGATES

1 9OOOlnlO-K%Jq= 1+ 128 A4 R6 N

1 CKa 1+Re

wh13rc3c-g9000h10J~ , a oonstant ind0pendent 128 &=n4 N

of orientation

85

(6)

tLnaseparation

of the

donor and ecoeptor dipoles. The angular function used for Ka depends on the fluorescence parameter involved, rend for transfer efficienoies the convenient expression is (Slater & Frank, 1947): Ka=(3cosaa+1)cosaj?

(6)

where a is the angle between the donor transition dipole moment and the line joining the donor and acceptor dipole centres, and p is the angle between the acceptor dipole moment and the electric vector of the donor field at the acceptor position. When the donor and acceptor dipoles rotate rapidly in all space during the donor lifetime, the fractional fiuorescence is given by integmtion, noting that a and /3 are independent variables : 1 s0 a so that $ = 0

(7)

wa (3cosaa+

4~

l)cosa~*sina*sin~*da*dfi

s o”

1 ; substituting 1 + 2C/3R6

Ri = $

where R. is the separation between rapidly rotating dipoles giving equal probability of emission and transfer, we have: F -= Fo

1

(9)

1 + (WV’

For an array of n independent donors having equal light absorption transferring to one acceptor, the fractional %uorescence is given by : F -=PO

1

11

1

n

1 1 + VWW

where R, is the distance from the acceptor of the &h donor, and R,, is aharaoteristic of the donor-acceptor pair. If all the donors have identical %uorescence parameters, then the R,, terms are constant, and F/F0 is cslculated from equation (9) by summing the contributions of all the donors over the whole range of R, values. If the donors can be assumed to be distributed uniformly within spherical or ellipsoidal volumes, the over-all fractional fluorescence is obtained by integrating the expression F -= FO

I+

VdR

s o 1 + WW

(11)

86

R. A. BADLEY

AND

F. W. J. TEALE

where V is the fractional volume of a spherical shell of thickness dR centred on the acceptor position, having radius R. For a spherical volume with one acceptor group in the surface, F

-=

FO

x8.ax s oX6+ WW 1

12

- 12

1

x9.ax

sox6

+ VLlW

(12)

where D is the sphere diameter and X = RID. The corresponding fractional lifetime is given by weighting the fractional lifetime of each donor by its fluorescence intensity, since for the ith donor ri/ro = FJF,; (13)

or replacing summation by integration, 12F,

7

70

we have :

= - F

x14.ax

l

(14)

s o [X6 + [R,/D]6]2’

The values of the integrals from equations (12) and (14) were obtained by digital computation for a wide range of R,lD values, and are shown graphically in Figure 9. A plausible modification of the spherical model relevant to enzyme function is the location of the acceptor site in the centre of the flat face of a truncated sphere. The fractional intensity calculated for this model is shown in Figure 10 for different

0

I

I

I

0.2

0.4

0.6

I

I

I

I

0.8

I.0

1.2

I.4

R. /D FIQ. 10. Computed curves for a truncated sphere with acceptor in the centre of the plane surface at distance KR from the oentre of the sphere, radius R. Fractional fluorescence against R,/D (D = 2R). For K = 1.0 (I), and K = 0.3 (2).

distances of the acceptor from the centre of the sphere. It will be seen that F/F0 does not change rapidly with acceptor position. Ellipsoidal volumes of revolution can have the acceptor located on either of the semiaxes, the polar and equatorial positions. For these two locations, the integration was performed numerically by digital computer, and in Figure 11 the results are shown for a prolate ellipsoid of axial ratio 2. As could be expected, the transfer efficiency is less to a polar acceptor than to the equatorial position.

ENERGY

TRANSFER

IN

PEPSIN

CONJUGATES

87

The average fractional fluorescence for a donor-acceptor pair in which the dipoles have random but fixed orientations can be calculated from equations (5) and (6).

0.2 0.4 06 0.8 I.0

0

I.2 I.4

R. ID Fro. 11. Computed ourves for a prolate ellipsoidal model (a = 2b), with one surface aooeptor, fractional fluorescenoe against RoD. D = equivalent sphere diameter. 0, Experimental points. Frsotional fluoresoenoe with aooeptor at the pole of the ellipsoid (l), and in an equatorial position

(2).

n/a npa

F Fo=

sso a

o

sin c&n t%dwdp 1 + C/R6 (3 cos2a + 1) cosa t3

(14

sin ~sin @da-d/l 1 + 3/2[R$16(3 cosa a + l)cosa$

Pf3)

B

ao that :

a

B

Values of F/F, were computed, after integration, for a wide range of RJR values, and the fractional 0uorescenoe intensity of a spherical array of donors wa8 obtained by dividing the sphere into 20 concentric shells centred on the acceptor group. Summation of the contributiona of all the shells gave the fractional fluorescence for a particular R, value. The variation of F/F, with R,/D for the rigid-random model is included in Figure 9. We acknowledge fruitful discussions with Dr R. E. Dale of this Department, and the assistance of Computer Services, Birmingham University. This work was supported by a research grant from the Scienoe Research Council, which is also thanked for a research studentship to one of us (R. A. B.).

REFERENCES Arnon, R. t Perlmann, G. E. (1963). J. Bid. Chem. 238, 063. de Boer, T. J. t Backer, J. H. (1963). Org. Syntheses CoZZ. vol. 4, p. 250. Chen, R. F. (1907). J. Biol. Chem. 242, 5813. Clement, G. E. (1967). Biochim. biophys. Ada, 140, 368. Davidson, B. E., Sajgb, M., Noller, H. F. & Harris, J. I. (1967). Nature, 216, 1181. Delpierre, G. R. & Fruton, J. S. (1965). Proc. Nat. Acud. Sci., Wash. 54, 1161. Delpierre, G. R. t Fruton, J. S. (1966). Proc. Nat. Acud. Sci., Wash. 56, 1817. Dopheide, T. A. A. & Jones, W. M. (1968). J. Biol. Chem. 243, 3906. Drenth, J., Jansonius, J. N., Koekoek, R., Swen, H. M. & Wolthers, B. G. (1968). Nature, 218, 929. Edelhoch, H. (1967). Biochemietry, 13, 1948.

88

R. A. BADLEY

AND

F. W. J. TEALE

Erlanger, B. F., Vratsenos, S. M., Wasserman, N. & Cooper, A. G. (1967). Rio&m. Biophys. Res. Comm. 28, 203. Fischer, E. (1907). Ber, 40, 3717. Fijrster, Th. (1959). Diet. Faraday Sot. 27, 7. Gebor, G. (1968). Biopolymers, 6, 809. Green, N. M. (1964). Biochem. J. 90, 564. Haugland, R. P. & Stryer, L. (1967). In Conformation of BiopoZymer8, ed. by G. N. Ramachandran, p. 321. New York: Academic Press Inc. Heller, W. (1945). Phys. Rev. 68, 6. Herskovits, T. T. & Sorensen, M. (1968). Biochemietry, 7, 2633. Konev, S. V., Bobrovich, V. P. & Chernitskii, E. A. (1965). Dokl. Biophys. 165, 205. L&t, S. A., Cheung, H. T. & Blout, E. R. (1966). J. Amer. C&m. Sot. 87, 995. Leggett-Bailey, J. (1962). Technique8 in Protein Chemistry, p. 165. Amsterdam: Elsevier. Lundblsd, R. L. & Stem, W. H. (1969). J. BioZ. Chem. 244, 164. Meckey, R. C., Pollack, S. A. & White, R. S. (1966). Rev. Sci. In&r. 36, 1715. Marvel, C. S. (1955). Org. Syntheses Co& vol. 3, p. 706. Perlmann, G. E. (1966). J. BioE. Chem. 241, 153. Perrin, F. (1929). Ann. de Phg8ique. 12, 169. Rajagopalan, T. G., Moore, S. & Stein, W. H. (1966). J. Biol. Chem. 241, 4940. Sanger, F. (1946). Biochem. J. 39, 607. Slater, J. C. & Frank, N. H. (1947). EZectromugnetGm, p. 158. New York: McGraw-Hill. Steiner, R. F., Roth, J. & Robbins, J. (1966). J. BioZ. Chem. 241, 660. Stryer, L. (1969). Biochim. biophye. Acta, 35, 242. Stryer, L. (1965). J. Mol. BioZ. 13, 482. Stryer, L. & Heuglend, R. P. (1967). Proc. Nat. Acad. Sci., Wash. 68, 719. Teale, F. W. J. (1961). Biochem. J. 80, 14P. Teale, F. W. J. & Weber, G. (1967). Biochem. J. 65, 476. van Duuren, B. L. (1961). J. Org. C&m. 26, 2964. Vazins, A. A., Lednev, V. V. & Lemazhikhim, B. K. (1966). Biokhimyia, 31, 720. Vollmann, H., Becker, H., Corell, M. & Streeck, H. (1937). Ann. Chem. 531, 106. Weber, G. (1952). Biochem. J. 51, 165. Weber, G. (1954). Trans. Faraday Sot. 50, 662. Weber, G. (1966). J. Opt. Sot. Amer. 46, 962. Weber, G. (1961). Nature, 190, 27. Weber, G. & Daniel, E. (1966). Biochembtry, 5, 1900. Weber, G. & Teale, F. W. J. (1957). Trans. Faraday Sot. 44, 646. Weber, G. & Teale, F. W. J. (1959). Disc. Faruduy Sot. 27, 134. Zeffren, E. & Kaiser, E. T. (1967). J. Amer. Chem. Sot. 89, 4204.