Proc. Nat. Acad. Sci. USAVol. 71, No. 10, pp. 4154-4158, October 1974

Room Temperature Phosphorescence and the Dynamic Aspects ofProtein Structure

(conformational fluctuations/liver alcohol dehydrogenase)

MARIA L. SAVIOTTI AND WILLIAM C. GALLEY

Department of Chemistry, McGill University, Montreal, Quebec

Communicated by Norman Davidson, June 27, 1974

ABSTRACT While the phosphorescence of. aromaticchromophores. in solution is normally quenched throughdiffusion of dissolved oxygen and other solvent-mediatedprocesses, the phosphorescence of some proteins in solu-tion is observed at room temperature. The tryptophanphosphorescence arises from residues which are hinderedfrom interaction with oxygen by the folding of the poly-peptide chains. Measurements of the phosphorescencelifetime of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) as a function of oxygenconcentration indicatethat internal tryptophan residuesare periodically exposed to oxygen. This permits the cal-culation of rate constants for conformational oscillationsin the enzyme. The present article illustrates the feasi-bility of employing phosphorescence in the stuedy of pro-teins in solution in general- and the utility of such ex-periments in probing the dynamic aspects of proteinstructure.

The properties of the triplet state of aromatic chromophoresmay be profitably exploited in the study of biomolecularstructure and dynamics. Phosphorescence measurements havebeen used in biochemical systems both to detect the proximityof aromatic groups (1-3) and to monitor long-range inter-actions (4, 5) in small molecule-biopolymer complexes. Inaddition tryptophan and tyrosine (6), and in favorable casesindividual tryptophan residues (7), can be resolved in proteinphosphorescence spectra.

Despite the molecular information which can be derivedfrom triplet-state spectroscopy, phosphorescence has beenemployed much less frequently than fluorescence as a bio-chemical tobl. This undoubtedly arises from the fact thatphosphorescence measurements have been made on bio-chemical samples in rigid glasses usually formed from glycerol-H20 or ethylene glycol-H20 mixtures at liquid nitrogentemperatures. While there is evidence to indicate that bio-polymers retain their native structures under these conditions,the inability to directly correlate biological activity withphosphorescence measurements has contributed to its in-frequent use.

Rigid media have been employed due to the normallyefficient quenching of the triplet state in fluid solution bydissolved oxygen and other solvent-quenching processes. Inthat aromatic amino acids residues are frequently buriedwithin the globular structure of proteins, it was anticipatedthat their triplet states would be less susceptible to quench-ing, the degree of protection afforded a particular aromaticresidue being a function of the location of the residue withinthe protein structure and the flexibility of the protein con-formation.

Our observations of protein phosphorescence with increas-inig temperature reveal that the folding of the polypeptidechains in protein molecules does hinder quenching of thetriplet states of internal tryptophan residues. Phosphores-cence from proteins is observed in fluid solution at tempera-tures where it cannot be observed from free chromophores,and in the present article preliminary data on the room tem-perature phosphorescence of horse liver alcohol dehydro-genase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) andEscherichia coli alkaline phosphatase (orthophosphoric-mono-ester phosphohydrolase, EC 3.1.3.1) are presented. The oxy-gen dependence -of the phosphorescence lifetimes observedat room temperature provides a measure of the kinetics ofconformational flexibility in proteins. The triplet lifetimesare also found to be sensitive to the binding of coenzymes andthe presence of protein stabilizing and destabilizing agents.We feel the present study not only indicates the usefulness

of phosphorescence in probing the dynamic aspects of proteinstructure, but suggests the feasibility of employing tripletstate experiments in general in the study of proteins in solu-tion.

MATERIALS AND METHIODSHorse liver alcohol dehydrogenase (LADH) was obtainedfrom Worthington Biochemical Co., as the 1 X crystallizedlyophilized product (lot no. 33E650 in most experiments), andE. coli alkaline phosphatase was obtained as an ammoniumsulfate suspension from Sigma Chemical Co. NADH was aCalbiochem product, ultra-pure puanidine . HCl was fromSchwarz/Mann and urea from the Fisher Chemical Co.Ethylene glycol arid glycerol we're ihi'omatoquality fromMatheson, Coleman, and Bell. Buffers used in the prepara-tion of enzyme solutions were made with double distilledwater..

In typical experiments, an enzyme concentration of 10-4 Mwas employed. Experiments with NADH involved the addi-tion of 2-fold excess of coenzyme to assure complete forma-tion of the binary complex.

In order to prepare enzyme solutions at a given concentra-tion of dissolved oxygen, 0.5 ml of buffer was equilibratedwith gas mixtures of known composition at 1 atm by bubblingthe gas through the buffer for 30 min at a rate of 200 cm'.min-'. The oxygen content of the ga's mixture was controlledby mixing the proper ratios of gases from tanks of knowncomposition of oxygen and nitrogen (Union Carbide CanadaLtd.) in a soap bubble flow meter. The concentration ofoxygen in the solution was calculated from the Henry's Lawconstant for oxygen in water at 250C (8). After equilibration,the tube containing the buffer was isolated from the gas flow

4154

Abbreviation: LADH, liver alcohol dehydrogenase.

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Phosphorescence of Proteins at Room Temperature 4155

and the environment. The buffer was poured into a side armcontaining a weighed amount of enzyme. The enzyme solutionwas transferred to a side arm which consisted of a quartz tube(5mm outside diameter) which served as the emission cell.Phosphorescence emission spectra were recorded with an

apparatus described previously (7). For phosphorescencemeasurements at room temperature, the intensity was en-hanced by removing the excitation monochromator and excit-ing with the full arc. Phosphorescence lifetimes were recordedwith an X-Y recorder for lifetimes longer than 0.1 sec, byphotographing the decay of individual phosphoroscope pulsesdisplayed on a Tektronix 546 oscilloscope or, averaging thedecay of 10-30 pulses with a C-1024 Varian Time AveragingComputer.

RESULTS AND DISCUSSION

1. Phosphorescence Spectra and Solvent Relaxation. Theinitial influence of increasing temperature on the phosphores-cence spectrum of LADH is depicted in Fig. 1. As has beenshown previously (7), the tryptophan phosphorescence spec-trum of LADH arises from the independent emission of twotryptophans; the components with peaks at 405 and 410 nmhaving been assigned as emission from a solvent-exposedresidue and an internal residue, respectively. One buried andone exposed tryptophan residue in each of the monomer unitsof LADH has been observed in the x-ray structure of theenzyme (C. -I. Brand6n, personal communication). Withdecreasing viscosity accompanying the increase in temperature,the tryptophan phosphorescence peak at 405-nm shifts tothe red between 1000K and 130'K in 1:1 ethylene glycol-H20. At the higher temperature it is hidden beneath the 410-nm peak and can no longer be recognized as a separate com-ponent. The temperature range over which the red shiftoccurs is similar to that observed for indole or tryptophans insmall peptides. A red shift in the fluorescence spectrum oftryptophan residues exposed to polar solvents is well known(9). It arises from solvent reorganization about the excitedsinglet state dipole moment (10). A corresponding, albeit

T=128' K

T= 103° K

LADH

Aex=280 nm

wavelength (nm)

FIG. 1. Phosphorescence spectra of LADH in a 1:1 ethyleneglycol-buffer glass as a function of temperature. The spectraillustrate the solvent relaxation red shift of the 405-nm peak asthe temperature is increased. Xex signifies the exciting wavelength.(The ordinate here and in Fig. 2 represents intensity in arbitraryunits.)

usually smaller, shift in phosphorescence spectra has beenobserved in our laboratory for a number of aromatic chromo-phores (11). The shift in the phosphorescence spectra occursat low temperatures where the viscosity is such that therelaxation time of the solvent is comparable to the tripletlifetime of the chromophores.The position of the tryptophan phosphorescence maximum

at 410 nm in LADH and 415 nm in alkaline phosphatase re-mains essentially unaltered with increasing temperature. Thisis consistent with the conclusion that emission maxima in thiswavelength region arise from tryptophan residues buriedwithin the polarizable core of the protein (7).

2. Oxygen Quenching of External Residues. At temperaturesof the order of 20'C above their respective glass transitiontemperatures, the ethylene glycol-H20 and glycerol-H20solvent mixtures become sufficiently fluid so that oxygen dif-fusion in the solution begins to influence triplet state life-times. At ambient oxygen concentrations, the lifetime of thetriplet state of indole or tryptophan methyl ester markedlydecreases and the phosphorescence is quenched at tempera-tures higher than 1830K with 1:1 ethylene glycol-H2O. Thephosphorescence lifetimes in this temperature region are afunction of the oxygen concentration of the solvent.At temperatures above 1730K (3:2 glycerol-H20) with air-

saturated solutions, phosphorescence from the externaltryptophan residue in LADH is quenched. While at thesetemperatures due to solvent relaxation, the externalresidue cannot be distinguished in the phosphorescence spec-trum of LADH; quenching is evidenced by the development ofa short-lived component in the phosphorescence decay*.With excitation at the red end of the enzyme absorptionband, only the internal residue is excited (7). When excitationoccurs at 305 nm, the short-lived component in the phos-phorescence decay is absent indicating that it arises from theexternal residue. The short-lived component can no longer beobserved in the emission at all exciting wavelengths as thetemperature is increased further.The tyrosine contribution to the phosphorescence spectrum

of LADH, which can be observed free of tryptophan at wave-lengths below 400 nm at 770K 'compare Fig. 1), is quenchedover the same temperature region as the external tryptophan.The quenching of these residues with increasing temperatureleaves the internal tryptophan residue in each of the monomerunits of LADH as the only phosphorescing center in the mole-cule. The observation that the tyrosine residues responsiblefor the phosphorescence are selectively quenched presumablybecause of their location at the surface of the protein, has alsobeen reported by McGlynn and McCarville (12) who ob-served the phosphorescence of bovine serum albumin between20 and 1530K.

3. Room Temperature Protein Phosphorescence. In contrastwith the behavior observed with indole or tryptophan methylester, the phosphorescence from internal tryptophan residuesin proteins is much less affected by increasing temperature ordecreasing solvent viscosity. The tryptophan phosphores-cence of proteins can be detected at much higher temperatures

* Both the temperature at which external tryptophan residuesundergo solvent relaxation and quenching by oxygen appear todepend on the effect of the local environment on solvent structure(J. G. Milton and W. C. Galley, unpublished data).

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4156 Chemistry: Saviotti and Galley

LADH

r,= 0.13 sec

350 400 450wavelength (nm)

500

ALKAUNE

PHOSPHATASE

To = 0.8 sec

350 400 450 500wavelength (nm)

FIG. 2. Room temperature tryptophan phosphorescencespectra of proteins. (a) 10-4 M horse liver alcohol dehydrogenase(LADH) in 0.032 M pyrophosphate buffer, pH 8.6; (b) E. colialkaline phosphatase in 0.05 M phosphate buffer, pH 7.5. Theoxygen concentration = 3.9 X 10-9 M.

and with some proteins is readily observed at room tempera-ture. The phosphorescence spectra of LADH and E. colialkaline phosphatase in buffer in the absence of glass-formingadditives and at room temperature are illustrated in Fig. 2aand 2b. The folding of the polypeptide chains with these twoproteins creates an environment for dne or more tryptophansin the protein which hinders quenching. As a result, the phos-phorescence of a chromophore in a complex structure is ob-served under conditions where it is normally impossible toobserve the phosphorescence of simple molecules.We have not been successful in observing room temperature

phosphorescence from several proteins we have looked at inspite of the fact that they contain tryptophan residues whichare buried within the globular protein structure. The phos-phorescence of a-chymotrypsin, bovine serum albumin, andbovine carbonic anhydrase was not observed under our presentconditions of oxygen removal in ethylene glycol-H20 mixturesat temperatures above -300C. The solvent viscosity at thispoint, however, is still many orders of magnitude lower thanit is at the temperature at which free tryptophan or indole arequenched by oxygen diffusion. Phosphorescence experimentson proteins in fluid solution, albeit at low temperatures, shouldalso prove to be of interest particularly in view of the recentstudies of Douzou and his coworkers on the slow rates ofenzymatic activity under these conditions (13).

4. The Dependence of Room Temperature Protein Phos-phorescence Lifetimes on Oxygen Concentration. The lifetimesof the room temperature tryptophan phosphorescence ofLADH and alkaline phosphatase depicted in Fig. 2 are 0.13and 0.8 sec, respectively, as compared with 5.5 see for bothproteins in glycerol- or ethylene glycol-H20 at 770K. It isnot possible to compare the room temperature lifetimes withknown oxygen-independent triplet state lifetimes under thesame conditions in that data for such comparisons do notexist. While triplet state lifetimes for aromatic hydrocarbons inrigid plastics are only reduced by factors of less than two ingoing from 770K to 2980K (14), the triplet states of aromaticresidues in proteins may be influenced by environmentalfluctuations not present in rigid matrices. The dependence ofprotein-triplet lifetime on the concentration of dissolved oxy-gen in the solvent was therefore investigated.The lifetimes of the tryptophan phosphorescence of both

LADH and alkaline phosphatase at room temperature aredependent on the concentration of oxygen in the buffer.

(i) Conformational Fluctuations and 02-Dependent TripletLifetimes. The phosphorescence lifetime of LADH in pyro-phosphate buffer at pH 8.6 tends to a limiting value of 0.13see in the absence of oxygen and decreases with increasing 02concentration. The triplet lifetime does not go to zero at highoxygen concentration but approaches a limiting finite value.If proteins possessed completely rigid conformations, thetriplet lifetimes of buried tryptophan residues would be in-dependent of the oxygen concentration in the solvent.Triplet states of aromatic chromophores in solution arequenched by oxygen at rates which are proportional to thefrequency of collisions between the aromatic molecule and thequencher (15), so that tryptophans unavailable to oxygenwould not be quenched. While the protein conformationmarkedly hinders quenching by dissolved oxygen, fluctuationsmust occur in the structure which periodically expose internaltryptophan residues to perturbation by oxygen.Two limiting dynamic models may be envisaged which

would permit the interaction of oxygen with internal trypto-phan residues in proteins. In the first model, small independentstructural fluctuations occur in the protein. The motions ofamino-acid side chains need only generate sufficient freevolume to permit an oxygen molecule to "diffuse" by smallrandom steps to the internal chromophores.

In the second model the structural oscillations tend tooccur in a more cooperative fashion. Interaction between aninternal tryptophan and oxygen can only occur subsequent toa structural change involving the "simultaneous" rearrange-ment of a number of intermolecular interactions.Data on the oxygen dependence of the tryptophan phos-

phorescence lifetime of LADH are consistent with a model ofthe latter type in which oscillations occur between two confor-mational states of the protein. The amplitude of the motion issuch that in the more stable state, the tryptophan residues areunavailable to oxygen while in the less stable "open" con-formation quenching by oxygen readily occurs. We assume atthis point that quenching in this latter state takes place at therate which would be observed for the free chromophore insolution. This can be represented in a kinetic scheme of thefollowing type:

top) k,

TrpCj Ti Trp0p + 02 -- quenchingI k, I

Proc. Nat. Acad. Sci. USA 71 (1974)

1 TOI /To

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Phosphorescence of Proteins at Room Temperature 4157

in which Trpcj and Trp., represent the triplet states of thetryptophans in the closed and open forms of the protein;k0, and keg the opening and closing rate constants, To thetryptophan lifetime, and kIc, the oxygen quenching rate con-stant for free tryptophan. In that the closed conformation ofthe protein is the stable one (k >> kp,), phosphorescence isobserved only for tryptophan in the closed form. The rateconstant for phosphorescence decay under conditions of steadystate illumination is given by:

0 2 4 6%o,, It x mole' x10-4

FIG. 3. The contribution of dissolved oxygen to the room-temperature phosphorescence lifetime of LADH at pH 8.6 as afunction of [oxygen]-T-ro, = (/T - l/ro)-1. In terms of theproposed model, the finite lifetime observed as [02] pro-

vides a measure of k., and the slope = kal/kop- kq.

The general conclusion from our data that conformationalfluctuations must occur in proteins in solution is consistentwith recent interpretations of tritium exchange data (16), andwith the conclusions of Lakowicz and Weber (17) who ob-served the influence of high concentrations of dissolved oxygenon the fluorescence of aromatic residues in proteins. In theirstudies, Lakowicz and Weber found that quenching of eveninternal tryptophan residues in a number of proteins occurredwith a quenching constant close to the diffusion-controlledone. Furthermore, the fluorescence lifetimes in their experi-ments tend toward zero with increasing oxygen concentration.The oxygen dependence of the triplet lifetime for LADH inthe present work results in an- effective oxygen quenchingconstant which is four orders of magnitude less than the tripletquenching rate constants of 109- 3 X 109 1 mole-1 sec' ob-served for aromatic molecules in solution (15), and the life-times do not extrapolate to zero. Comparison between our datawith LADH and alkaline phosphatase and the fluorescenceresults of Lakowicz and Weber for a number of other proteins,suggests that globular proteins differ markedly in the rate atwhich they undergo conformational fluctuations. In order toclearly resolve this question, experiments in which fluores-cence and phosphorescence quenching by oxygen are mea-sured in the same proteins will be needed since singlet andtriplet quenching appear to occur through different mecha-nisms and with different rate constants (15).We do not as yet have sufficient data to attempt to corre-

late the rate constants for conformational oscillations inLADH observed in these studies with enzyme activity. It isof interest that isomerization of the enzyme-coenzyme com-plex has been proposed (18) to occur in LADH with a rateconstant (11 sec-') essentially equal to the opening rate con-stant in our studies. More detailed studies in the presence ofcoenzyme and substrate will be required to ascertain whetherthe structural motions monitored by this technique are in-volved in the mechanism of enzyme catalysis.

1.

Oa)

[1]

The contribution of the oxygen dependence to the lifetime canbe expressed in the form:

[2]

Values of r, are computed from the measured lifetimes andvalues for ro, the oxygen-independent lifetime, are determinedat low oxygen concentrations. Eq. [2] predicts that To, shouldbe a linear function of the reciprocal of the oxygen concentra-tion in the solution. As the 02 concentration goes to infinity,the lifetime is determined by ko,, the rate constant for thetransition from the "closed" to the "open" conformation.The oxygen dependence is governed by an "effective" quench-ing constant, kg - kp/k,1, which is the quenching constant for afree chromophore modified by kIp!k1,, the ratio of the closingto opening rate constant for the conformational isomerization.A significant feature of the model is that at high oxygen con-centration, the triplet lifetime of the internal residue tends to afinite value. Quenching under these conditions is rate-limitedby the structural change which exposes the chromophore tooxygen. In a diffusional type quenching, the lifetime of thechromophore would go to zero at infinite 02 concentration.A plot of To,, obtained from the tryptophan triplet lifetime

of LADH in pyrophosphate buffer at 298'K, as a function ofthe reciprocal of the oxygen concentration appears in Fig. 3.A linear relationship is observed and the lifetime extrapolatesto a finite value as the oxygen concentration goes to infinity.The intercept at infinite 02 concentration results in a value ofkop = 12 sec', and the slope leads to an effective quenchingconstant of 6.5 X 104 1 mole-1 sec'. The oxygen tripletquenching constant for tryptophan in water at room tempera-ture has not been determined to date. Employing an averagevalue of 109 1 mole-' sec-' [obtained from the data of Gijze-man, Kaufman, and Porter (15) who have recently deter-mined quenching constants for aromatic chromophores insolution at room temperature] results in a k, I of 1.8 X 105 sec-l.The ratio of kp/kel is 6.7 X 10-5 indicating that the equi-librium lies far in the direction of the closed conformation, sothat the protein exists for only a small fraction of the time inthe more open structure. The calculation of keg is based on theassumption that the tryptophan in the more "open" conforma-tion is quenched at the rate observed for aromatic chromo-phores in solution. Quenching in the conformational state inwhich the tryptophan can interact with oxygen will dependon its extent of exposure in this state. Quenching at the diffu-sion-controlled rate represents an upper limit and thus thecalculated value for ke1 must be regarded as a maximum.

I= - + kop (1 k+-k[02])

T To k, k [02

1 1 kc, 1TO, = = +1 1 /cop kop.k0 [021

T To

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4158 Chemistry: Saviotti and Galley

TABLE 1. Phosphorescence lifetimes of LADHunder various experimental conditions

(Lot no 33 E 650)Enzyme concentration = 10-4 M T = 250C'

[021 < 10-6 MPhosphorescence

Experimental conditions lifetimes (msec)

pH 8.6* 134pH 7.5, 0.05 M phosphate buffer 90NADH:enzyme (3:1), pH 8.6 706 M Urea, pH 8.6 971 M Guanidine* HCI, pH 8.6 60Ethylene glycol: buffer (1: 1) 260Glycerol: H20 (3:2) 240

* Unless otherwise stated the buffer used in the experimentswas 0.032 M pyrophosphate buffer at pH = 8.6

While the present data indicate that a periodic loosening ofthe protein structure must occur in the region of the internaltryptophan in LADH, they do not establish the amplitude ofthe oscillations nor the extent to which these are delocalizedover the protein. Triplet quenchers that have a variety ofmolecular diameters may help to resolve the former question,but the charge, chemical nature, and hydration must also beconsidered in accessing their effectiveness as quenchers.

Phosphorescence studies of the dynamic aspects of thestructure of LADH will become more meaningful when exam-ined in the light of the x-ray structure of the enzyme in thecrystalline form (19, 20). The room temperature phosphores-cence observed in these studies is undoubtedly from Trp 314 inthe sequence (21) which from the x-ray data of C. -1. Bran-ddn et al. (personal communication) is buried in a hydro-phobic environment close to the contact region between thetwo subunits in the enzyme. Trp 15 which is partially exposedto solvent correlates well with the residue whose phos-phorescence properties reflect an external environment.Analysis of the x-ray data particularly in the region of Trp 314should aid in elucidating the structural basis of the conforma-tional fluctuations which we appear to be monitoring in thesestudies.

(ii) Oxygen Independent Triplet Lifetimes. The Orinde-pendent lifetime for the tryptophan residue in LADH of 0.13see is markedly shorter than the 5.5 see lifetime typicallyobserved from the phosphorescence at 770K. It is also con-siderably shorter than the 0.8 sec 02-independent tryptophanphosphorescence lifetime observed for alkaline phosphatase.At very low 02 concentration, the triplet lifetime for trypto-phan residues in a protein is clearly influenced by the partic-ular environment in which the chromophore is located as wellas by the temperature. The Orindependent triplet lifetime ofLADH was observed to be extremely sensitive to perturba-tions to the protein structure. The lifetime measured at roomtemperature was reproducible from day to day with a givenlot of enzyme, but both the enzyme activity and the lifetimevaried between 80 and 200 msec from one lot number to the

next; the enzyme batches with the shorter lifetimes appearingto be the most active. With a given lot of enzyme, the trypto-phan triplet lifetime of LADH at room temperature is in-fluenced by changes in solvent, pH, and the addition of co-enzyme and denaturants. The influence of variations in theexperimental conditions of the 02-independent phosphores-cence lifetime of LADH are summarized in Table 1. Ourknowledge of the factors affecting radiationless transitions inaromatic chromophores is inadequate at the present to inter-pret these lifetimes in terms of particular environmentalperturbations. It is likely that time-dependent perturbationsarising from fluctuations in the protein structure and/orperiodic exposure to a mobile polar solvent may play a role indetermining the lifetime. More detailed studies of this typewhich include comparisons with x-ray structural data shouldhelp to establish the factors which influence triplet-singletradiationless transitions at ambient temperatures, not only forchromophores in proteins but for molecules in general. Inorder to determine in a more definitive way the influence ofthese various experimental conditions on the rate of conforma-tional fluctuations in the protein a study of the oxygen depen-dence in each case will be required.

We are indebted to G. Strambini for helpful discussions of thiswork and to J. Milton for assistance with low temperature ex-periments. This work was supported by a grant from the NationalResearch Council of Canada.

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