Photochemistry and Photobiology, 1976, Vol. 23, pp. 147-154

Pergamon Press. Printed in Great Britam

PHOTOCHEMISTRY OF MODIFIED PROTEINS BENZOPHENONE-CONTAINING BOVINE SERUM ALBUMIN PATRICK S. MARIANO: GEDRGE I. GLOVER* and TIMOTHY J. WILKINSON Department of Chemistry, Texas A & M University, College Station, TX 77843, U S A (Received 17 July 1975; accepted 16 October 1975) Abstract-The results of exploratory and mechanistic studies of the photochemistry of poly-p-benzoylacetimido-bovine serum albumin, a modified protein containing photoreactive and photosensitizing groups, are reported. Specifically described are our recent findings concerning (1) the synthesis and characterization of a modified bovine serum albumin that contains benzophenone-like moieties, (2) the photochemistry of this modified protein which appears to involve photoreductive coupling of the benzophenone chromophores to the protein backbone, and (3) triplet energy transfer from modified bovine serum albumin to small molecule acceptors resulting in quenching of the photoreaction.

INTRODUCTION

modified proteins containing covalently-bound, conjugated carbonyl moieties and the study of their photochemistry and utility as macromolecule photosensitizers. These studies have resulted in several rather interesting observations that we are reporting in this and the succeeding paper. In this paper we describe our results concerning (1) the synthesis and characterization of a modified bovine serum albumin (BSA) that contains benzophenone-like moieties, (2) the photochemistry of this modified protein which appears to involve photoreductive coupling of the benzophenone chromophores to the protein backbone, and (3) triplet energy transfer from modified BSA to small molecule acceptors resulting in quenching of the protein photoreaction and sensitization of the acceptor triplet photoreaction.

Recent studies in our laboratories (Glover et al., 1974) have focused on several aspects of electronic excited state reactions of suitably modified proteins. One facet of this effort results from our interest in both the effect and utility of irradiation of proteins which have been modified with reagents containing conjugated carbonyl chromophores. The possibility that the photochemistry, which results from selective excitation of the phenone groups and involves reaction of the excited phenones and protein backbone, might yield novel methods for photoaffinity labeling is of particular interest. In addition, complementary studies on methods to quench these intra-protein excited state reactions could lead to a more thorough exploration of the mechanism of intermolecular energy transfer from modified proteins to small molMATERIALS AND METHODS ecule quenchers in solution. Another important aspect of our studies concerns the potential utility of approGeneral. Bovine serum albumin (BSA) (A-4278, Sigma) priately modified proteins as photosensitizers in novel was used without further purification since gel filtration revealed that only monomer and a small amount of dimer methods for chiral photochemical synthesis. present. All buffered solutions were made with the In our previous studies we have demonstrated that were same pH 8.0 buffer, consisting of 0.05 M KH,PO, and phenacyl- and naphthacyl-modified cr-chymotrypsin 0.1 M KC1 adjusted to pH 8.0 with 5 N KOH. trans-Cinhave absorption characteristics that allow selective namic acid (Sigma) was twice recrystallized prior to use. excitation of the aryl ketone chromophores. In addi- All other chemicals were commercially available and were tion, we observed that the interesting photochemistry of reagent grade or better. Ultraviolet (UV) spectra were recorded using a Beckman of these enzymes is a result of reactions involving Acta I11 UV-visible spectrophotometer.NMR spectra were cleavage of the aracyl a-carbon methionine-192 car- determined with a Varian T-60 spectrometer using TMS bon-sulfur bond leading to photoregeneration of (tetramethylsilane)or TSP-deuterated (sodium 3-trimethylnative cr-chymotrypsin and a new, as yet unidentified, silylpropionate-2,2,3,3-d4)as internal standards. All melting points were determined in capillary tubes with a Melphotoaffinity labeled enzyme. Although these reac- Temp melting point apparatus and are reported uncortions are most probably occurring from the triplet rected. Simultaneous irradiations of multiple samples were excited states of the aracyl moieties, the efficiency of carried out using an apparatus consisting of a merry-gothe process is unchanged in the presence of known round at the center of which was located a 450-W Hanovia triplet quenchers. More recent investigations in this medium-pressure lamp surrounded by a uranium glass filter (T330nm = 5%, T350nm = 45%, T370nm = 76%) in a area have been directed at the preparation of other water cooled quartz immersion well. Single sample irradiations were done using an immersion apparatus, consisting of a water-cooled quartz well and appropriate glass filters, * Authors to whom correspondence should be sent. 147 I’.A.P.

2313-

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PATRICK

s. MARIANO, GEORGE I. GLOVERand TIMOTHY J. WILKINSON

within an outer flask containing the solution. All photolysis solutions were degassed in vucuo using a water aspirator and saturated with nitrogen prior to irradiation. Howcvcr. we have found that dissolved oxygen has no effect upon the rate of the modified-protein photoreaction. Microanalysis was conducted by Galbraith, Knoxville, Tennessee. Methyl p-henzoylphenylacetimidatehydrochloride (1). The method utilized was similar to that reported for preparation of methyl 2-phenylpropanimidate hydrochloride (McElvain and Stevens, 1947). p-Benzoylphenylacetonitrile (2) (Zderic et al., 1961) was used as starting material. The yield of this reaction was 70% and the crystalline product, mp 81--82"C(dec), has the following NMR spectral properties; 6 7.73 (2H, ABd, o-aromatics on disubstituted phenyl), 7.48 (2H, ABd, m-aromatics on disubstituted phenyl), 7.4 8.0 (SH, m, monosubstituted phenyl, 4.32 (3H, s, NCH,) and 4.19 (2H, s, CH,). N-Methyl p -benzoylphenylacetamidine hydrochloride (3). A procedure similar to that of Hand and Jencks (1962) for preparation of N-methylbenzamidine was employed. A solution of 1, 0.8 g (2.8 mmol), in 24 m/ water and 12 m/ of methanol containing 1.8 g (26 mmol) methylamine hydrochloride and 0.6 g (9.1 mmol) KOH was stirred at room temperature for 12 h. Workup followed the published procedure and gave a crystalline product, 0.60 g (75%). which was very hygroscopic and had an indefinite melting point of ca. 85-90"C, dec. NMR (D,O); 6 7.5-8.0 (9H, m, aromatic), 4.2 (2H, s, -CH,-) and 3.2 (3H, s, N C H , ) ; UV ( H 2 0 ) :1,,,259 nm, E 18,700. Anal. Calcd. for C,,H1,N,0CI.1/2 H,O: C, 64.53; H, 6.05; N, 9.40. Found: C. 64.26: H, 5.94; N, 9.61. p-Benzoylphenylacetimido-bovine serum albumin (ModBSA). A procedure similar to that of Wofsy and Singer (1963) for acetimidation of BSA was used. BSA, 1.0 g, was dissolved in 100 ml deionized water and the pH of the solution was adjusted to 9.2 with 1 N KOH. The imido ester hydrochloride, 1, 0.21 g (0.73 mmol) was dissolved in 12 m/ of cold methanol. After adding 12 m/ of cold water and adjusting the pH to 9.2 with 5 N KOH, the solution was rapidly mixed with the cold BSA solution. The resulting mixture was stirred in an ice-bath for 5 h. The pH of the mixture was then adjusted to 8 with 1 M HC1 and a small quantity of precipitated material was removed by centrifugation and Millipore filtration (0.45 ppore size). Gel-filtration of the filtrate on a 5 x 70 cm column of Sephadex (3-25 (fine) was conducted; elution was with deionized water. Fractions (10 m/) were collected at a flow rate of 20 m/ per h and the eluent was monitored at 254 nm. The modified protein-containing fractions (9-31) were combined and lyophilized to yield 860 mg of mod-BSA. Determination of the number of covalently hound benzophenone moieties per Mod-BSA molecule. The titrimetric method of Hunter and Ludwig (1962) was used to determine the number of covalently modified lysines per protein molecule to be 7. An independent method utilizing UV spectroscopic measurements on mod-BSA, BSA and the model amidine 3 was also employed. The number of modifications per mod-BSA molecule, ca. 6, was determined by dividing the difference between molar extinction coefficients of mod-BSA and BSA at 260 nm by that of the model acetamidine hydrochloride, 3. Cis-Cinnamic acid. A mixture of cis- and trans-cinnamic acid was prepared by the standard photochemical method and separated to give the pure cis-isomer by the new method employing Sephadex G-25 chromatography (Glover, 1975). Exploratory photolysis of mod-BSA. Mod-BSA, 100 mg, n ' of pH 8 buffer, 7.2 pM, was irradiated in the in 200 t preparative apparatus, using a uranium glass filter. Two m" aliquots of this solution were removed at the time intervals indicated in Fig. 3 and the UV spectrum scanned

from 200-360 nm. During the irradiation, the absorbance at 260 nm decreased from its initial value of 0.988, A , and after ca. 6 h leveled off at a value of 0.490, A , . The mole fraction of the benzophenone chromophores remaining at time t is expressed as A,-A/A,-A,, where A is the vs time absorbance at time t. A plot of In (A,-A/A,-A,) is given in Fig. 3. Solutions of mod-BSA in pH 8 buffer (72, 43, 29, 14 and 7.2 pM) were irradiated using the merry-go-round apparatus and monitored in the same manner as described above. When plotted as in Fig. 3, the observed mod-BSA photoreaction velocity was independent of protein concentration. Extensively irradiated protein was purified by gel filtration on Sephadex G-25 (fine); the eluent was monitored at 254 nm. The UV spectrum of the lyophilized protein fractions is shown in Fig. 4. Irradiations of mod-BSA in the presence of varying concentrations of potential quenchers. Stock solutions of the following potential quenchers were prepared in pH 8 buffer: sodium trans-cinnamate (59 mM), sodium octanoate (59 mM) and sodium 3-phenylpropionate (59 mM). Aliquots of these solutions were diluted with appropriate amounts of buffer to give a series of solutions of varying molarities. One m t portions of these solutions were mixed with 2 naP of a mod-BSA solution (65 p M in pH 8 buffer) to give final solutions containing the varying concentrations of the three potential quenchers as indicated in Fig. 5. These solutions along with a control consisting of mod-BSA alone were simultaneously irradiated for 2 h in the merry-go-round apparatus using uranium glass filtered light. One naP of each photolysate was gel filtered on Sephadex G-25 (fine) to separate quencher from protein. The protein-containing peaks were combined and diluted to 10.0 mf with pH 8 buffer. The absorbancies at 260 nm of these solutions were measured and used to obtain the relative observed mod-BSA reaction velocities. which are a function of the relative disappearance of absorbance at 260 nm for mod-BSA alone and mod-BSA in the presence of quenchers after 2 h irradiations. Ratios of the unquenched to quenched relative velocities as a function of quencher concentrations are plotted in Fig. 5. Mod-BSA photosensitized isomerization of sodium cis- and trans-cinnamate Seven 2.5 r d solutions each containing 5 mg of mod-BSA (28 pM)and 20 mg of sodium trans-cinnamate (4.7 mM) in pH 8 buffer, and seven 2.5 mf solutions each containing 5 mg of mod-BSA (28 p M ) and 20 mg of sodium cis-cinnamate (4.7 mM) in pH 8 buffer were individually placed in pyrex tubes and simultaneously irradiated in the merry-go-round apparatus using uranium glass-filtered light. Two controls each containing 20 mg of cis- and trans-cinnamate, respectively, and 5 mg of BSA in 2.5 mt of pH 8 buffer were also irradiated. In addition, dark controls containing both cinnamate isomers and mod-BSA were prepared. Tubes were removed from the irradiation apparatus at 1, 2, 3, 4, 5, 6 and 8 h intervals. A 1.9 aliquot from each tube was chromatographed on a Sephadex (3-25 (fine) column (1.1 x 70 cm) using pH 8 buffer as eluent, and monitoring eluate at 254 nm. The cis- and trans-cinnamate-containing peaks were independently collected and diluted to exact volumes. Quantitative determinations of the amounts of cis- and trans-isomers were accomplished by UV spectroscopy using as the molar absorptivity for the cis-isomer har= 11,500, and for the trans-isomer, hmax = 20,000. The results of this experiment are summarized in Fig. 6 which shows a plot of the mol % of cis-isomer vs irradiation time. In addition, spectroscopic analysis of the cinnamate fractions from the chromatographed photomixtures which contained BSA and the individual cinnamate isomers and the corresponding dark controls demonstrated the complete absence of isomerization in these cases.

Photochemistry of benzophenone-containing BSA Methyl p-benzoylphenylacetamidine hydrochloride sensitized isomerization of cis- and trans-cinnamic acid. Solutions containing the same concentrations of the sodium cinnamate isomers and amidine hydrochloride, 3, as sensitizer (based on 6 modifications per molecule of mod-BSA) as used in the mod-RSA sensiti7ed isomerij.ations described abovc. wcrc utilized. Specifically. seven 3 m/ solutions each containing the amidine hydrochloride (0.1 1 mg per m/, 0.037 mM) and sodium cis-cinnamate (0.77 mg per m/, 5.2 mM) in buffer and seven 3 ml solutions each containing the amidine hydrochloride (0.11 mg per &, 0.037 mM) and sodium trans-cinnamate (0.77 mg per mC, 5.2 mM) in pH 8 buffer were simultaneously irradiated using the merry-go-round described above. Tubes containing each of the sensitizer-cinnamate solutions were removed at various time intervals during the irradiation. The contents were acidified and ether extracted. The ethereal extracts were extracted with pH 8 buffer. Sephadex G-25 (fine) chromatography on a 1.1 x 70 cm column of the buffer extracts was used to separate the cis- and trans-cinnamate isomers and the exact quantities of each isomer were determined by UV spectroscopy using the same procedure as described above for analogous mod-BSA sensitized runs. A plot of the % cis-isomer composition vs irradiation time is found in Fig. 6. Phosphorescence spectra. Phosphorescence spectra were recorded, using a Fluorispec SF-100 (Baird Atomic) equipped with a quartz Dewar flask and rotating shutter, at 77 K. Samples were prepared by dissolving the protein and/or quenchers in N,-saturated deionized water and diluting with an equal volume of ethylene glycol. The solutions in quartz tubes were then degassed by repeated freezethaw cycles at reduced pressure. Spectra were recorded on the following solutions; mod-BSA (0.14 mM) and sodium acetate trihydrate (48 mM); mod-BSA (0.14 mM) and sodium a-napthylacetate (48 mM); sodium a-naphthyl acetate (48 mM); and extensively irradiated mod-BSA (0.14 mM) (see Fig. 7). In all cases the excitation wavelength was at 366 nm (except for a-naphthylacetate at 313 nm) while recording emission from 370 to 650 nm. The excitation spectra of the phosphorescence bands in all cases matched closely those of the species excited.

RESULTS

Preparation of p-henzoylphenylacetimicio-bovine serum albumin (Mod-BSA). The modification reaction sequence (see Fig. 1) used to attach benzophenone moieties to the BSA backbone was analogous to that developed by Wofsy and Singer (1963) who observed that ethyl acetimidate undergoes smooth reaction with BSA, resulting in modifications of ca. 62-100% of the e-amino groups of lysine. The percent modificat

0

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HCl 2

R

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CHfC-NHCH3 Mod-ESP.

Figure I . Synthetic sequence utilized for the preparation of mod-BSA and the model amidine 3.

149

tion was found to be dependent on reaction time and concentration of the imidate reagent. Accordingly, the modification reagent, methyl p-benzoylphenylacetimidate hydrochloride (I), was prepared from the known substituted acetonitrile 2 (Zderic et al., 1961) by treatment with methanolic hydrogen chloride and was characterized on the basis of its spectral properties; its NMR spectrum displayed the characteristic phenone and para-disubstituted phenyl aromatic proton resonances along with those of the acetimidate methylene and methyl ester protons. This imidate hydrochloride proved to be a labile compound and, thus, resisted accurate elemental analysis. However, its N-methyl amidine hydrochloride derivative 3 was easily derived by treatment of 1 with methylamine, and analyzed yielding an elemental composition and spectral data in excellent agreement with its assigned structure. Derivatization of 1, as well as offering support for its structure, established the feasibility of using 1 as an imidinating reagent with compounds containing free amino groups. The modification reaction to prepare p-benzoylphenylacetimido-BSA (mod-BSA) occurs smoothly upon mixing BSA and the imido ester 1 in aqueous methanol at pH 9.2. Mod-BSA was purified by gel filtration of the crude reaction mixture on Sephadex G-25. The UV spectrum of mod-BSA, recorded in Fig. 2, displayed an expected enhanced absorbance over that of BSA at 260 nm and above 300 nm, wavelengths at which benzophenone and the model amidine 3 have maxima. UV spectral data of the modified protein was used to obtain the approximate number (m) of p-benzoylphenylacetimido residues per molecule of BSA introduced in the modification reaction. Accordingly, m was determined to be ca. 6 from the division of the difference between the molar absorptivity of mod-BSA and BSA at 260 nm by that of the model N-methylacetamidine 3. Determination of m, after mod-BSA was subjected to the defatting procedure of Chen (1967) followed by gel filtration, gave the same result as above. This offered a firm assurance that all benmphenone containing moieties were covalently linked to the protein and not strongly reversibly bound due to a possibly large association constant (Flanagan and Ainsworth, 1968). Another estimate of the number of covalent modifications of lysines in mod-BSA was obtained using the titration method developed by Hunter and Ludwig (1962). This method gave an estimate of 7 modified lysine residues per mod-BSA molecule. Photochemistry of the modijied bovine serum albumin. The UV spectra of mod-BSA and BSA along with the difference spectrum between the two proteins, recorded in Fig. 2, clearly indicate that irradiation with light of wavelengths greater than 320 nm results in selective (> 99.9%) excitation of the benzophenone chromophores of mod-BSA. Irradiation of mod-BSA (7.1 pA4) in aqueous buffered solution at pH 8.0 using uranium glass filtered light (A > 320 nm) was conducted while monitoring the reaction by UV

I50

PATRICK S. MARIANO, GEORGEI. GLOVER and TIMOTHY J. WILKINSON

Wavelength,

nm

Figure 2. UV spectra of mod-BSA (-), BSA (---) and the amidine hydrochloride 3 (-.-.) in pH 8 buffer. The mod-BSA BSA difference spectrum (. ...) was recorded with equal concentrations of BSA in the reference and mod-BSA in the sample compartment.

spectroscopy. During the irradiation, the absorbance at 260 nm rapidly decreased. A plot of In (Ao-A/Ao-A,) vs t, as shown in Fig. 3, indicates that the reaction is essentially pseudo first-order throughout. The apparent rate is also independent of modBSA concentration in the range of 7.2-72 pM. A control irradiation of BSA under identical reaction conditions did not result in an observable change in the ultraviolet spectral properties of the protein, thus, confirming its expected photostability under these reaction conditions (see McLaren, 1970). After extensive irradiation, the photolysate was gelfiltered on Sephadex G-25 in order to separate protein from any small molecule fragments that may have been liberated during photolysis. No low-molecular weight compounds were detected in the column eluant by monitoring at 254 nm. Additionally, the recovered protein had an UV spectrum identical to that before gel-filtration; the molar absorptivity at 260 nm was 6.61 x lo4 compared to 13.2 x lo4 for mod-BSA (see Fig. 4). Since native BSA showed no change in UV absorbance on irradiation, its molar absorptivity can be subtracted from that of mod-BSA

P Figure 4. UV spectra of recovered, purified protein after extended irradiation of mod-BSA (-), and of benzhydrol (-----),

before and after irradiation to obtain the components of the 260 nm molar extinction coefficients which correspond to benzophenone moieties in mod-BSA (10.5 x lo4) and the new chromophores present in irradiated mod-BSA (3.95 x lo4). The per chromophore absorptivity, calculated on the basis of 6 modifications per protein molecule, at 260 nm of irradiated mod-BSA (7.05 x lo3) is much larger than expected if all benzophenone moieties are transformed upon irradiation into, for example, benzhydrol-like chromophores (note Fig. 4). In order to determine if the remaining absorbance of the extensively irradiated modified protein was due to unreacted benzophenone chromophores, phosphorescence spectral analyses were conducted. No phosphorescence from the recovered protein was detected under conditions in which one benzophenone moiety per five protein molecules would have been detected. Quenching of the mod-BSA photoreaction. In an attempt to more fully understand the nature of the photoreaction occurring upon irradiation of modBSA at wavelengths greater than 320 nm, and to determine the types of compounds that affect the rate of this process, exploratory quenching experiments were conducted. In Fig. 5 are summarized the results of these experiments in which the observed velocity --I of the mod-BSA photochemical reaction was measured as a function of concentrations of the sodium salts of trans-cinnamic, octanoic, and 3-phenylpropionic acids. Plotted in Fig. 5 are the 4 ratios of the observed photochemical reaction velocities in the presence and absence of these compounds (VJV,) vs their concentrations, [Q]. Mod-BSA as a triplet photosensitizer, In order to determine if the observed quenching by sodium trans0 40 80 120 I60 200 240 280 cinnamate of the mod-BSA photoreaction is due to Irradlotlon time. min triplet energy transfer, we have investigated the phoFigure 3. Plots of the disappearance of mod-BSA absortosensitized cis-trans isomerization of cinnamate bance. expressed as % absorbance loss at 260 nm (0)and the In of the ”/, absorbance loss (A)vs irradiation time (Bregman et al., 1964) using mod-BSA and the model amidine 3 as sensitizers. Irradiations, using conditions using uranium glass filtered light. Y

Photochemistry of benzophenone-containing BSA

151

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Figure 6. Mod-BSA (&A) and amidine hydrochloride 3 sensitized photoisomerization of sodium cis- and Figure 5 . Plots of mod-BSA photoreaction quenching effi- trans-cinnamates. A plot of cis-isomer composition vs irraciencies (V,,/ VJ vs quencher (Q) concentration using trans- diation time. Open symbols refer to isomerizations starting cinnamate ,).( octanoate (0)and 3-phenyl-propionate (0) with cis- and closed symbols with trans-cinnamic acid. as quenchers. Quencher

concentration,

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in which light is absorbed only by the amidine 3 vations concerning the photochemistry of benzo(uranium glass-filtered light), led to cis-trans isomeri- phenone containing systems and the excited state zation of both the isomeric acids (Fig. 6).When mod- reactions of benzophenone with di- and polypeptides. The first aspect that requires attention involves the BSA was used as the photosensitizer, cis-trans isomerization of the cinnamic acids was again observed (Fig. location of the excitation energy in the reactive 6). Control irradiations under identical conditions in excited state of mod-BSA. Intraprotein excitation the absence of either amidine 3 or mod-BSA and in migration via singlet and triplet energy transfer the presence of BSA did not result in isomerization mechanism is a well studied phenomenon in protein photochemistry (Cassen and Kearns, 1969). Thus, our of either of the cinnamic acids. Phosphorescence spectroscopy was employed as an observation that the phosphorescence spectrum of additional method for demonstrating the nature of the mod-BSA contains only emission maxima characterquenching effect. The phosphorescence emission spec- istic of benzophenone is important in that it strongly trum of mod-BSA, excited at 366 nm, closely resem- indicates that the triplet benzophenone moieties, bles that of benzophenone. Excitation of mod-BSA formed by rapid intersystem crossing from their inin the presence of sodium a-naphthylacetate results itially populated singlets, are the locus of the exciin a substantially reduced intensity for the benzo- tation energy and the sites of reaction in the excited phenone-like emission and a simultaneous and over- mod-BSA molecule, Further support for this conclulapping sensitized a-naphthylacetate phosphorescence sion derives from the fact that those molecules which spectrum as is shown in Fig. 7. quench the benzophenone-like phosphorescence of mod-BSA also quench its photoreaction (vide inpa). The nature of the mod-BSA photoreaction must then resemble that of the well studied benzophenone, DISCUSSION which is known to undergo a highly efficient photoreMod-BSA photoreaction. The observations reported duction to benzpinacol in the presence of hydrogen above appear to indicate that the p-benzoylphenylace- atom donating solvents (Turro, 1965), and photoretimido derivative of BSA undergoes a novel intraproductive additions across carbon hydrogen bonds tein photochemical reaction upon selective excitation resulting in the production of benzhydrol derivatives of the covalently bound benzophenone moieties. (Arnold et al., 1964; Bradshaw, 1966). The first of Although the exact structural outcome of this photo- these reaction types seems an unlikely possibility for process is unknown at this time,* a reasonable the modified protein system due to two factors; the rationale for our observations in terms of the prob- quantum yield for photoreduction of benzophenone able mechanism of the excited state reaction along to benzpinacol in water is low (Beckett and Porter, with the local structure about the transformed benzo1963)and the intramolecular separation between benphenone moieties can be suggested. Our interpre- zophenone moieties is most probably large and tation is aided by several recent and pertinent obser- rigidly fixed by the protein backbone. On the other hand, the second reaction type appears as a reason*The locations of the lysine groups in the three dimen- able and likely possibility for the covalently bound sional structure of BSA and, thus, of the benzophenone triplet excited benzophenone moieties, born in an enmoieties of mod-BSA are not known. As a result, an exact structural analysis of the product from the mod-BSA pho- vironment consisting mainly of water and the polytoreaction would be extremely difficult to make at this peptide chain. It is interesting that the literature holds several reports of observations on simpler molecular time.

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PATRICK S . MARJANO, GEORGE I. GLOVER and TIMOTHY J. WILKINWN

Wavelength,

nm

Figure. 7. Phosphorescence spectra of mod-BSA (-1. sodium a-naphthylacetate (----) and a mixture of modBSA and sodium a-ndphthylacetate (-.-.-). systems, which appear directly related to the photoreactions occurring in the more complex protein (vide inpa). Accordingly, our experimental findings can be conveniently rationalized on the basis of intraprotein reactions, resulting from hydrogen abstractions by triplet benzophenone groups followed by coupling of the formed radicals and leading to conversion of benzophenone chromophores into those of substituted benzhydrols (see Fig. 8). The net structural effect of this process would be the introduction of cross-linkages into the protein chain through the bridging p-(phenylhydroxymethyl)phenylacetimido groups. The UV and emission spectral properties of the protein recovered after irradiation are consistent with structure 4, which schematically represents the local structure about the original benzophenone groups. Although the results that serve as a basis for our conclusions are limited, they are in accord with pertinent observations in the literature on the specific nature of this reaction. For example, Breslow (1972)

has utilized the efficient photoreductive addition reactions of benzophenone derivatives in methods fof remote intramolecular functionalization of steroids and fatty acids. In the case of benzophenone-containing esters of fatty acids and esters of steroidal alcohols, hydrogen abstraction by the triplet excited aromatic ketone carbonyl occurs predominantly at sites within close spacial proximity to the carbonyl oxygen. In addition, the result of the primary photochemical reactions in these cases is the formation of cyclized benzhydrol derivatives, e.g. the transformation of the cholestanol ester 5 (Breslow and Baldwin, 1970) (see Fig. 9). We can assume that the modified protein photoreaction would have its course governed by similar factors, i.e. that the sites of hydrogen abstraction and ensuing carbonxarbon bond formation would be determined by the locations of the triplet excited benzophenone carbonyls. Although proximity would be of prime importance, another feature that should be influential in determining the sites of crosslinking in the polypeptide chain is the nature of the proximal carbon-hydrogen bonds. A priori, one would expect that the wCH bonds of the amino acid residues along with the benzylic- and pseudo-benzylic-CH bonds of phenylalanine, tyrosine, histidine and tryptophan would be most labile to radical abstraction. Pertinent in this regard are the interesting results of Schwarzberg et al. (1973) which demonstrate that hydrogen abstractions by triplet acetone and oxy-radicals, quite surprisingly, display selectivity for the cr-CH bonds of glycine in glycine-containing di- and polypeptides. This selectivity exists even when tyrosine and phenylalanine residues are also present. The recent results of Galardy et al. (1973) are analogous to those of Schwarzberg and serve as direct precedence for our proposal for the nature of the mod-BSA photoreaction. They have found that irradiation of benzophenone in the presence of the methyl ester of N-acetylglycine (6)results in production of the diphenylhydroxymethyl derivative, 7, of the blocked amino acid (see Fig. 9). R

R

1

H obttroct.

5

R

x

P

hJ C H ~ C - N H - C H & - O C ~ L CH$-NH-CHC-OCH~

R

I

P h2C 0

PhT-Ph

OH

6

Figure 8. Proposed mechanistic pathway and partial photoproduct structure for the mod-BSA intraprotein photochemical reaction.

7

Figure 9. Reported photochemical reactions involving additions of benzophenone across carbon-hydrogen bonds in steroidal esters and blocked amino acids.

Photochemistry of benzophenone-containing BSA

Thus, the excited state reaction of mod-BSA, which results in destruction of the selectively excited benzophenone groups, can be conveniently rationalized on the basis of photoreductive additions of the triplet excited benzophenone groups to the or-carbons of glycine or other amino acid residues proximal to the carbonyl moiety. The near pseudo-first order disappearance of UV absorbance at 260 nm throughout the entirety of the mod-BSA photoreaction on first thought may not appear abnormal, since under the reaction conditions all photons of light are not absorbed. However, since each mod-BSA molecule contains more than one benzophenone modification, the near first order observation appears to indicate that all benzophenone moieties react with approximately the same efficiency. Another point worth mentioning concerns the observation that the molar extinction coefficient of extensively irradiated modBSA is higher than that predicted if all benzophenones are converted to benzhydrol-like derivatives as a result of the reaction type proposed. An explanation of this, based upon incomplete reaction of all benzophenone moieties, appears insufficient since no phosphorescence from the recovered protein was detected under conditions in which one benzophenone moiety per five protein molecules would have been detected. A possible, yet untested, rationale might be found if one or more glycine residue are functionalized as schematically represented in the partial structure 8. A secondary dehydration reaction is possible, due to the labile /I-hydroxycarbonyl linkage, and would yield the highly UV absorbing a-aminocc,fl-unsaturated amide linkages, represented in 9 (see Fig. 10). It is important to note, however, that Galardy and co-workers (1973) did not observe products of this nature in their study of model systems. Quenching of the mod-BSA photoreaction. Results from experiments designed to measure the effect of a selected variety of organic salts on the efficiency of the mod-BSA photoreaction have led to several interesting conclusions. As the data plotted in Fig. 5 indicate, the salt of trans-cinnamic acid quenches the mod-BSA photoreaction with modest efficiency, while those of octanoic and 3-phenylpropionic acid appear to slightly enhance the modified protein photoreaction. It is evident from these observations that only the salt possessing a chromophore of lower triplet energy than benzophenone (285 kJ/mol) is capable of quenching the mod-BSA reaction and, therefore, that the benzophenone moieties within the protein

153

must be reacting from their triplet excited states. The absence of quenching when photoreactions are conducted in the presence of 3-phenylpropionate and octanoate, compounds which should possess high binding affinities to mod-BSA [Peters, 1970; Reynolds et al., 1965; and Spector et at., 19711, rules out the possibility of non-photophysical quenching mechanisms that might have been operable. For example, reductions in the observed rates of the modBSA photoreaction could have been attributed to conformational changes of the protein, induced by binding of the organic salts (Nozaki et at., 1974), which change the relative spacial locations of the triplet excited carbonyls and abstractable hydrogens. Indeed, the small enhancement of the observed rate when octanoate and 3-phenylpropionate are present might be due to such a phenomenon. On the basis of the above, we can draw the preliminary conclusion that the observed quenching must be photophysical in nature and that it is the result of triplet energy transfer from the excited benzophenone groups. Further documentation for this conclusion is found in our studies of the photosensitized isomerization of sodium cis- and trans-cinnamate and of the photosensitized phosphorescence of sodium cc-naphthylacetate using mod-BSA. Our observation that mod-BSA serves as an effective photosensitizer for the familiar cis-trans isomerization of sodium cinnamate verifies our assignment of the triplet multiplicity to the protein photoreaction and energy transfer as the mechanism responsible for quenching. Also, we have shown that native BSA does not act as a photosensitizer for this cis-trans isomerization process under reaction conditions identical to those used with the modified protein. The isomerization, starting with either of the isomeric acid salts, does not proceed to the photostationary state even after extended sensitized irradiation (see Fig. 6). This observation is attributable to competitive photodestruction of the sensitizer over long irradiation periods. The phosphorescence spectrum of mod-BSA, excited at 366 nm, contains emission bands characteristic of benzophenone (Fig. 6). As mentioned above, this observation has led to our conclusion that the lowest energy and reactive excited states of mod-BSA must be of triplet multiplicity and have the excitation located on covalently attached benzophenone groups. The observation that sodium a-naphthylacetate quenches the triplet emission of mod-BSA in concert with the appearance of sensitized phosphorescence band identical to those of the naphthalene moiety of the acid salt is significant. Since a-naphthylacetate is not absorbing light directly under these condition+ proven by control runs-its triplet excited state must be populated by triplet energy transfer from the modBSA excited states. The accumulated results from these studies 8 9 designed to explore the nature of the mod-BSA phoFigure 10. Possible secondary dehydration reactions from toreaction, all point to the fact that the modified protein undergoes a novel photochemical reaction from initial products of mod-BSA photoreaction.

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PATRICK S. MARIANO, GEORGE I. GLOVER and TIMOTHY J. WILKINSON

the triplet excited states of the covalently-attached benzophenone groups and that the reaction is quenched by low molecular weight organic salts, which contain chromophores of the triplet energy below 285 kJ/mol, via triplet energy transfer. An interesting and perhaps significant aspect of these observations is that this protein photochemical process represents another example of the novel method of photoaffinity labeling, which utilizes as the reagent a reactive excited-state moiety covalently bound to known sites in the protein backbone. As we have discussed earlier (Glover et al., 1974) this photoaffinity labeling process would derive its advantage from the fact that the location of the photogenerated reagent

on the protein backbone is fixed and, therefore the site of functionalization is insured to be close by. Studies are continuing to explore and apply various aspects of the protein photochemistry we have reported.

Acknowledgments-The Robert A. Welch Foundation (Grants A-512 and A-501), the National Science Foundation (Grant No. 34245) and the donors of the Petroleum Research Fund administered by the American Chemical Society are gratefully acknowledged for their generous financial support of this research. T.J.W. would like to thank the R. A. Welch Foundation for a graduate fellowship.

REFERENCES

Arnold, D. R., R. L. Hinman and A. G. Glick (1964) Tetrahedron Letters 1425-1430. Beckett, A., and G. Porter (1963) Trans. Faraday Soc. 59, 2038-2050. Bradshaw, J. S. (1966) J . Ory. Chem. 31, 237-240. Bregman, J., K. Osaki, G. Schmidt and F. Sonntag (1964) J . Chem. Soc. 2021-2030. Breslow, R., and S. W. Baldwin (1970) J . Am. Chem. Soc. 92, 732-734. Breslow, R. (1972) Chem. Soc. Rev. 1, 553-580. Cassen, T., and D. R. Kearns (1969) Biochim. Biophys. Acta 194, 203-212. Chen, R. F. (1967) J . Biol. Chem. 242, 173-181. Flanagan, M. T., and S. Ainsworth (1968) Biochim. Biophys. Acta 168, 16-26. Galardy, R. E., L. C. Craig and M. P. Printz (1973) Nature (New Bid) 242, 127-128. Glover, G. I., P. S. Mariano, T. J. Wilkinson, R. Hildreth and T. Lowe (1974) Arch. Biochem. Biophys. 162, 73-82. Glover, G. I., P. S. Mariano and T. J. Wilkinson (1976) Separations Sci. (in press). Hand, E. S., and W. P. Jencks (1962) J . Am. Chem. Soc. 84, 3505-3514. Hunter, M. J., and M. L. Ludwig (1962) J . Am. Chern. SOC.84, 3491-3504. McElvain, S . M., and C. L. Stevens (1947) J . Am. Chem. Soc. 69, 2663-2666. McLaren, A. D. (1970) In Photochemistry of Macromolecules (Edited by R. F. Reinisch), Plenum Press, New York. Nozaki, Y., J. A. Reynolds and C. Tanford (1964) J . Bid. Chem. 249, 4452-4459. Peters, J., Jr. (1970) Adu. Clin. Chem. 13. 37-111. Reynolds, J. A., S . Herbert, H. Polet and J. Steinhardt (1965) Biochemistry 6, 937-947. Schwarzberg, M., J. Sperling and D. Elad (1973) J . Am. Chem. Soc. 95, 6418-6726. Spector, A. A,, J. E. Fletcher and J. D. Ashbrook (1971) Biochemistry 10, 3229-3232. Turro, N. J. (1965) Molecular Photochemistry, Chapt. 6, Benjamin, New York. Wofsy. L., and S. J. Singer (1963) Biochemistry 2, 104-116. Zderic, 2. A., M. J. Kubitschek and W. A. Bonner (1961) J . Org.Chem. 26, 1635-1637.

Photochemistry of modified proteins. Benzophenone-containing bovine serum albumin.

Photochemistry and Photobiology, 1976, Vol. 23, pp. 147-154 Pergamon Press. Printed in Great Britam PHOTOCHEMISTRY OF MODIFIED PROTEINS BENZOPHENONE...
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