Photochemical protease: Site-specific photocleavage of hen egg lysozyme and bovine serum albumin

Abstract

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 ± 0.3 × 10⁵ M⁻¹ and 6.5 ± 0.4 × 10⁷ M⁻¹, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at ≈460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

Reagents that can cleave the protein backbone with a high specificity (chemical proteases) can be useful as biochemical tools (1–6). Structure–activity studies of proteins, investigation of protein structural domains, and studies of the proximity of specific residues in a given protein are some applications of chemical proteases. Rapid proliferation of chemical nucleases for DNA cleavage studies suggests how small-molecule-based chemical reagents can be of use as tools in molecular biology (7–10). Chemical proteases, analogously, could be useful for the manipulation of proteins (1–6, 11–14). Determining the exposure of selected residues of membrane-bound proteins to the aqueous environment, design of new therapeutic agents, and converting large proteins into smaller fragments that are more amenable to sequencing are additional applications of chemical proteases.

Chemical proteases can be activated by light, and novel photochemical proteases (15) are described here that have (i) high affinities for selected sites of proteins, (ii) strong absorption bands in the near visible region, (iii) long-lived singlet excited states that are convenient to initiate photoreactions, and (iv) a chromophore that has been used to probe microenvironments. Use of light as a reagent for protein cleavage has distinct advantages. Photoreactions can be started and sustained with light, providing a sharp control for initiation and termination of the reaction. Visible light is perhaps the least toxic reagent for environmentally benign chemical approaches. Photoreactions are amenable for time-resolved mechanistic studies to delineate the elementary steps. By controlling the wavelength of excitation, specific chromophores can be selectively activated to high energies, thereby minimizing side reactions.

Vanadate, for example, has been used as a photoprobe to investigate phosphate-binding sites of proteins (16). Vanadate competes for phosphate-binding sites of proteins (17), and it induces photocleavage of proteins with high preference for serine residues (17–19). Applicability of vanadate is limited to phosphate-binding proteins (18) and, hence, developing alternatives to vanadate is challenging.

Two approaches for the design of chemical proteases can be distinguished: (i) affinity-based design (1, 5) and (ii) covalent linking of redox-active metal complexes to specific residues of the protein, followed by activation of the metal complex to achieve protein cleavage (2–4, 11–14). Coupling chiral recognition elements with a suitable chromophore has been our approach to design photochemical proteases. N-(l-Phenylalanine)-4-(1-pyrene)butyramide (Py-Phe; see Fig. 1), for example, induces site-specific photocleavage of hen egg lysozyme (Lyso), bovine serum albumin (BSA), and carboxypeptidase A (CpA), under mild conditions (15). Advantages of using the 4-(1-pyrene)butyroyl (Py) chromophore in the design are as follows. (i) Py has high extinction coefficients, and hence, low concentrations of the probe can capture a significant fraction of the incident light. (ii) Py has a long-lived (≈110-ns), high-energy (≈70 kcal/mol) singlet excited state that can drive a number of photochemical reactions. Short-lived excited states are more likely to waste excitation by rapidly decaying to ground state without a fruitful chemical event. (iii) Py has been used to probe the microenvironments of micelles, proteins, DNA, and other organized media (20). (iv) Considerable mechanistic information regarding Py excited states and related intermediates (such as Py⨥, PyH⁺, and Py⨪) is available. (v) Py-Phe shows strong induced circular dichroism bands in the 300- to 400-nm region, and these spectra are convenient to monitor the interaction of Py-Phe with proteins.

Figure 1.

Structure of protein probe with a specific recognition element. The pyrenyl moiety can be photoactivated in the presence of suitable electron acceptors.

Lyso, BSA, and CpA have been chosen as model proteins for photocleavage studies. Lyso is a small (≈14-kDa) water-soluble enzyme whose activity can be readily monitored. The x-ray crystal structure of Lyso and its substrate-binding site are known (21). BSA is a plasma protein that has many physiological functions. Hydrophobic ligands such as fatty acids, steroids, and bilirubin bind to BSA (22). Because of its affinity for a variety of biological ligands, the interactions of BSA with specific ligands have been extensively investigated. Several C-terminal phenylalanine peptides are known to be substrates for CpA (23). Phenylalanine, therefore, is chosen as the chiral recognition element. Site-specific photocleavage of these proteins by Py-Phe and mechanistic details of protein photocleavage are presented here.

MATERIALS AND METHODS

Lyso (M_r = 14,300), BSA (M_r = 66,267), and CpA (M_r = 35,250) were purchased from Sigma. Protein solutions were prepared by dissolving the appropriate amounts of the protein in 50 mM Tris⋅HCl, pH 7.0. Py-Phe was synthesized as described previously (15). All probe solutions were prepared fresh, and calibration graphs were constructed by using Beer’s law. Probe concentrations were restricted to the linear region of the graph. The molar extinction coefficient of Py-Phe was estimated to be 42,600 M⁻¹⋅cm⁻¹ at 343 nm.

Photochemical Protein Cleavage.

Photocleavage reactions were carried out at room temperature in 50 mM Tris⋅HCl buffer, pH 7.0. The mixtures of protein (15 μM) and Py-Phe (15 μM) in the presence of cobalt(III) hexammine trichloride [Co(NH₃)₆Cl₃; CoHA] (1 mM), in a total volume of 100 μl, were irradiated at 344 nm by using a 100-W xenon lamp attached to a PTI (South Brunswick, NJ) A1010 monochromator. A UV cut-off filter (WG-345; 78% transmission at 344 nm) was used to remove stray UV light. Irradiated samples were dried for gel electrophoresis experiments.

Electrophoresis.

SDS/polyacrylamide gel electrophoresis (SDS/PAGE) experiments were performed as reported (24). Samples were heated for 3 min in the presence of SDS (7%, wt/vol), glycerol (4%, wt/vol), Tris (50 mM), 2-mercaptoethanol (2%, vol/vol), and bromophenol blue (0.01%, wt/vol), adjusted to pH 6.8 with HCl. Gels (12% polyacrylamide for Lyso and 8% for BSA) were run by applying 60 V until the dye passed through the stacking gel. The voltage was then increased to 110 V, and electrophoresis was continued for an additional 2.5 hr for Lyso or 1.5 hr for BSA. The gels were stained with Coomassie blue for 4 hr, and destained in acetic acid (10%, vol/vol) for 4 hr. The gels were scanned with a Hewlett–Packard scanner (ScanJet 4C) and the images were processed by using Adobe PhotoShop/NIH-Image software to quantitate band intensities and band positions. Molecular weight markers were used in each gel for calibration and the molecular weights of the fragments were estimated from their mobilities. For quantum yield measurements, various concentrations of Lyso and BSA were electrophoresed and calibration graphs were constructed to estimate the intensities of the protein fragments.

Peptide Sequencing and Mass Spectrometry.

Protein bands from the gels were electroblotted onto Gore-Tex, manually excised, and transferred to a sequencing cartridge. Chemical sequencing was performed on an automated protein sequencer (G1005A, Hewlett–Packard). At least five cycles were performed from the N terminus, while a single cycle for the C terminus was attempted on each band.

The entire reaction mixture was desalted and concentrated by using HPLC (Ultra-Plus MicroLC system, Micro-Tech Scientific, Sunnyvale, CA). A short reversed-phase column (Hypersil BDS C-18, 1.0 mm × 20 mm, Keystone Scientific, Bellefonte, PA) was used with increasing acetonitrile concentration from 5% (in aqueous 0.1% trifluoroacetic acid) to 75% (vol/vol). The peak representing the unreacted protein as well as any fragments, as detected by UV absorption at 215 nm, was collected and completely desolvated under vacuum. The sample was resuspended in 5% acetic acid and analyzed for intact molecular weight by using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on a LaserMat instrument (FinniganMat, San Jose, CA) with sinapinic acid (Sigma) as the matrix.

Quantum Yield Measurements.

A ferrioxalate actinometer (25) was used to determine light intensities at specific wavelengths for quantum yield measurements. The entire experiment was carried out in a dark room lit only by a yellow safelight. The actinometer solution (0.006 M) was prepared by dissolving potassium ferrioxalate [K₃Fe(C₂O₄)₃⋅3H₂O, 0.14 g] in water (45 ml) and sulfuric acid (0.5 M, 5 ml). Actinometer solutions (200 μl) were irradiated at 344 nm for various lengths of time (0, 5, 10, and 15 min). Aliquots (150 μl) of irradiated actinometer solutions were mixed with excess 1,10-phenanthroline (50 mg/50 ml of H₂O, total 1 ml), and concentration of the ferrous ion was estimated from absorbance at 510 nm. Light intensities were measured before and after each irradiation and an average of two measurements was recorded (within ±5%). Quantum yields for the photocleavage were estimated from three separate runs.

Laser Flash Photolysis.

Light pulses (355 nm, ≈8 mJ per pulse, 8 ns) from a Continuum Surelite I Nd-YAG laser were used to excite the samples. A pulsed 150-W xenon lamp, combined with an ISA (Metuchen, NJ) H10 monochromator, served as the monitoring system. Signals from a Hamamatsu R928 photomultiplier tube were terminated into 50 ohms and digitized by using a Tektronix 380 (400 MHz) digitizer. The entire experiment was controlled with a Macintosh Quadra 650 using labview 3 software as described earlier (26). Transient absorption spectra were produced by obtaining full kinetic traces at various wavelengths and by sampling the change in optical density at a fixed delay after the laser pulse. The change in absorption was then plotted as a function of wavelength at a given delay time subsequent to laser excitation.

RESULTS

Site-specific photocleavage of Lyso and BSA by Py-Phe is demonstrated here. Detailed spectroscopic investigations of Py-Phe binding to these proteins will be described separately.

Protein Cleavage Studies.

Irradiation of Py-Phe/Lyso mixture at 344 nm results in protein cleavage with high efficiency and specificity as monitored in SDS/PAGE experiments. Photocleavage of Lyso (35% yield, Fig. 2) yields only two sharp bands of lower molecular weights (≈3,300 and ≈11,000) than that of Lyso (14,300). If the photocleavage were random, one would have observed a smear in these lanes. The molecular weights of the photocleavage products were estimated by using the markers in lane 1. Molecular weights of the daughter fragments add up to that of the parent protein, suggesting a single cut in the Lyso backbone. Irradiation of Lyso and CoHA (1 mM) without Py-Phe did not yield any fragmentation (lane 9). The photoreaction requires an electron acceptor such as CoHA. Irradiation of Lyso without CoHA, in the presence or absence of Py-Phe, did not result in protein cleavage (lanes 8 and 7, respectively). Control experiments, thus, establish that Py-Phe, CoHA, and light are essential for the protein photocleavage.

Figure 2.

Gel electrophoresis pattern of Lyso under various conditions. Lane 1, molecular weight markers; lane 2, Lyso (15 μM); lane 3, Lyso (15 μM), Py-Phe (15 μM), and CoHA (1 mM), dark control; lanes 4–6, same as lane 3 but after 5, 10, and 20 min of irradiation at 344 nm, respectively; lane 7, Lyso (15 μM) after irradiation for 20 min; lane 8, Lyso (15 μM) and Py-Phe (15 μM), after irradiation for 20 min; lane 9, Lyso (15 μM) and CoHA (1 mM) after irradiation for 20 min. The samples were run top to bottom and the new bands in lanes 4, 5, and 6 correspond to the lower molecular weight fragments.

BSA (15 μM) was also successfully cleaved by Py-Phe (15 μM), resulting in two fragments of molecular weights 40,800 and 28,400 (344-nm irradiation, 1 mM CoHA, Fig. 3). Electron acceptors such as chloropentammine cobalt(III) chloride, tris(bipyridine) cobalt(III) chloride [Co(bpy)₃³⁺], and cobalt(III) sepulchrate trichloride all resulted in the same cleavage fragments as CoHA (Fig. 3) although with various efficiencies. No photocleavage was observed in the absence of light (lane 2) or the electron acceptor. Thus, the cleavage most likely occurs at the Py-Phe binding site, irrespective of the Co(III) species.

Figure 3.

Gel electrophoresis pattern of BSA with four different cobalt(III) complexes. Lane 1, molecular weight standards, as marked (×10⁻³); lane 2, BSA (15 μM), Py-Phe (15 μM), and CoHA (1 mM), dark control; lane 3, BSA (15 μM), Py-Phe (15 μM), and tris(bipyridine) cobalt(III) chloride (1 mM), after irradiation for 60 min; lane 4, BSA (15 μM), Py-Phe (15 μM), and Co(III) sepulchrate trichloride (1 mM), after irradiation for 60 min; lane 5, BSA (15 μM), Py-Phe (15 μM), and chloropentammine cobalt(III) chloride (1 mM), after irradiation for 60 min; lane 6, same as lane 2, but after irradiation for 60 min. The irradiation was performed at 344 nm.

Yields of the photoproducts increase steadily with irradiation time (Fig. 4). The yields of Lyso cleavage products increased rapidly with time and saturated at ≈35%, whereas the yields of BSA products increased linearly at short times. Irradiations for longer than 20 min increased the product yields only marginally, with maximum conversions of 35% for Lyso and 21% for BSA. The photocleavage of Lyso was found to be more rapid, with better yields than those of BSA. Perhaps the flexible structure of Lyso plays an important role in exposing the bound pyrenyl moiety to CoHA to initiate the photoreaction. Surface binding of the probe to Lyso would also permit facile access of the pyrenyl chromophore to CoHA and can result in higher yields.

Figure 4.

Percent photoproduct yields of BSA and Lyso vs. time of irradiation. The yields of the photoproducts were obtained from quantitated intensities of the product bands on the gel.

Absorption and CD spectra of Lyso or BSA (200- to 300-nm region) showed no evidence of protein structural changes in the presence of CoHA (1 mM). Irradiation of heat-denatured BSA in the presence of Py-Phe and CoHA did not result in any photoproducts, suggesting the importance of the tertiary structure of the native protein for the photoreaction.

Enzyme Activity Studies.

Activity of Lyso was followed as a function of the progress of the photoreaction to gain further insight. Glycol chitin was used as a substrate for Lyso (27), and Lyso activity was unchanged as the photocleavage progressed, indicating no deactivation of the enzyme prior to or after chain scission. Fragmented Lyso is nearly as active as the parent, or the fragments reassociate to form the active enzyme. Cleavage of Lyso with retention of the key residues required for activity is indicated by these studies.

Chemical Sequencing Studies.

Sequencing of the photoproducts was carried out to determine the cleavage site. The N-terminal sequence of the ≈3-kDa fragment from Lyso was found to be Val-Ala-(Trp)-Arg. Interestingly, the tryptophan residue was not directly observed, but appeared as a peak whose retention time on the reverse-phase column had decreased, indicating that the photocleavage process in some way modified this residue, reducing its hydrophobicity. The C-terminal sequencing of the 3-kDa fragment yielded a single Leu residue, consistent with the known C terminus of Lyso. The ≈11-kDa fragment was observed to have an N-terminal sequence of Lys-Val-Phe-Gly, consistent with the N-terminal sequence of Lyso. The C-terminal sequencing of the ≈11-kDa fragment did not yield an interpretable signal, most likely because the expected Trp residue was modified by the photocleavage chemistry, and thus not amenable to sequencing. The cleavage site was determined from the known sequence of Lyso and from the N- and C-terminal sequence data to be between Trp-108 and Val-109 (Fig. 5).

Figure 5.

Cleavage patterns for Lyso and BSA. The cleavage of Lyso was found to be between Trp-108 and Val-109, and the cleavage of BSA was observed between Leu-346 and Arg-347.

Mass spectrometry of the desalted reaction mixture from Lyso showed the presence of three species, peaks at mass to charge ratio (m/z) of 14,550, 11,460, and 3,057. A standard Lyso (Sigma) sample showed a protonated mass of 14,314 Da. The highest m/z peak (14,550) from the reaction mixture was determined to be Lyso that had been modified, presumably by Py-Phe. Therefore, a mass increase of 236 Da is indicated (Py-Phe has a mass of 435 Da). The experimental error under these conditions is about ±1.0%, or a mass of ±15 Da at this m/z. Addition of pyrenyl moiety to this fragment (201 Da) can partly explain the increased molecular mass. The average theoretical masses of the unmodified 3-kDa and 11-kDa fragments were 2,489 Da and 11,840 Da, respectively. These also appeared at higher masses with differences of 568 Da and 380 Da, respectively.

Chemical sequencing of the BSA fragments was similarly performed on the electroblotted and excised bands. The N-terminal sequence of the ≈28-kDa band was found to be (Arg)-Leu-Ala-Lys-Glu. This is a sequence internal to BSA, and Arg was not directly observed, but was shifted to lower retention time, indicating that it had been modified. The C-terminal sequencing of the 28-kDa band showed an Ala residue, consistent with the known C terminus of BSA. The N-terminal sequencing of the ≈40-kDa band yielded a sequence of Asp-Thr-His-Lys-Ser, as expected from the known N terminus of BSA. The C-terminal sequencing of this fragment did not produce an interpretable signal, again indicating modification of the residue adjacent to the cleavage site. From the preceding data and the known sequence of BSA, the cleavage site was determined to be between Leu-346 and Arg-347 (Fig. 5).

The mass spectra of BSA photoproducts showed peaks at m/z values of 66,890, 39,930, 33,420, and 26,860. The mass spectrometer had been calibrated immediately prior to this experiment with unmodified BSA, the singly charged protein at m/z 66,430 and the doubly charged species at 33,220. Therefore, the uncleaved BSA in the reaction mixture had been modified in some way to increase its mass by 460 Da. The ≈28-kDa band seen in the gel and chemically sequenced was most likely the 26,860 m/z peak, a deviation from its average theoretical mass by 100 Da. The peak in the mass spectrum at m/z 33420 resulted from the doubly charged intact protein. The remaining peak in the mass spectrum at m/z 39,930 then must correspond to the ≈40-kDa fragment, and it deviates from its average theoretical mass by 240 Da. These deviations are within ±1% of instrumental error. The chemical sequencing and mass spectral data, thus, confirmed the high specificity of the photocleavage. Sequencing data also revealed modification of a few residues during the photoreaction. Despite these modified residues, six of eight termini were sequenced successfully.

Flash Photolysis Studies.

The protein photocleavage was investigated in flash photolysis experiments to gain insight into the photocleavage mechanism. Excitation of Py-Phe (30 μM), Lyso (30 μM), and CoHA (3 mM) by 355-nm Nd-YAG laser pulses (full width at half maximum, 8 ns) resulted in a strong transient absorption spectrum with maxima at 390, 425, 470, and 510 nm (Fig. 6A). Similarly, excitation of Py-Phe (30 μM), BSA (30 μM), and CoHA (3 mM) resulted in transient absorption bands at 405, 420, 460, 490, and 510 nm (Fig. 6B). The transient signals observed in case of Lyso share similarity with those observed for BSA.

Figure 6.

Transient optical absorption spectra of aqueous Py-Phe solutions (30 μM) recorded at 2–17 μs after the laser pulse. In addition, the solutions contained: Lyso (30 μM) and CoHA (3 mM) (A); BSA (30 μM) and CoHA (3 mM) (upper spectrum), or CoHA (3 mM) (lower spectrum) (B); Lyso (30 μM) (C); or BSA (30 μM) (D).

Quenching of the Py-Phe excited state (Py-Phe*) by the proteins resulted in specific transient signals (in the absence of CoHA). A weak transient spectrum with maximum at 450–470 nm was observed when a mixture of Py-Phe (30 μM) and Lyso (30 μM) was excited with laser pulses (Fig. 6C). In contrast, a strong transient absorption spectrum with a maximum at ≈420 nm was observed with Py-Phe (30 μM) and BSA (30 μM) (Fig. 6D). The 420-nm transient was later identified as pyrene triplet (³Py). Py⨥ was generated independently by quenching Py-Phe* with CoHA (3 mM). This resulted in a transient with a strong absorption at 460 nm (Fig. 6B, lower spectrum).

The kinetics of various transients produced were monitored at their respective maxima. The 460-nm transient produced by the quenching of Py-Phe* by CoHA decayed with a lifetime of ≈50 μs (Fig. 7A) and roughly corresponds to the recovery of ground state bleaching monitored at 340 nm (≈63 μs, Fig. 7B). The 460-nm transient was assigned to Py⨥ on the basis of literature reports (λ_max at 457 nm) (28) and additional evidence (discussed below). The kinetic traces observed for Py-Phe (30 μM) in the presence of Lyso (30 μM) and CoHA (3 mM) at 390, 425, 470, and 510 nm all decayed with lifetimes similar to the lifetime of the Py-Phe cation radical (data not shown). The transient decay traces observed with Py-Phe, BSA, and CoHA at the corresponding peak maxima (Fig. 7 C and D) differed from each other, indicating the formation of several transients.

Figure 7.

Kinetic traces after laser flash photolysis of aqueous Py-Phe solutions (30 μM) recorded at different wavelengths in solutions containing CoHA (3 mM) without the protein (A and B) or CoHA (3 mM) and BSA (30 μM) (C and D).

DISCUSSION

Design and synthesis of chemical proteases is challenging. Use of chemical proteases complements protein structural methods such as x-ray diffraction, NMR, light scattering, and gel electrophoresis. Developing new and highly selective chemical proteases (as biochemical tools) could also contribute to our basic understanding of how small molecules may interact with proteins. In this context, chromophores linked with specific recognition elements were synthesized and tested in our laboratory for their interaction with proteins (15, 29, 30). These probes show high affinities for various proteins (29, 30), and length of the linker separating the chromophore from the recognition element plays an important role in the binding behavior (29).

A molecular probe such as Py-Phe, consisting of hydrophobic and hydrophilic groups, is likely to bind such that the hydrophobic Py chromophore is away from the protein surface/solvent, while the polar COOH group is interacting with polar residues at the surface (29–31). Simultaneous binding to sites that can accommodate both these structural features is expected to predominate. Binding of the probe will be most favored at a site where the array of microscopic environments of the binding site is complementary to that of the probe. The binding free energy for such a multisite binding model is expected to be lower than for binding of Py-Phe to only hydrophobic or to only polar functions of the protein. Detailed spectroscopic studies indicate binding of Py-Phe to Lyso at the protein surface, whereas binding to BSA may occur at an interior site with binding constants in the range of 10⁶ to 10⁷ M⁻¹ (15). Current sequencing data support such a binding model.

Photocleavage.

Although the pyrenyl chromophore or its singlet excited state (S¹) do not cleave proteins, irradiation of probe–protein complexes in the presence of an electron acceptor such as CoHA results in protein cleavage. The number of products, product molecular weights, and sequencing data indicate a single cut in the protein backbone. The photoproduct formation with Lyso was rapid and reached a maximum of ≈35%, whereas in the case of BSA the photoproduct yield reached a maximum of 21% at longer times (>20 min). These results clearly indicate the high efficiency of the Lyso reaction compared with that of BSA. The quantum yield of photocleavage, accordingly, for Lyso was found to be 0.26, in contrast with 0.0021 for the BSA reaction. Fluorescence quenching studies indicate greater accessibility of Py-Phe bound to Lyso compared with Py-Phe bound to BSA, consistent with the above quantum yield measurements.

Characterization of the Cleavage Site.

Successful use of the probes for protein cleavage (as reagents in molecular biology) demands that the sequence at which protein cleavage occurs be identified and that the fragments be amenable to sequencing. Analysis of at least five N-terminal cycles and one C-terminal cycle of chemical sequencing indicated the cleavage sites to be at residues 108–109 for Lyso and at residues 346–347 for BSA. Of the eight peptide termini, six have been successfully sequenced. The newly generated N-terminal sequences were successfully sequenced, whereas the newly produced C-termini could not be sequenced. Oxidative cleavage of proteins, in some cases, may block the N or C termini for sequencing (11, 12). Present data, thus, indicate the viability of photofragments for sequencing, albeit with some limitations.

Molecular modeling studies using the known crystal structure of Lyso indicate that the cleavage site of Lyso is at the hydrophobic cleft, which is surrounded by tryptophan residues (Trp-62, -63, -111, and -123). Arginine residues (Arg-112 and -114) are also located near the cleavage site of Lyso. Lyso activity studies indicate that the photocleaved Lyso is active, again consistent with the sequencing data. Although the crystal structure of BSA is unknown, the observed cleavage site is at the hydrophobic binding site of BSA in subdomain IIA.

Only two fragments are produced for each protein with a high specificity. The MS data suggest that Py-Phe or its derivatives are covalently attached to the protein at specific sites. Sequencing data confirm the modification of nucleophilic residues such as Trp and Arg near the cleavage site. The presence of Arg residues at the cleavage sites suggests salt bridge formation between the carboxyl group of the Py-Phe with Arg side chains. The methyl ester of Py-Phe (not capable of salt bridge formation with Arg side chains) does not cleave either Lyso or BSA. These observations are consistent with anchoring of Py-Phe at specific sites on protein, perhaps by means of salt bridge formation with Arg residues (Fig. 8). In addition to salt bridge formation, hydrophobic interactions of the pyrenyl group with Trp and Phe residues may be significant.

Figure 8.

Binding modes. (A) Anchoring of the hydrophilic part of the probe at the protein surface. (B) Binding of the probe at the interface. The hydrophobic moiety of the probe is buried in a nonpolar region of the protein, while the hydrophilic function of the probe interacts with charged residues at the protein surface.

Mechanistic Studies.

The photoreaction requires both Py-Phe and Co(III) species. Since Co(III) complexes have a weak broad absorption in the 300- to 400-nm region, the reaction could be due to light absorption by Co(III) or Py-Phe. The charge transfer states of CoHA are not accessible by excitation at 344 nm, and pyrenyl fluorescence is quenched by CoHA at diffusion-controlled rates. We propose that only excitation of the pyrenyl probe results in the photoreaction. This proposal was tested by plotting photoproduct yields as a function of irradiation wavelength (action spectrum, Fig. 9). The plot resembles the absorption spectrum of Py-Phe and is distinguishable from the broad weak absorption bands of CoHA. Therefore, light absorption by Py-Phe is essential for the reaction. These results are consistent with photocleavage studies using various Co(III) complexes, where the same product formation was indicated, regardless of the Co(III) complex used.

Figure 9.

Action spectrum obtained from the photocleavage of BSA (15 μM) with Py-Phe (15 μM) and CoHA (1 mM) at various wavelengths. The plot of percent yield of the photoproduct formation as a function of wavelength of irradiation results in a pyrene-like absorption spectrum.

Direct evidence for Py⨥ intermediacy was obtained from flash photolysis studies, which yielded the following observations. (i) The rapid formation of the cation radical is consistent with a fast quenching of the Py-Phe singlet excited state by CoHA (k_q = 1.8 × 10¹⁰ M⁻¹·s⁻¹). (ii) The 460-nm transient assigned to Py⨥ was not quenched by oxygen, which is typical for cation radicals (32). (iii) The absorption spectrum of pyrene cation radical was reported to have a strong, sharp maximum around 445 nm at 77 K in argon matrix (33). The small differences in the peak positions observed here could be due to the substituent, temperature, and matrix effects. (iv) The extinction coefficient (ɛ₄₆₀) of the 460-nm transient estimated from the ground state bleaching is 9.3 × 10⁴ M⁻¹⋅cm⁻¹, comparable to the reported extinction coefficient of pyrene cation radical (9.6 × 10⁴ M⁻¹⋅cm⁻¹) (34).

Laser flash photolysis of Py-Phe/CoHA systems in the presence of Lyso or BSA (Fig. 6 A and B, respectively) indicates the absorption maxima corresponding to that of the pyrene cation radical. Py⨥, thus, is produced under the photocleavage conditions. The cation radical may be a key intermediate in the photocleavage mechanism.

To test the intermediacy of Py⨥ in the protein photocleavage, the effect of electron donors such as triethylamine and ethanolamine on the protein cleavage yields were evaluated. The long lifetime of Py⨥ (≈50 μs, Fig. 7A) makes it possible to intercept this intermediate without affecting the much shorter-lived singlet excited state. Ethanolamine, for example, quenches the photocleavage yield of BSA (K_sv = 60 M⁻¹) without significantly quenching the Py-Phe singlet excited state (K_sv = 0.06 M⁻¹, τ_f = 110 ns). Therefore, interception of cation radical by ethanolamine is indicated. Analogously, k_q for quenching of Py⨥ by ethanolamine estimated from the flash photolysis experiments was 1.3 × 10⁶ M⁻¹⋅s⁻¹ with an estimated K_sv (K_sv = k_qτ) value of 60 M⁻¹. Therefore, Py⨥ is a key intermediate in the photocleavage. Singlet oxygen is not involved in these reactions, as degassing of the reaction mixture did not alter the site of photocleavage or reduce the photoproduct formation. Addition of azide (2 mM), a singlet oxygen quencher, had no effect on the photocleavage yield or specificity.

Flash photolysis studies of protein–CoHA solutions (30 μM protein, 3 mM CoHA) without Py-Phe did not produce the transient absorption at 460 nm, consistent with the assignment of 460 nm band to Py⨥. Transient absorption spectra of radicals derived from aromatic amino acid side chains (Trp^⋅, Tyr^⋅, His^⋅ as well as the corresponding radical ions) may be responsible for the additional peaks observed in the transient spectra. The absorption maxima of Tyr^⋅ and Trp^⋅ are reported to be at 405 and 510 nm, respectively (28, 32–35). Oxidative quenching of Py-Phe* by disulfide links may result in RSSR⨪ (absorption maximum at 400 nm) (35). The relative yields of the reactive intermediates derived from various amino acid side chains are expected to depend upon their reactivities, number of these residues present at the probe binding site, and their proximity to Py-Phe.

The assignment of all the observed transients is beyond this report, but the data clearly indicate the formation of Py⨥ as a key intermediate. The 420-nm transient observed with BSA/Py-Phe mixture, in the absence of CoHA, (Fig. 6D) decayed with a lifetime of ≈18 μs (air-saturated solutions) characteristic of pyrene triplet. This transient is quenched by oxygen with k_q = 1.3 × 10⁸ M⁻¹⋅s⁻¹, consistent with the value obtained for the oxygen quenching of pyrene triplet state in BSA solution (λ_max (triplet) = 415 nm; k_q = 1.0 × 10⁸ M⁻¹·s⁻¹) (36).

Low diffusional mobility of Py⨥ compared with that of the hydroxyl radical can localize the reactive intermediate. High affinity of Py-Phe for selected sites on proteins may be important in restricting the reagent to selected sites and result in high specificity. Two distinct contributions to the specificity of the photocleavage can be considered. (i) Upon photoexcitation, Py⨥ reacts with specific residues and causes cleavage at these sites (reactivity model). (ii) The probe binds to a unique site in the protein matrix and reacts with only the neighboring side chains, resulting in a high specificity (recognition model). A combination of recognition and reactivity is proposed to be responsible for the observed high specificity. This model is consistent with the lack of a consensus sequence for photocleavage. On the other hand, salt bridge formation with Arg side chains may play a significant role in cleavage site recognition. Photocleavage may proceed via hydrogen atom (H^⋅) abstraction by Py⨥ from the α-C atom of selected amino acid residues (Fig. 10). Subsequent addition of O₂ to the C-centered radical to form a hyperoxyl radical (—C—OO^⋅) and peptide cleavage may occur as reported in the literature (37).

Figure 10.

Possible mechanism for the photocleavage reaction. The initial step is hydrogen atom abstraction at the α-carbon by Py⨥, followed by the addition of O₂ to form hydroperoxyl radical. The cleavage of the peptide backbone may then occur after mild hydrolysis.

Current results clearly indicate high specificity for protein photocleavage. Further studies with probes carrying specific recognition elements will help us in the rational design of chemical reagents to target specific sites of proteins. In addition to biochemical applications, such reagents may be of interest for therapeutic purposes.

ABBREVIATIONS

Py

4-(1-pyrene)butyroyl

Py-Phe

N-(l-phenylalanine)-4-(1-pyrene)butyramide

Lyso

hen egg lysozyme

CpA

carboxypeptidase A

CoHA

cobalt(III) hexammine trichloride