Reaction Dynamics of Plant Phenols in Regeneration of Tryptophan from Its Radical Cation Formed via Photosensitized Oxidation

Abstract

Photooxidation imposes structural damage on proteins, and the amino acid tryptophan (Trp) is a key target for protein oxidation. The Trp radical cation (Trp^•⁺), as an oxidative product, can be reduced by plant phenols (φ-OH), a category of dietary phytochemicals essential for human health. This work is intended to investigate the efficacy of φ-OH regeneration of Trp from Trp^•⁺ as a function of φ-OH concentration and environmental pH. We have examined, by using laser flash photolysis, six different kinds of φ-OH in the aqueous system consisting of Trp and riboflavin as a photosensitizer. Taking syringic acid (Syr) as an example, upon systematically varying the pH from 2 to 10, the partition of Syr phenolate, Syr-O²⁻, increases from 0% to 70% and, accordingly, the rate of Trp regeneration increases from 4.8 × 10⁶ M⁻¹·s⁻¹ to 1.7 × 10⁸ M⁻¹·s⁻¹. It is found that the regeneration rate correlates with the driving force of the electron transfer (ET) reaction between φ-OH and Trp^•+, which can be well accounted for by Marcus’s ET theory (R² = 0.89). The λ = 0.43 ± 0.08 eV for the reorganization energy for ET from the plant phenols to the Trp^•⁺. The effects of φ-OH concentration, environmental pH, and ET driving force on the Trp regeneration reaction herein revealed are significant for enlightening further study of protein (anti)oxidation.

1. Introduction

The spoilage of protein-rich foods poses significant challenges, primarily affecting shelf life, color, flavor, and overall nutritional value [1,2]. By understanding the mechanisms behind these changes, it is possible to predict, control, and regulate reactions during food production, thus reducing or eliminating spoilage’s adverse effects on human health.

Protein oxidation is a major spoilage in protein-based foods like milk and cheese. Addressing protein oxidation involves understanding the oxidative pathways within food proteins and their impact on food quality. This knowledge can then be applied to enhance antioxidant defenses, thereby improving protein functionality [3,4]. Riboflavin (Rib) is also known as vitamin B₂, which is essential for energy metabolism, immune system support, skin and eye health, and maintaining nervous system function in the human body [5]. As a vital nutrient in the food system, Rib is abundantly present in dairy products, meats, leafy vegetables, and other foods, helping to fulfill daily nutritional requirements. However, Rib can also act as an efficient photosensitizer. Upon absorbing visible light, it reaches a singlet excited state and undergoes intersystem crossing to a highly reactive triplet state [6,7], as shown in Equation (1). In dairy products, Rib can oxidize proteins, forming low-molecular-weight sulfur compounds, such as dimethyl disulfide, which imparts an undesirable burnt feather aroma [8,9]. The above oxidative processes are key factors in protein spoilage. While Rib is commonly present in food systems and is essential for providing nutritional value, it also poses a risk of oxidizing amino acids in biological activities.

Photooxidation is an important pathway for protein oxidation. Amino acids, such as tryptophan, tyrosine, phenylalanine, and cystine, are primary targets of photodegradation. Under light exposure, electrons are transferred from amino acids to oxygen molecules, initiating an oxidation reaction. Common photooxidative products of amino acids include dehydrogenation products, phenolic compounds, free radicals, and peroxides, among others [10]. These oxidative products can affect the original physicochemical properties and may even impact the intrinsic value of proteins. Within proteins, tyrosine and phenylalanine can transfer their excited-state energy to tryptophan (Trp), which, despite its relatively low abundance, has the highest molar absorptivity with an indole ring. Consequently, Trp becomes the primary participant in the photodegradation pathway [10]. Trp is highly oxidized and degrades into a variety of products during manufacturing, storage, and processing. As described in detail in the literature [11], many physical and chemical processes cause tryptophan degradation, primarily through oxidation or cleavage of highly active indole rings. The cation radical of Trp (Trp^•+), with an extinct excited state absorption feature (λ_max) at 550–560 nm, is the major oxidation product of Trp. It has a lifetime of approximately microseconds and reduces the original nutritional value [12], as shown in Equation (2). Therefore, preventing the oxidation of Trp is essential for maintaining the nutritional value of proteins in food.

$$\mathrm{R}\mathrm{i}\mathrm{b}\stackrel{hv}{\to }{[}^1{\mathrm{R}\mathrm{i}\mathrm{b}}^{\ast }]\stackrel{\mathrm{I}\mathrm{S}\mathrm{C}}{\to }{}^3\mathrm{R}\mathrm{i}\mathrm{b}^{\ast }$$

(1)

$${}^3\mathrm{R}\mathrm{i}\mathrm{b}^{\ast }+\mathrm{T}\mathrm{r}\mathrm{p}\to {\mathrm{T}\mathrm{r}\mathrm{p}}^{•+}+\mathrm{R}\mathrm{i}\mathrm{b}/{\mathrm{R}\mathrm{i}\mathrm{b}}^{•-}/{\mathrm{R}\mathrm{i}\mathrm{b}\mathrm{H}}^{•}$$

(2)

$${\mathrm{T}\mathrm{r}\mathrm{p}}^{•+}+{\phi -\mathrm{O}}^{-}\to {\phi -\mathrm{O}}^{•}+\mathrm{T}\mathrm{r}\mathrm{p}$$

(3)

Plant phenols play a crucial biological role, including acting as free radical scavengers to protect organisms from oxidative stress. Plant phenols exert antioxidant effects through a reduction–regeneration mechanism [13]. The ability of plant phenols to regenerate lipophilic carotenoids from their radical cations at water/lipid interfaces is considered a key factor in the antioxidant synergism frequently observed between carotenoids and polyphenols in heterogeneous food systems. This process is particularly important in the context of food systems, where the interaction between these compounds enhances the overall antioxidant capacity, providing greater protection against oxidative stress [14,15]. The regeneration of carotenoids by plant phenols thus plays a crucial role in improving the stability and effectiveness of antioxidants in complex food matrices. This study examines the reaction between plant phenols and tryptophan radical cation, as shown in Equation (3). The results, based on real-time kinetic methods, utilize nanosecond laser flash photolysis combined with transient absorption spectroscopy to excite Rib to its triplet state, subsequently generating amino acid radical cations. Plant phenols can effectively restore oxidized amino acid radical cations, thereby regenerating the amino acids. This mechanism holds potential for application in the production of protein-based foods, where it may help mitigate protein damage during processing, protect amino acids from degradation, and thus enhance the functionality, flavor, and nutritional value of food proteins.

2. Materials and Methods

2.1. Chemicals

Riboflavin was purchased from Shanghai Sharing Technologies CO., LTD (Shanghai, China). Tryptophan was purchased from Beijing OUTE Technology Co., Ltd., Beijing, China. Plant phenols (salicylic acid, m-hydroxyl-benzoic acid, vanillic acid, syringic acid, caffeic acid, and catechin) were analytical grade and were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Water was prepared by using a Mingchet-D 24UV water purification system (Merck Millipore Corp., Shanghai, China).

2.2. Ultraviolet–Visible (UV-Vis) Absorption Spectroscopy

Optical absorption spectra were measured with a Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA), using 1.0 cm quartz cells.

2.3. Laser Flash Photolysis and Transient Absorption Spectroscopy

The nanosecond laser flash photolysis and transient absorption (TA) spectroscopy apparatus was as described with the following details. Excitation laser pulses (7 ns, 10 Hz) at the wavelength of 445 nm were supplied by an optical parametric oscillator (OPO) driven by an Nd: YAG laser (Quanta-Ray Pro-Series, Spectra Physics Lasers Inc., Mountain View, CA, USA), and pulse energy was attenuated to 0.12 mJ/pulse. The desired probe wavelengths (400–900 nm) were provided by a laser-driven white light source (LDLS-EQ-1500, Energetic Technology, Inc., Woburn, MA, USA). Kinetics were detected with a photodiode (model S3071, Hamamatsu Photonics, Hamamatsu, Japan) attached to a spectrograph (SP2500i, Princeton Instruments, Trenton, NJ, USA) when both the excitation light and the probe light were focused on the optical sample cuvette during the experiment. The signals were stored and averaged with a digital storage oscilloscope (bandwidth of 500 MHz, Teledyne LeCroy HDO 4054, Chestnut Ridge, NY, USA) connected to a personal computer. The original collected data provided single wavelength kinetics, and parameter fitting was based on MATLAB 5.3 (MathWorks) software.

Unless specified, the concentrations used in this study were as follows: riboflavin (1 × 10⁻⁵ M), tryptophan (1.0 × 10⁻⁴ M), and plant phenols (2.0 × 10⁻⁴ M), all prepared in 0.01 M phosphate-buffered saline (PBS). All measurements were carried out in a thermostated room (23 ± 1 °C).

3. Results

3.1. UV-Vis Steady-State Absorption Characterizations

Riboflavin (Rib) is widely present in foods and has been shown to act as a photosensitizer of many biological substrates [1,16,17,18,19]. Figure 1A shows the absorption spectra of Rib, tryptophan (Trp), syringic acid (Syr), and vanillic acid (Van) in PBS solution at pH 7.2. The absorption peak of Trp is typically located around 280 nm, which is caused by the π→π* transitions of the aromatic ring [10]. Plant phenols generally exhibit absorption below 350 nm, which is caused by the π→π* and n→π* transitions of the aromatic ring [20]. The absorption peaks of Rib typically occur in the range of 250–450 nm, with the strongest absorption peak usually around 370 nm. This peak primarily corresponds to the π→π* transition, while the range of 350–450 nm may also include some n→π* transitions. Rib has a distinct absorption band at 445 nm. Thus, we chose to use 445 nm excitation light to selectively excite Rib without affecting other substances. Upon excitation at 445 nm, Rib is promoted to its singlet state, followed by intersystem crossing to the triplet state. The triplet state of Rib (³Rib*) has a high oxidation potential (1.77 V vs. NHE) [7], enabling it to oxidize tryptophan (1.015 V vs. NHE) to form the tryptophan radical cation [10].

3.2. The Oxidation of Trp by Triplet State of Rib Probed by ns-TA Spectroscopy

The irradiation of Rib solutions at 445 nm excites Rib to its lowest singlet excited state, which undergoes efficient intersystem crossing to produce the highly reactive ³Rib* species. This triplet state is critical in various photobiological processes [21,22]. The following oxidation processes of tryptophan by this triplet state of Rib were monitored using nanosecond time-resolved absorption spectroscopy. As illustrated in Figure 2, panels (A) and (B) present the complete transient absorption spectra for Rib and Rib upon Trp addition, respectively, displayed at various delay times. Figure 2A shows the transient absorption spectrum of the ³Rib* in PBS solution at pH 7.2. The 400–500 nm wavelength range feature corresponds to the ground-state bleaching signal of Rib, with a negative absorption peak value at 450 nm. The broader absorption band from 500 to 800 nm is attributed to the triplet state of the Rib, reaching maximum absorption at ~720 nm [19]. In the presence of oxygen, the ³Rib* decays completely within 12 μs, as displayed in the insert panel of Figure 2A, which is similar to the transient spectra reported for Rib in the literature [19]. In the addition of Trp, this approach to the reactive triplet state of ³Rib* enables real-time monitoring of its reaction with Trp via transient absorption spectroscopy (Figure 2B). The triplet lifetime of the Rib at 720 nm dropped to 2.7 μs with a mono-exponential fitting to the decay curve (see Figure 2C). Compared to Figure 2A, a smaller absorption intensity of the ³Rib* can be observed in Figure 2B with an equivalent excitation condition, indicating that Trp exhibits a quenching effect on the ³Rib*. In the absence of Trp, ³Rib* fully decays within 12 μs (Figure 2A). However, upon the addition of Trp, a prominent long-lived excited state emerges at 12 μs within the 500–600 nm range, as shown in the inset panel of Figure 2B, and remains up to 150 μs. This state is attributed to the long lifetime of Trp radical cation (Trp^•+) [10,11,12]. The corresponding kinetics at characteristic wavelengths are illustrated in Figure 2D. Specifically, the kinetic profile at 460 nm indicates that the ground-state bleaching of Rib is not fully restored at a time scale of 20 μs, which is likely due to the formation of RibH^• with transient features at ~320 and 520 nm through a rapid protonation process [9]. The distinct feature of the Trp^•+ at 560 nm was further confirmed by the sulfate radical anion with a significantly stronger oxidant (E⁰ = 2.43 V vs. NHE) [23], displaying a similar spectral feature and kinetics (Figure S1). Notably, we chose a relatively low Rib concentration of 1.0 × 10⁻⁵ M in this study; when the concentration of Trp is below 1.0 × 10⁻⁴ M, Rib does not effectively sensitize the formation of long-lived Trp^•+ (Figure S2). The decay of triplet Rib at 720 nm followed first-order exponential kinetics, both in the absence and presence of Trp, indicating that ³Rib* can transfer electrons to Trp. Consistently, the presence of certain amino acids accelerated the decay of ³Rib*, with the decay rate increasing proportionally to the concentration of these amino acids [24]. The removal of oxygen showed a slower decay of ³Rib* but did not affect both the formation and the decay of Trp^•+ (Figure S3). This suggests that oxygen does not interact with Trp^•+, likely due to Rib efficiently competing with oxygen in its reaction with Trp^•+.

3.3. Concentration Effect of Syr and Van for Regeneration of Trp from the Trp^•+

Syr and Van are commonly used, naturally occurring reducing agents. The kinetics analyses at their characteristic wavelengths demonstrate their ability to regenerate the Trp from its radical cation (Trp^•+), as shown in Figure 3A,B. Moreover, the reduction rate of Trp^•+ is dependent on the concentration of Syr and Van, as illustrated in Figure 3C,D. The observed pseudo-first-order rate constant was found to depend on the excess concentration of plant phenols according to the Stern–Volmer equation [25]:

$${\displaystyle \frac{{\tau }_0}{\tau }}=1+k_{\mathrm{q}}{\tau }_0\left[Q\right]$$

(4)

where τ₀ and τ are the Trp^•+ lifetimes in absence and presence of plant phenols, respectively, and [Q] is the concentration of plant phenols. The quenching rate k_q thus determined. From the kinetics monitored at 560 nm, the second-order reaction rate constants were determined from the pseudo-first-order rate constants for various Syr and Van concentrations within the concentration of 50 μM to 150 μM. The obtained results for Syr and Van are qualitatively in agreement with the reported results for the reaction of Trp with β-Car^•+ [13]. The rate constants display linear dependence on the concentration for Syr (Figure 3C) and Van (Figure 3D), which suggests that the reaction between the Trp^•+ and plant phenols follows second-order kinetic characteristics. The Stern–Volmer equation yields a quenching rate constant on Trp^•+ at pH 7.2, for Syr of 4.6 × 10⁷ M⁻¹·s⁻¹, and for Van of 1.4 × 10⁷ M⁻¹·s⁻¹ (

Table 1. Observed pseudo-first-order rate constants (k_obs/s⁻¹) for reduction of Trp^•+ by Syr and Van as measured at 560 nm.

	[Syr]			[Van]
	50 μM	100 μM	150 μM	50 μM	100 μM	150 μM
kobs/s−1	5.0 × 103	7.0 × 103	1.0 × 104	3.2 × 103	4.2 × 103	5.0 × 103
kq/M−1·s−1	4.7 × 107	4.3 × 107	4.9 × 107	1.1 × 107	1.5 × 107	1.6 × 107
kq (average)	4.6 × 107			1.4 × 107

).

3.4. Higher pH Enhanced Syr and Van for Regeneration of Trp from the Trp^•+

In the decay of Trp^•+ and the regeneration of Trp by Syr and Van, an intriguing behavior was observed, with the reaction rate constants showing a pronounced dependence on pH. The pH dependence of the rate constant for Syr and Van in the pH range from 2 to 10 could be described by a single acid–base equilibrium corresponding to the following pH dependence for the observed rate constant in Equation (5) [9], $k_{\mathrm{o}\mathrm{b}\mathrm{s}}$:

$$k_{\mathrm{o}\mathrm{b}\mathrm{s}}=k_{\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}}\left[{10}^{-\mathrm{p}\mathrm{H}}/\left({10}^{-\mathrm{p}\mathrm{H}}+{10}^{-{pK}_a}\right)\right]+k_{\mathrm{b}\mathrm{a}\mathrm{s}\mathrm{e}}\left[{10}^{-\mathrm{p}\mathrm{H}}/\left({10}^{-\mathrm{p}\mathrm{H}}+{10}^{-{pK}_a}\right)\right]$$

(5)

The results obtained for the Syr and Van showed that the kinetics of deactivation by plant phenols were similar to what was observed for the free amino acids (Figure 4A,B). Furthermore, a significant pH dependency on Syr and Van was observed as shown in Figure 4C. Under alkaline conditions, Syr exhibits a stronger pH dependence than that of Van.

This trend follows the deprotonation curve associated with the pKa value, allowing the determination of the proportion of each deprotonated group at different pH environments, as summarized in

Table 2. Percentages of deprotonated forms of plant phenols in Syr (and Van) from pH 2 to 10 ^a.

pH	2	5.6	7.2	9	10
φ-OH	98.84% (99.31%)	2.1% (3.47%)	0.05% (0.09%)	0.00% (0.00%)	0.00% (0.00%)
φ-O−	1.16% (0.69%)	97.88% (96.53%)	99.5% (99.79%)	78.02% (93.18%)	26.20% (57.74%)
φ-O2−	0.00% (0.00%)	0.01% (0.00%)	0.44% (0.12%)	21.98% (6.82%)	73.80% (42.26%)

^a Calculated with Marvin Calculator Plugins. Marvin 18.24.0 2018, ChemAxon (http://www.chemaxon.com, accessed on 10 January 2024).

. As pH increases, the proportion of φ-O²⁻ increases, enhancing the reducing ability of plant phenols and accelerating the reduction rate. Specifically, as the pH shifts from acidic to alkaline, the proportion of φ-O⁻ in the environment exceeds 70%. Consequently, the bimolecular reaction rate of Syr with Trp^•+ increases from 4.8 × 10⁶ M⁻¹·s⁻¹ at pH 2 to 1.7 × 10⁸ M⁻¹·s⁻¹ at pH 10. Similarly, for Van, the reaction rate rises from 1.4 × 10⁶ M⁻¹·s⁻¹ at pH 2 to 4.6 × 10⁷ M⁻¹·s⁻¹ at pH 10.

3.5. E Dependence on the Regeneration of Trp from Trp^•+

We further examined the reducing capacities of various plant phenols toward tryptophan cations. To explore the effect of driving force (∆E) on the reduction of Trp^•+ by plant phenols, we selected six phenols with differing ∆E values: salicylic acid (Sal, ∆E = −0.13 V), m-hydroxyl-benzoic acid (m-hyd, ∆E = −0.02 V), Van (∆E = 0.08 V), Syr (∆E = 0.32 V), caffeic acid (Caf, ∆E = 0.36 V), and catechin (Cat, ∆E = 0.62 V). The kinetic curves of Trp^•+ at 560 nm are shown in Figure 5. As shown in Figure 5A, the rate constants listed in

Table 3. Rate constants (k₂/M⁻¹·s⁻¹) for reduction of Trp^•+ by plant phenols with different driving forces (∆E) as measured at 560 nm.

	Sal	m-hyd	Van	Syr	Caf	Cat
∆E	−0.13	−0.02	0.08	0.32	0.36	0.62
k2/M−1·s−1	2.20 × 107	2.18 × 107	5.03 × 107	8.73 × 107	1.26 × 108	7.50 × 107

cover a broad range. These values indicate that tryptophan participates in a second-order reaction, with rate constants consistent with a diffusion-controlled bimolecular process.

The second-order rate constants for electron transfer (ET) from the plant phenols to the Trp^•+ increase with the driving force, approaching rates near the diffusion limit for bimolecular reactions [26]. At pH 9, the reaction rate with Trp^•+ increases from 2.2 × 10⁷ M⁻¹·s⁻¹ for Sal to 1.3 × 10⁸ M⁻¹·s⁻¹ for Caf. However, as the driving force continues to increase, the rate constants for ET with Trp radical cation (Trp^•+) decrease. For instance, the reaction rate with Trp^•+ for catechin is 7.5 × 10⁷ M⁻¹·s⁻¹. This bell-shaped dependence of ET rates on the driving force is consistent with predictions from Marcus theory [27], which was later experimentally confirmed for various processes, including ET from phenolate ions [28,29,30,31,32]. In accordance with this framework, we analyzed the rate data using Marcus theory [27,32], which describes the ET rate as being controlled by the driving force, a matrix coupling element (V), and the reorganization energy (λ) for the reaction: [32,33]

$$k_{\mathrm{E}\mathrm{T}}=\sqrt{{\displaystyle \frac{4{\mathsf{\pi }}^3}{h^2\lambda RT}}}{V}^2\exp -\left\{{\displaystyle \frac{{\left(\mathsf{\Delta }{E}^0+\lambda \right)}^2}{4\lambda RT}}\right\}$$

(6)

The plot of Figure 5B is based on a reorganization of Equation (6) and yields λ = 0.43 ± 0.08 eV for the reorganization energy for ET from the plant phenols to the Trp radical cation at pH 9. The matrix coupling element has the value of V = 3.75 ± 0.35 cm⁻¹ for the plant phenols. The values for the relative coupling elements represent the coupling between each of the Trp radical cations and the plant phenolates investigated.

4. Discussion

³Rib* has a strong oxidizing potential (1.77 V vs. NHE) capable of oxidizing Trp (1.015 V vs. NHE) to form Trp^•+. The rate at which plant phenols regenerate Trp from Trp^•+ depends on conditions such as plant phenols concentration, the pH of the environment, and the driving force of the reaction. The following aspects will be discussed in detail below.

The efficiencies of Syr and Van in regenerating Trp from Trp^•+ are concentration-dependent, as shown in Figure 3. Within a certain range, the reduction rate increases with a higher concentration of plant phenols, a phenomenon that aligns with the Stern–Volmer equation (Equation (4)). Similar trends have been observed in other studies, such as the reduction of carotenoid radical cations by plant phenols. For instance, as previously reported for the puerarin dianion reacting with carotenoid radical cations, the second-order rate constants were consistent for β-carotene, which was found to increase linearly with plant phenols concentration [24]. However, at higher concentrations of plant phenolate, deviations from a simple pseudo-first-order reaction were observed, with indications of rate saturation at elevated phenol concentrations.

The reduction potential decreases with increasing pH, as observed in the recovery of Trp by Syr and Van. The standard reduction potential for the Trp radical is 1.015 V at pH 7 in water but decreases to 0.65 V at pH 13 [10]. Concurrently, the proportion of φ-O²⁻ increases with rising pH, from 0% at pH 2 to 74% at pH 10 for Syr and from 0% at pH 2 to 42% at pH 10 for Van (Table 2). This increase enhances the reducing ability of plant phenols and accelerates the reduction rate.

The proportion of deprotonated phenol radicals is strongly influenced by pH due to the dissociation equilibrium of hydroxyl groups (–OH) in plant phenol molecules. The primary mechanisms are as follows [34,35]: (i) Changes in dissociation equilibrium. Hydroxyl groups in plant phenols can deprotonate to form phenoxy radicals (φ-O^•) under alkaline conditions. At low pH (acidic conditions), the high concentration of H⁺ favors the protonated state, resulting in fewer phenoxy radicals. In contrast, high pH facilitates deprotonation, significantly increasing the formation of phenoxy radicals. (ii) Role of pKa. The acid dissociation constants (pKa) of hydroxyl groups in plant phenols determine the balance between protonated and deprotonated forms. As pH approaches the pKa, the equilibrium shifts, leading to noticeable changes in the proportion of deprotonated radicals. (iii) Radicals stability. The stability of phenoxy radicals varies with pH. Under alkaline conditions, delocalization of the negative charge across the aromatic ring via conjugation stabilizes deprotonated phenoxy radicals. (iv) pH and redox reactions. pH also affects the rate and equilibrium of redox reactions involving plant phenols, modulating the formation and consumption of deprotonated radicals.

The regeneration of Trp is further influenced by the driving force, as calculated from the standard reduction potentials. Optimal regeneration conditions align with the reorganization energy of the donor–acceptor complex, as described by Marcus theory [26,36]. Caffeic acid, with ∆E= 0.36 V, achieves an ideal match for charge distribution in the donor–acceptor transition state, resulting in a maximal rate constant of k₂ = 1.3 × 10⁸ L·mol⁻¹·s⁻¹.

Plant phenols with both lower and higher reducing powers exhibit slower electron transfer rates, in agreement with Marcus theory. This observation further supports the notion that plant phenols with excessively high reducing power fall within the inverted region predicted by Marcus theory [26]. Syr, with ∆E = 0.32 V, has a smaller driving force than caffeic acid and reduces Trp^•+ slightly less efficiently with k₂ = 8.7 × 10⁷ L·mol⁻¹·s⁻¹. Similarly, catechin with ∆E = 0.62 V, has a larger driving force than caffeic acid but reduces Trp^•+ less efficiently with k₂ = 7.5 × 10⁷ L·mol⁻¹·s⁻¹, due to entering the ‘inverted region’ predicted by Marcus theory. The formation of a precursor complex between the Trp radical cation and the phenolate anion appears to be critical for establishing the donor–acceptor transition state, where a match in reorganization energy ensures the maximal electron transfer rate, according to the Marcus theory for electron transfer [27]. Beyond this kinetic factor, the resonance stabilization of the phenoxyl radical following electron transfer serves as an important thermodynamic factor. As illustrated in Figure 5B, the relationship between electron transfer rate constants and ΔE demonstrates the dependence of rate constants on driving force. The rate constants obtained for Trp regeneration support the free energy relationship predicted by the Marcus theory for electron transfer involving plant phenols [24].

In future applications, plant phenols could serve as additives to mitigate oxidative damage during the processing and preservation of protein-based foods. The incorporation of plant phenols may extend the shelf life of protein-rich foods, enhance their antioxidant capacity, and ensure that the protein content remains bioavailable and nutritionally effective for consumers. This approach holds the potential for better preserving the structural integrity and nutritional value of proteins, thereby improving the overall stability and health benefits of protein-based food products.

5. Conclusions

This study investigates the protective effects of plant phenols on Trp. Rib, an effective photosensitizer, possesses a triplet excited state with strong oxidizing power capable of oxidizing Trp to its radical cation (Trp^•+). Plant phenols, known for their antioxidant properties, can reduce the Trp^•+ and regenerate Trp. The efficiency of Trp regeneration depends on the concentration of plant phenols, pH, and the driving force of the reaction. The pseudo-first-order rate constants for Trp, calculated by exponential fitting, were found to increase linearly with the plant phenol concentration. As the pH shifts from acidic to alkaline, the bimolecular reaction rate of Syr with Trp^•+ rises from 4.8 × 10⁶ M⁻¹·s⁻¹ at pH 2 to 1.7 × 10⁸ M⁻¹·s⁻¹ at pH 10. Similarly, the rate for vanillic acid rises from 1.4 × 10⁶ M⁻¹·s⁻¹ at pH 2 to 4.6 × 10⁷ M⁻¹·s⁻¹ at pH 10, corresponding to an increase in the proportion of φ-O⁻ and φ-O²⁻ from 0% to nearly 100%. Furthermore, the reduction rate of Trp^•+ by φ-OH is influenced by the driving force of the electron transfer reaction between φ-OH and Trp^•+. Plant phenols with moderate reducing power exhibit the fastest reduction rate for Trp^•+ (1.7 × 10⁸ M⁻¹·s⁻¹). These findings are significant for advancing our understanding of protein oxidation and antioxidant mechanisms. The rate of Trp regeneration from Trp^•+ by plant phenols is influenced by factors such as concentration, pH, and the driving force. Additionally, the reverse reaction between plant phenols and Trp may play a critical role in a protective mechanism via rapid electron transfer. This mechanism helps reduce or prevent the oxidation and degradation of protein-based foods, thereby contributing to the protection of human health.