A mechanistic approach towards the formation of bityrosine in proteins by ionizing radiation – GYG model peptide
Introduction
Protein-based nanoparticles represent a potential group of therapeutic agents applied for biomedical research. Globular proteins and proteolytic enzymes, e.g. bovine serum albumin and papain, have been studied at the nanoscale (Varca et al., 2014; Queiroz et al., 2016) and demonstrated high potential for drug delivery due to the tunable nanoparticle size, biological affinity among other biopharmaceutical advantages (Amri and Mamboya, 2012) which are not observed nor expected in the case of inorganic molecules and more advanced than in polymeric materials meant for biomedical applications.
For instance, papain (EC 3.4.22.2) is a proteolytic enzyme extracted from the latex of Carica Papaya Linnaeus with a well-defined structure (Kamphuis et al., 1984) and a high potential for drug delivery (Varca et al., 2016; Fazolin et al., 2020), as this enzyme holds desirable properties for wound treatment (Amri and Mamboya, 2012) due to its anti-inflammatory and antitumoral characteristics (Müller et al., 2016).
The use of ionizing radiation for the synthesis of polymer-based nanoparticles has been demonstrated over the years and proven effective for a wide variety of substrate molecules (Ulański et al., 1998; Ulanski and Rosiak, 2004; An et al., 2011; Dispenza et al., 2012; Varca et al., 2014; Duygu Sütekin and Güven, 2019; Matusiak et al., 2020). When it comes to proteins and protein-like structures, the use of high-energy radiation combined with cosolvents like ethanol and methanol may promote crosslinking and sterilization simultaneously, depending upon the dose, and has been successfully applied towards achieving protein-based nanoparticles with controllable particle size and preserved bioactivity or biological function (Soto Espinoza et al., 2012; Queiroz et al., 2016; Fazolin et al., 2020).
On the other hand, high-energy radiation may affect the biomolecules by direct or indirect effects and can impair the biological function of proteins as well as their tridimensional structure (Saha et al., 1995). In principle, biomolecules are prone to attack by radiation via indirect effects to a major extent through the action of free radicals generated as a function of water/solvent radiolysis. This exposition leads to damage and depending upon the conditions may trigger the formation of several protein-protein crosslinks which contribute significantly to the conformational changes and the newly acquired structure within the protein. Among those, disulfide bridges, bityrosines, and other linkages have been studied, identified, and hypothesized (Giulivi et al., 2003; Houée-Levin and Bobrowski, 2013; Bian and Chowdhury, 2014; Hägglund et al., 2018).
The mechanism of bityrosine formation, perhaps one of the most relevant cross-linking types identified in papain (Varca et al., 2014) and BSA (Queiroz et al., 2016), occurs via three main stages: abstraction of hydrogen (with the formation of phenoxyl radical), recombination and isomerization (Giulivi et al., 2003). The formation of Tyr-Tyr linkages occurs when the oxidizing species derived from water radiolysis, e.g. hydroxyl radical, lead to protein crosslinking mainly via tyrosine residues (Queiroz et al., 2016). The hypothesis behind the mechanism involved in the nanoparticle formation is evidence of crosslinking formation by C–O–C bonds induced by the hydroxyl radical (Heinecke et al., 1993; Saha et al., 1995). The nature of such linkages plays an important role in the nanoparticle formation, whereas the intermolecular bonding is characterized by the increase in molecular weight due to the linkages with another molecule and intramolecular one does not present any change (Ulański et al., 2002).
The formation of phenoxyl radicals is the first step in the process of tyrosine-mediated crosslinking. In aqueous solutions of proteins and peptides, phenoxyl radicals on tyrosine (TyrO•) are formed as one of the products of reaction with hydroxyl radicals (Fig. 1). Most of the hydroxyl radicals reacting with tyrosine undergo addition to aromatic ring in meta- (reaction 1) and ortho- (reaction 2) positions, forming dihydroxyhexadienyl radicals (•TyrOH). A small fraction of hydroxyl radicals is known to cause the formation of phenoxyl radicals without observable intermediate product (reaction 3). The main route of phenoxyl radicals formation is proton-catalyzed water elimination (reaction 4). This reaction is known to occur only from ortho-adducts (o-•TyrOH). Another product of tyrosine solutions radiolysis is dihydroxyphenylalanine (DOPA), being a product of disproportionation and recombination reactions (reactions 5, 6), that compete with the water elimination process. Kinetics of mentioned processes and factors influencing them seem of great importance in terms of bityrosine formation (Solar et al., 1984). Those three processes amount to about 90% of radicals formed on tyrosine in reaction with hydroxyl radical (Getoff, 1992), other processes being primarily relatively slow reactions with main-chain of the amino acid. While possible, other adducts to side-chain group of tyrosine (on C1 and C4) are not observable spectroscopically and therefore it is not possible to experimentally confirm their contribution to reactions in this system.
Cysteine bonding is another linkage that was also postulated to take place in the crosslinking process via disulfide bridges (Gaber, 2005). The Cys-Cys and cysteine thiyl are derivates of the free cysteine residues attacked by oxidizing species and depending upon the condition may bind to another thiol to build up a disulfide bridge (Houée-Levin and Bobrowski, 2013).
Regardless of several types of crosslinking which have been postulated, it does vary from protein to protein and the crosslinking method. When it comes to radiation-induced reaction pathways, there is limited knowledge not allowing a proper understanding of the radiation chemistry aspects and pathways for the formation of such crosslinks even in terms of what is the major type of crosslinking and the possible role of each one of them.
Although special attention to this matter has been given in the last decade, the mechanism of nanoparticle formation has not been thoroughly established yet and remains a theme of future studies. Within this context, this paper aims to provide a radiation-chemistry approach for better understanding of the mechanism beneath the formation of such crosslinks, particularly directed towards the synthesis of papain-based nanoparticles. H-Gly-Tyr-Gly-OH (GYG) tripeptide was chosen as low-molecular-weight model for more complex protein systems, being more realistic analogue of the protein system than tyrosine itself and allowing to follow selectivity of the reaction with tyrosine residue, but on the other hand reducing the complexity of multitude of reactions and transients encountered when complete protein molecule is irradiated. Such an approach was also intended to indicate if replacing the carboxyl and amino terminal functions in tyrosine by peptide bonds does influence the reaction pathways and kinetics known from earlier works on pure tyrosine. Kinetic study of this compound in conditions similar to the ones used for proteins in synthesis of nanoparticles provided insight into processes involved in radiation- induced crosslinking of proteins via bityrosine formation, especially regarding the influence of phosphate buffer on formation of phenoxyl radicals on tyrosine. Kinetic simulations have been applied to confirm the mechanistic scheme.
Section snippets
Materials and samples preparation
Peptide H-Gly-Tyr-Gly-OH (GYG, M = 295.3 g mol−1) (>99%, Bachem, Germany) was used as received. Peptide solutions were prepared with deionized water (conductivity ≈ 10 μS/m, produced with TKA-Millipore system). Peptide solutions in water were confirmed to have pH = 5.4 in the used range of concentrations. More acidic conditions were obtained by the addition of perchloric acid (Sigma-Aldrich). The phosphate buffer used in the experiments was obtained by dissolving dibasic sodium phosphate
Absorption spectra of transient species
Previous observations on tyrosine indicated that the main route of tyrosyl radical formation involves water molecule elimination from hydroxyl radical adduct to the aromatic ring in the tyrosine side chain (the dihydroxycyclohexadienyl radicals, •TyrOH, reaction 4) (Land and Ebert, 1967; Lynn and Purdie, 1976). While •OH forms adducts at a high rate both in meta- and ortho-positions (reactions 1 and 2, respectively), water elimination has been reported only from the o-adducts. The reported
Conclusions
Hydroxyl radicals react with GYG tripeptide mainly by addition to the tyrosine ring. Replacing the carboxyl and amino terminal functions in tyrosine by peptide bonds does not significantly influence the reaction pathways and kinetics known from earlier works on pure tyrosine. Pulse radiolysis experiments confirmed that phenoxyl radicals on tyrosine residues are formed primarily through water elimination from hydroxyl radical adduct to the ring, which is catalyzed by the presence of H+. Changes
Author statement
Sebastian Sowiński: investigation, formal analysis, visualization, methodology, writing (original draft preparation); Gustavo H. C. Varca: conceptualization, funding acquisition, resources, writing (reviewing & editing); Sławomir Kadłubowski: validation, writing (reviewing & editing); Ademar B. Lugão: supervision, writing (reviewing & editing); Piotr Ulański: methodology, supervision, writing (reviewing & editing).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank Sao Paulo Research Foundation - FAPESP for a scholarship (No. 2010/10935-9 2015-13979-0), the International Atomic Energy Agency IAEA (CRP F22064) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) (project no. 402887/2013-1) for financial support.
References (39)
- et al.
Radiation-induced synthesis of poly(vinylpyrrolidone) nanogel
Polymer
(2011) - et al.
A compilation of specific bimolecular rate constants for the reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals with inorganic and organic compounds in aqueous solution
Int. J. Appl. Radiat. Isot.
(1967) - et al.
Application of radiation for the synthesis of poly(N-vinyl pyrrolidone) nanogels with controlled sizes from aqueous solutions
Appl. Radiat. Isot.
(2019) Effect of γ-irradiation on the molecular properties of bovine serum albumin
J. Biosci. Bioeng.
(2005)A general method for numerically simulating the stochastic time evolution of coupled chemical reactions
J. Comput. Phys.
(1976)- et al.
The use of the methods of radiolysis to explore the mechanisms of free radical modifications in proteins
J. Proteom.
(2013) - et al.
The effect of oxygen, antioxidants, and superoxide radical on tyrosine phenoxyl radical dimerization
Free Radic. Biol. Med.
(1989) - et al.
Structure of papain refined at 1.65 Å resolution
J. Mol. Biol.
(1984) - et al.
Pulse radiolysis system based on ELU-6E Linac—II. Development and upgrading the system
Int. J. Radiat. Appl. Instrum. C Radiat. Phys. Chem.
(1992) - et al.
Some pulse and gamma radiolysis studies of tyrosine and its glycyl peptides
Int. J. Radiat. Phys. Chem.
(1976)
Radiation-synthesized protein-based drug carriers: size-controlled BSA nanoparticles
Int. J. Biol. Macromol.
Reactions of phosphate radicals with substituted benzenes
J. Photochem. Photobiol. Chem.
Radiation-induced inactivation of enzymes—a review
Radiat. Phys. Chem.
Radiation synthesis of seroalbumin nanoparticles
Radiat. Phys. Chem.
Radiation formation of polymeric nanogels
Radiat. Phys. Chem.
Synthesis of poly(acrylic acid) nanogels by preparative pulse radiolysis
Radiat. Phys. Chem.
Synthesis of papain nanoparticles by electron beam irradiation – a pathway for controlled enzyme crosslinking
Int. J. Biol. Macromol.
Radiation synthesized protein-based nanoparticles: a technique overview
Radiat. Phys. Chem.
Papain, a plant enzyme of biological importance: a review
Am. J. Biochem. Biotechnol.
Cited by (5)
Radiation-processed silk fibroin micro- /nano-gels as promising antioxidants: Electron beam treatment and physicochemical characterization
2022, Colloids and Surfaces A: Physicochemical and Engineering AspectsCitation Excerpt :The search for new signals was performed to gain more detailed information on the transformations taking place upon irradiation. The formation of bityrosine bridges upon irradiation of Tyr-rich proteins is now well-documented process explaining their cross-linking into macrogels, when irradiated as a concentrated solution [81] or micro- of nano-gels upon treatment of dilute solutions by irradiation [82–84] or other chemical pathways such as the Fenton [85] and photo-Fenton reactions [86]. Based on available 13C NMR data on SF [87] and bityrosine [88,89], spectroscopic evidence of the formation of this dimeric form could arise from the presence of the following signals in the spectral zone corresponding to aromatic carbon atoms: (i) in the 154–155 ppm range, corresponding to C(ζ)-OH of the phenyl ring next to the one of Tyr, (ii) by 118–122 ppm, the C(η)-H vicinal to carbon of the phenol group in bityrosine, (iii) at about 129 ppm for the C of the phenyl connected to the other Tyr moiety, yet difficult to observe by 13C NMR, since it is a quaternary carbon present at low concentration.
Radioprotective Protein of Tardigrades Dsup (Damage Suppressor) is Resistant to High Doses of Ionizing Radiation
2024, Moscow University Physics BulletinSpontaneous and Ionizing Radiation-Induced Aggregation of Human Serum Albumin: Dityrosine as a Fluorescent Probe
2022, International Journal of Molecular SciencesStrengthened Silk-Fibroin/Poly(ethylene oxide) Nonwoven Nanofibers: A Dual Green Process Using Pure Water for Electrospinning and Electron Beam-Assisted Cross-Linking
2022, ACS Sustainable Chemistry and Engineering