Dityrosine

FIGURE 9.3 Cross-linked recombinant resilin. (a) Structure of the dityrosine adduct, (b) A cross-linked moulded rod. (c) HPLC analysis of acid-hydrolyzed uncross-Unked recombinant resilin and cross-linked recombinant resilin. (From Elvin, C.M., Carr, A.G., Huson, M.G., Maxwell, J.M., Pearson, R.D., Vuocolo 1, T., Liyon, N.E., Wong, D.C.C., Merritt, D.J., and Dixon, N.E., Nature, 437, 999, 2005.)... 

Amino acid analysis, by reverse-phase HPLC, of acid-hydrolyzed uncross-linked recombinant resilin and cross-linked recombinant resilin clearly shows the presence of dityrosine in the cross-linked sample (Figure 9.3c). Further evidence of the presence of dityrosine was obtained by UV irradiation (Xmax,ex 315 nm Xmax,em 409 nm). Dityrosine endows natural resilin with pH-dependent blue fluorescence [38] on UV irradiation. The cross-linked recombinant resilin material was similarly fluorescent, strongly suggesting dityrosine cross-links. 

Other clues to the self-association of recombinant resilin in solution, and thus a degree of defined stmcture, include the propensity of the monomer proteins to covalently cross-link very rapidly through dityrosine side chains using a mthenium-based photochemical method [29]. Proteins which do not naturally self-associate do not form biomaterials when exposed to the Ru(ll)-based photochemical procedure (Elvin, C.E. and Brownlee, A.G., personal communication). Furthermore, Kodadek and colleagues showed that only intimately associated proteins are cross-linked via this zero-A photochemistry procedure [45]. 

Andersen, S.O., The crosshnks in resihn identified as dityrosine and trityrosine, Biochim. Biophys. Acta, 93, 213-215, 1964. 

Malencik, D.A. and Anderson, S.R., Dityrosine formation in calmodulin Cross-linking and polymerization catalyzed by Arthromyces peroxidase. Biochemistry, 35, 4375 386, 1996. 

Fig. 19 Dityrosine and trityrosine crosslinks found in resilin. The R groups connect to the backbone of the polypeptide chain...

Briza R, Ellinger A., Winkler G. Breitenbach M. (1990) Characterization of a D, L-dityrosine-containing macromolecule from yeast ascospore walls. JB/o/Cfteni, 265, 15118-15123. 

Lassandro, F., Sebastiano, M., Zei, F. and Bazzicalupo, P. (1994) The role of dityrosine formation in the cross-linking of cut-2, the product of a 2nd cuticlin gene of Caenorhabditis elegans. Molecular and Biochemical Parasitology 65,147-159. 

Dordick, 1991). In addition, Fancy et al. (1996) as well as Fancy and Kodadek (1997, 1998) have applied the oxidation of tyrosine to form dityrosine to the study of protein-protein interactions using nickel-chelated 6 X His tagged fusion proteins in oxidative environments. 

On the other hand, in accord with the free radical mechanism peroxynitrite is dissociated into free radicals, which are supposed to be genuine reactive species. Although free radical mechanism was proposed as early as in 1970 [111], for some time it was not considered to be a reliable one because a great confusion ensued during the next two decades because of misinterpretations of inconclusive experiments, sometimes stimulated by improper thermodynamic estimations [85]. The latest experimental data supported its reliability [107-109]. Among them, the formation of dityrosine in the reaction with tyrosine and 15N chemically induced dynamic nuclear polarization (CIDNP) in the NMR spectra of the products of peroxynitrite reactions are probably the most convincing evidences (see below). 

Probably, the most convincing proof of free radical mechanism of peroxynitrite reactions is the formation of dityrosine [117,118]. It has been suggested [118] that the nitric dioxide radical is responsible for the formation of both 3-nitrotyrosine and dityrosine (Figure 21.1), however, hydroxyl radicals (which were identified in this system by ESR spectroscopy [119]) may also participate in this process. Pfeiffer et al. [118] proposed that dityrosine is predominantly formed at low fluxes of superoxide and nitric oxide, which corresponds to in vivo conditions, however, this observation was not confirmed by Sawa et al. [117],... 

The reaction of peroxynitrite with the biologically ubiquitous C02 is of special interest due to the presence of both compounds in living organisms therefore, we may be confident that this process takes place under in vivo conditions. After the discovery of this reaction in 1995 by Lymar [136], the interaction of peroxynitrite with carbon dioxide and the reactions of the formed adduct nitrosoperoxocarboxylate ONOOCOO has been thoroughly studied. In 1996, Lymar et al. [137] have shown that this adduct is more reactive than peroxynitrite in the reaction with tyrosine, forming similar to peroxynitrite dityrosine and 3-nitrotyrosine. Experimental data were in quantitative agreement with free radical-mediated mechanism yielding tyrosyl and nitric dioxide radicals as intermediates and were inconsistent with electrophilic mechanism. The lifetime of ONOOCOO was estimated as <3 ms, and the rate constant of Reaction (42) k42 = 2 x 103 1 mol 1 s 1. 

ONOO- + C02 => ONOOCOO ONOOCOO- + tyrosine => dityrosine + 3-nitrotyrosine... 

Heinecke et al. [191] studied the oxidation of L-tyrosine by the H202-MP0 system and showed that the main product of this reaction is dityrosine. They have also found that tyrosine successfully competed with chloride as a substrate for MPO that points out at the possibility of in vivo oxidation of tyrosine by MPO even in the presence of big physiological concentration (0.10-0.1 mol l-1) of chloride in human blood. It was also suggested that the tyrosyl radical formed at the catalytic oxidation of tyrosine by peroxidases may interact with... 

Marquez and Dunford [193] have studied the kinetics of L-tyrosine oxidation by MPO. They measured the rate constants for the reactions of MPO compounds I and II with tyrosine and dityrosine and found out that, comparing with HRP, LPO, and TPO, MPO is the most effective catalyst of tyrosine oxidation at physiological pH (Table 22.1). Furthermore, the rate constant for Reaction (9) with tyrosine turns out to be comparable with that for Reaction (16), confirming the possibility for tyrosine to compete in blood plasma with chloride, which is considered to be the major MPO substrate and a potent oxidizing agent against invading bacteria and viruses. 

HOCl-mediated protein oxidation accelerates under pathophysiological conditions. Thus, proteins from extracellular matrix obtained from advanced human atherosclerotic lesions contained the enhanced levels of oxidized amino acids (DOPA and dityrosine) compared to healthy arterial tissue [44], It was also found that superoxide enhanced the prooxidant effect of hypochlorite in protein oxidation supposedly by the decomposition of chloramines and chlor-amides forming nitrogen-centered free radicals and increasing protein fragmentation [45], In addition to chlorination, hypochlorite is able to oxidize proteins. The most readily oxidized amino acid residue of protein is methionine. Methionine is reversibly oxidized by many oxidants including hypochlorite to methionine sulfide and irreversibly to methionine sulfone [46] ... 

R Lygo, Enantioseledive Synthesis of Dityrosine and Isodityrosine via Asymmetric Phase-Transfer Catalysis , Tetrahedron Lett. 1999, 40,1389-1392... 

The fluorescence of purified histones has been studied by several different groups, 90 95) with the most detailed studies being on calf thymus histone HI. Histone HI, which binds to the outside of core particles, contains one tyrosine and no tryptophan. This protein exhibits a substantial increase in fluorescence intensity in going from a denatured to a folded state.<90) Collisional quenching studies indicate that the tyrosine of the folded HI is in a buried environ-ment.(91) Libertini and Small(94) have identified three emissions from this residue when in the unfolded state with peaks near 300, 340, and 400 nm. The 340-nm peak was ascribed to tyrosinate (vide infra), and several possibilities were considered for the 400-nm component, including room temperature phosphorescence, emission of a charge transfer complex, or dityrosine. Dityrosine has the appropriate spectral characteristics, but would require... 

In testing the possibility of proton transfer as a quenching mechanism of tyrosine in oligopeptide/polynucleotide complexes, Brun et a/.(102) compared the fluorescence emission spectra of the tyrosine and O-methyltyrosine tripeptides. They noted that, in the complex, the O-methyltyrosine tripeptide had a unique secondary emission near 410 nm. Whether this emission is related to that observed by Libertini and Small(94) is an important question. While one must consider the possibility that two tyrosine side chains could be converted to dityrosine, (96) which has a fluorescence at 400 nm, another intriguing possibility is ambient temperature tyrosine phosphorescence. This could happen if the tyrosine side chain is in a rigid, protective environment, very effectively shielded from collisions with quenchers, particularly oxygen. 

R. Amado, R. Aeschbach, and H. Neukom, Dityrosine In vitro production and characterization, Methods Enzymol. 107, 377-388 (1984). 

The 328/378- and 370/440 fluorescences are characteristic for pentosidine and Maillard products, respectively (Dyer et al., 1991b) while the 317/407 fluorescence indicates dityrosine formation (Huggins et al., 1993). 

Emission spectra of blank (buffer, A), standards (D, 0.28 pM dityrosine at 317 nm P, 3.3 nM pentosidine at A,. 328 nm), digested sound dentin (B), and carious dentin (C) of a tooth from series 1. Note the increase of fluorescence in carious dentin (4-j. 

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