Chemical Modifications of Globular Proteins Phototriggered by an Endogenous Photosensitizer

Abstract

The main goal of the present work was to investigate the damages photoinduced by pterin (Ptr), an endogenous photosensitizer present in human skin under pathological conditions, on a globular protein such as ubiquitin (Ub). A particular attention has been paid on the formation of covalent adducts between Ptr and the protein that can behave as photoantigen and provoke an immune system response. Here, a multifaceted approach including UV/visible spectrophotometry, fluorescence spectroscopy, electrophoresis, size exclusion chromatography and mass spectrometry is used to establish the Ub changes triggered by UVA irradiation in the presence of Ptr. Under anaerobic conditions, the only reaction corresponds to the formation of a covalently bound Ptr-Ub adduct that retains the spectroscopic properties of the free photosensitizer. A more complex scheme is observed in air-equilibrated solutions with the occurrence of three different processes ie. formation of a Ptr-Ub adduct, dimerization and fragmentation of the protein. General Chemicals. Pterin (Ptr, purity > 99%, Schircks Laboratories, Switzerland) and ubiquitin from bovine erythrocytes, whose sequence is identical to the human protein, (Sigma-Aldrich, St. Louis, MO) were used without further purification after checking for impurities by HPLC. Sodium dodecyl sulfate (SDS, ~ 99 %), glycerol, 2mercaptoethanol, bromophenol blue, glycine (Gly, > 99% titration), ammonium persulphate (> 98%), N,N,N´,N´-tetramethylethylene-diamine (TEMED, ~ 99 %), Cytochrome c (Cyt) from horse heart, E-3,5-dimethoxy-4-hydroxycinnamic acid (E-SA), α-cyano-4-hydroxycinnamic acid (CHCA), bradykinin (1-7), insulin, angiotensin I and II and guanidine chloride were provided by Sigma Aldrich. Methanol was provided by Laboratorios Cicarelli. Trifluoroacetic acid (TFA) was provided by Merck. Acetic acid was provided by Anedra. Coomassie Brilliant Blue G was provided by Fluka. Acrylamide, N´,N´-methylene-bis-acrylamide and trishydroxymethylaminomethane (Tris) were purchased by Genbiotech. NaCl and acetonitrile were provided by J. T. Baker. Aqueous solutions were prepared using ultrapure water from Milli-Q ® purification system (Millipore Corporation, USA). Measurements of pH. The pH measurements were performed using a pH-meter sensION+ pH31 GLP combined with a pH electrode 5010T (Hach) or microelectrode XC161 (Radiometer Analytical). The pH of the aqueous solutions was adjusted by adding drops of HCl and NaOH solutions from a micropipette. The concentration of the acid and the base used for this purpose ranged from 0.1 to 2 M. UV⁄vis analysis. Electronic absorption spectra were recorded on a Shimadzu UV-1800 spectrophotometer. Measurements were made using quartz cells of 0.4 or 1 cm optical pathlength. The absorption spectra of the solutions were recorded at regular intervals of irradiation time.

Role of Tryptophan Residues in the Toxicity and Photosensitized Inactivation of Escherichia coli α-Hemolysin

“…28 Several studies have demonstrated that pterin (Ptr), the parent and unsubstituted compound of oxidized pterins (Figure 1B), can photosensitize proteins, such as albumin 29,30 and ubiquitin. 31 The studies performed using human serum albumin (HSA) revealed that the photosensitizer induces chemical changes in the protein in, at least, two amino acids: W and tyrosine (Y), 30 the latter being responsible of the oligomerization of the protein through the formation of tyrosine dimers (Y 2 ). The photooxidation of W and Y does not significantly affect the secondary and tertiary structures of HSA.…”

“…It is known that, under UV-A irradiation, Ptr can produce the oxidation of W and Y residues, and it also induces the dimerization of the latter. 30,31,42 HlyA contains four W residues and forty Y residues in its sequence, 5 so it is expected that HlyA will be affected by the photosensitization process. On the contrary, alkyl groups, because they are saturated, 43 will not be oxidized by photosensitization.…”

“…When the fact that the lamp emits quasi-monochromatic radiation was taken into account, the q n,p 0,V value was converted into the UV irradiance of the lamp (E UV L ). 31 A value of 30 ± 3 W m −2 was obtained for E UV L . Protein Isolation.…”

“…Aromatic or oxidized unconjugated pterins are natural heterocyclic compounds that absorb UV-A radiation and can act as photosensitizers through both type I and type II mechanisms . Several studies have demonstrated that pterin (Ptr), the parent and unsubstituted compound of oxidized pterins (Figure B), can photosensitize proteins, such as albumin , and ubiquitin . The studies performed using human serum albumin (HSA) revealed that the photosensitizer induces chemical changes in the protein in, at least, two amino acids: W and tyrosine (Y), the latter being responsible of the oligomerization of the protein through the formation of tyrosine dimers (Y 2 ).…”

Photosensitized Dimerization of Tyrosine: The Oxygen Paradox†

“…Ptr-photoinduced one-electron oxidation of Tyr moiety leads to dimerization of free Tyr (36) and cross-linking of peptides (40,41) and proteins (42,43) (Fig. 1b).…”