Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2).
Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.
Smirnova
Julia
J
Institute of Gene Technology, Tallinn Technical University, 12618 Tallinn, Estonia.
Zhukova
Liliya
L
Witkiewicz-Kucharczyk
Aleksandra
A
Kopera
Edyta
E
Oledzki
Jacek
J
Wysłouch-Cieszyńska
Aleksandra
A
Palumaa
Peep
P
Hartwig
Andrea
A
Bal
Wojciech
W
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013997:Time Factors; D051760:Xeroderma Pigmentosum Group A Protein / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology*
C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D006861:Hydrogen Peroxide; D015032:Zinc; D003545:Cysteine