Abstract
The hydrolysis of the iron-binding blood plasma glycoprotein transferrin (Tf) has been examined at pH = 7.4 in the presence of a series of Zr-substituted polyoxometalates (Zr-POMs) including Keggin (Et2NH2)10[Zr(PW11O39)2]∙7H2O (Zr-K 1:2), (Et2NH2)8[{α-PW11O39Zr-(μ-OH) (H2O)}2]∙7H2O (Zr-K 2:2), Wells-Dawson K15H[Zr(α2-P2W17O61)2]·25H2O (Zr-WD 1:2), Na14[Zr4(α-P2W16O59)2(μ3-O)2(μ-OH)2(H2O)4]·57H2O (Zr-WD 4:2) and Lindqvist (Me4N)2[ZrW5O18(H2O)3] (Zr-L 1:1), (nBu4N)6[(ZrW5O18(μ–OH))2]∙2H2O (Zr-L 2:2)) type POMs. Incubation of transferrin with Zr-POMs resulted in formation of 13 polypeptide fragments that were observed on sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), but the hydrolysis efficiency varied depending on the nature of Zr-POMs. Molecular interactions between Zr-POMs and transferrin were investigated by using a range of complementary techniques such as tryptophan fluorescence, circular dichroism (CD), 31P-NMR spectroscopy, in order to gain better understanding of different efficiency of investigated Zr-POMs. A tryptophan fluorescence quenching study revealed that the most reactive Zr-WD species show the strongest interaction toward transferrin. The CD results demonstrated that interaction of Zr-POMs and transferrin in buffer solution result in significant secondary structure changes. The speciation of Zr-POMs has been followed by 31P-NMR spectroscopy in the presence and absence of transferrin, providing insight into stability of the catalysts under reaction condition.
Highlights
Proteins play fundamental and crucial roles in most biological processes such as catalysis, signaling transduction, DNA and RNA synthesis, transport and immune response [1]
Human serum transferrin is a glycoprotein consisting of 679 amino acids (AA) with an approximate molar mass of 80 kDa and two N-linked and one O-linked glycan chains
This study has shown that transferrin, a relatively large glycoprotein, was selectively hydrolyzed by a series of Zr-POMs under physiological pH conditions
Summary
Proteins play fundamental and crucial roles in most biological processes such as catalysis, signaling transduction, DNA and RNA synthesis, transport and immune response [1]. “Bottom-up” protein analysis refers to the characterization of proteins analyzing peptide fragments which are derived from proteolytic digestion of intact proteins [4]. This method employs different enzymes, among which trypsin is the most frequently used. Trypsin cleaves peptide bonds at the carboxyl side of arginine and lysine amino acids, which generates many peptide fragments that are less than six residues long. These short peptides often cannot be uniquely identified by MS, which leads to an incomplete coverage of the examined
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