Abstract
Development of catalysts for the selective hydrolysis of proteins is challenging, yet important for many applications in biotechnology and proteomics. The hydrolysis of hydrophobic proteins is particularly challenging, as due to their poor solubility, the use of surfactants is often required. In this study, the proteolytic potential of catalyst systems based on the Zr(IV)-substituted Keggin polyoxometalate (Et2NH2)10[Zr(PW11O39)2] (Zr-K 1:2) and three different surfactants (ionic SDS (sodium dodecyl sulfate); zwitterionic Zw3-12 (n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate); and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)), which differ in structure and polarity, has been investigated. Hydrolysis of ovalbumin (OVA) was examined in the presence of Zr-K 1:2 and surfactants by sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), which showed the appearance of new polypeptide fragments at lower molecular weight, indicating that selective hydrolysis of OVA took place for all three catalyst systems. The same fragmentation pattern was observed, showing that the selectivity was not affected by surfactants. However, the surfactants influenced the performance of the catalyst. Hence, the interactions of OVA with surfactants and Zr-K 1:2 were investigated using different techniques such as tryptophan fluorescence, Circular Dichroism, and Dynamic Light Scattering. The speciation of the catalyst in surfactant solutions was also followed by 31P Nuclear Magnetic Resonance spectroscopy providing insight into its stability under reaction conditions.
Highlights
Membrane proteins are some of the most important molecules in living organisms and have a wide range of biological roles [1]
The proteolytic potential of catalyst systems based on the Zr(IV)-substituted Keggin polyoxometalate (Et2NH2)10[Zr(PW11O39)2] (Zr-K 1:2) and three different surfactants (ionic SDS; zwitterionic Zw3-12 (n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate); and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)), which differ in structure and polarity, has been investigated
The reactivity and selectivity of the Zr(IV)-substituted Keggin polyoxometalate towards the hydrolysis of ovalbumin has been investigated in the presence of different surfactants typically used for protein solubilization
Summary
Membrane proteins are some of the most important molecules in living organisms and have a wide range of biological roles [1]. The affinity constant of Zr-K 1:2 towards OVA is an order of magnitude higher in comparison to the binding constant of Hf-WD 1:2 [9] This might be due to the larger size and charge of Hf-substituted POM, since it is possible that Zr-K 1:2 being smaller makes it easier for it to approach the Trp residues on the protein surface in comparison with the larger and more rigid Hf-WD 1:2 species. In the presence of 0.5 wt% SDS (Figure S3), the affinity constant of Zr-K 1:2 towards OVA is 3 orders of magnitude lower compared to when there is no added surfactant This is most likely due to the presence of negatively charged polar heads of SDS, which, after binding to the protein, can repel the negatively charged POM and this results in a lower amount of POM molecules that can approach the protein surface. The POM is able to interact more with the protein in the presence of zwitterionic surfactants than in the presence of SDS due to its anionic nature
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