A comparison study on ribonuclease A modifications induced by substituted p-benzoquinones

• RNase modifications were induced by a series of substituted p-benzoquinones.
• Quinone substituents impacted RNase modifications.
• RNase aggregation was detected by SDS–PAGE and confocal microscopy.
• Structural changes in RNase were detected by fluorescence spectroscopy and ESI+-MS.

In this paper, we present our investigation on ribonuclease A (RNase) modifications induced by 1,4-benzoquinone (PBQ), 2-methyl-1,4-benzoquinone (MBQ), and 2-chloro-1,4-benzoquinone (CBQ). The goal of the study was to evaluate quinone-induced protein modifications as well as substituent effects, utilizing several techniques such as SDS–PAGE, fluorescence spectroscopy, microscopy, and LC-ESI+-QTOF-MS. SDS–PAGE experiments revealed that all quinones modify RNase through oligomerization as well as polymeric aggregation; with CBQ functioning as the most efficient quinone while MBQ was least efficient. The fluorescence emission was found to be less intense and the anisotropy values were found to be slightly higher for the modified RNase compared to the unmodified RNase. UV–Vis spectroscopy indicated that all three quinones formed adducts in which they were covalently linked to RNase. Confocal imaging analysis showed that the presence of CBQ resulted in massive RNase aggregation, while PBQ-treated RNase formed much smaller aggregates. MBQ-treated RNase exhibited micrographic features that closely resembled those of the unmodified RNase. LC-ESI+-QTOF-MS studies indicated the nature of PBQ- and CBQ-induced RNase modifications are complex mainly due to simultaneously occurrence of both adduct formation and oligomerization. Kinetic studies on quinone reactivity toward lysine revealed the rank order of CBQ > PBQ ≫ MBQ, based on the second-order rate constants. We also utilized scanning electron microscopy in order to investigate the effect of modified RNase on the biomineralization of salts.

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