Monitoring Binding of Protamine with DNA Using Protein\nCharge Transfer Spectra

In\nthis work, novel intrinsic electronic absorption (250–400\nnm) with a molar extinction coefficient of 752 M<sup>–1</sup>cm<sup>–1</sup> at 250 nm, arising from photoinduced electron\ntransfer involving charged amino acid side chains and the polypeptide\nbackbone, along with luminescence (300–500 nm) with quantum\nyield of 0.011 from subsequent charge recombination, was observed\nin salmon sperm Protamine (PRM). The absorption of PRM was attributed\nto the previously identified Peptide Backbone-to-Side chain Charge\nTransfer (PBS-CT) from the polypeptide backbone to the abundant cationic\nheadgroups of Arginine in PRM, while the luminescence was believed\nto originate from charge recombination within the charge-separated\nexcited states of PRM. Remarkably, since Arg is the only charged residue\nin the PRM sequence, the PRM Protein Charge Transfer Spectra (ProCharTS)\nis both totally and uniquely Arg specific. Interestingly, the peak\nof PRM luminescence emission spectrum was independent of the excitation\nwavelength, unlike other proteins such as human serum albumin, displaying\nunconventional luminescence. Aggregation-induced effects on PRM absorbance\nand luminescence were ruled out, as both PRM absorbance and luminescence\nincrease maintained linearity with increasing concentration in the\n25–150 μM range. Nucleoprotamine complex formation, resulting\nfrom the binding of PRM with calf-thymus genomic DNA (gDNA), was monitored\nthrough increased scattering by the nucleoprotamine complex, a decrease\nin gDNA/PRM absorbance, a decrease in gDNA/PRM ellipticity, and shifts\nof nucleoprotamine complex band upon agarose gel electrophoresis.\nUpon binding with gDNA (700 μM base pair concentration), PRM\nProCharTS absorbance at 260 nm decreased by 72%. This decrease was\nattributed to the formation and subsequent precipitation of nucleoprotamine\ncomplex upon PRM-gDNA binding. The application of ProCharTS absorbance\nto <i>indirectly</i> monitor DNA–protein binding\nin a <i>label-free</i> approach was thus demonstrated.