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  • The following is the supplementary data


    The following is the supplementary data related to this article.
    Funding This work was supported by departmental funding to Prof. Mühl. Funding derived from the State of Hessia to the Institute of General Pharmacology and Toxicology at the pharmazentrum frankfurt (Head: Prof. Josef Pfeilschifter).
    Transparency document
    Introduction Nanosecond electric pulses (nsEP) evoke a high voltage in specific cellular structures without producing any heat, thereby triggering responses such as membrane poration and DNA damage [1], [2], [3]. nsEP can be used to enhance chemotherapy and ablate tissues non-thermally. However, the electrochemical effect of nsEP on proteins remains unclear. Studies using molecular dynamics simulations suggest that nsEP can alter the free-energy profile of the side-chain orientation of histidine of aquaporin 4, and induce the unfolding of myoglobin [4], [5]. However, as yet there is no direct experimental evidence. Carboxypeptidase G2 (CPG2) is a zinc-dependent homodimer. The molecular mass of the Diphenyleneiodonium chloride sale is 41.4–41.8kDa, and each monomer has two zinc ions [6]. This quaternary structure suggest that those determinants of the function of a protein (i.e., intactness and conformation of each monomer, the assembly of subunits and the prosthetic group) can be assayed simultaneously, providing a better model for exploring the protein's response to nsEP experimentally. Thus, the electrochemical effect of nsEP on CPG2 was investigated in this study.
    Results and discussion The activity of CPG2 decreased with increasing exposure time at each pulse duration, and decreased to <20% when the total exposure time was ≥120s; values at 240s were 6.5±7.4% (8-ns), 1.7±2.9% (16-ns) and 7.7±9.5% (24-ns), demonstrating complete deactivation. The 16- and 24-ns pulses caused a lower activity at 20 and 40s, compared with 8-ns pulses (Fig. 1). These data indicated that nsEP inhibited CPG2 enzyme activity in a pulse duration-dependent manner. The depolymerization of CPG2 would lead to the loss of activity. The molecular mass of control CPG2 was 80.3kDa in SEC, confirming that the active form was a dimer. No monomer was detected in nsEP-treated CPG2, and the peak area of dimer was not decreased (Fig. 2). These data showed that nsEP did not disassemble the CPG2 protein. The integrity of the CPG2 monomer was necessary for the preservation of protein function. No lower-molecular fragments were observed in both reducing and non-reducing SDS-PAGE, demonstrating the intactness of monomer (Fig. 3A–B). Considering the lower sensitivity of SDS-PAGE to small peptides, we then performed MS analysis. The molecular mass of control CPG2 was 41,571Da, and 41,571, 41,570 and 41,571Da in 8-, 16- and 24-ns pulse-treated CPG2, respectively. The SDS-PAGE and MS data indicated that nsEP did not break the CPG2 monomer. The CPG2 monomer was also assayed by RP-HPLC. No new peak occurred in nsEP-treated CPG2 (Fig. 3C–F). The RP-HPLC behavior depended on the structure and conformation of a protein. Further, there was no difference in the CD spectrum between the samples (Fig. 4). Overall, these data suggested that the CPG2 subunit was not affected by nsEP. We assayed the levels of zinc, as it was necessary for catalysis [6]. There was no difference in the activity of CPG2 before and after dialysis (29.1±1.4% vs. 26.0±3.3% in CPG2 exposed to 90s of 24-ns pulses, p=0.3159), suggesting that zinc was the prosthetic group of CPG2. Zinc levels were 3.30μg/mg protein in control CPG2 (corresponding to 4.2 zinc ions per mol CPG2). By contrast, after 90 and 240s of nsEP exposure, zinc levels decreased to 1.74 and 0.40, 1.60 and 0.12, and 1.42 and 0.38μg/mg protein, at 8-, 16- and 24-ns pulses, respectively. These findings suggested that nsEP inhibited CPG2 via removal of zinc: Next, zinc was reloaded via direct addition or redialysis. The activities of CPG2 exposed to 8-, 16- and 24-ns pulses recovered to 81.1±3.9%, 70.0±4.4% and 67.9±3.6% (p=0.1258, 0.0011, 0.0002) after direct addition, and to 94.7±3.4%, 84.0±5.2% and 81.7±7.0% (p=0.0853, 0.0741, 0.0668) after redialysis, respectively. These findings confirmed that zinc loss was the mechanism of CPG2 deactivation following nsEP. Dialysis was a dynamic equilibrium process, thus favoring the rebinding of zinc ions.