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l-Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys45 residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion–enriched human arginase I mutant (HAI) was Co2+ > Ni2+ ≫ Mn2+. The overall kcat/KM values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40 kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13 mg/kg and Ni-HAI-PEG20L at 15 mg/kg was able to maintain a L-Arg level below its detection limit for over 120 h after one injection. The body weights of mice could return to normal levels within 5 days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. Key Points: • Mono-PEGylation applied on human arginase I mutant (HAI) successfully. • The catalytic efficiency of Co- and Ni-enriched HAI was higher than the wild type. • At least eight types of cancer cell lines were inhibited by Co- and Ni-HAI-PEG20L. • Co- and Ni-HAI-PEG20L were able to achieve weekly depletion of L-Arg. [ABSTRACT FROM AUTHOR]

Abstract: The interaction between l-Arg and calf thymus DNA (ctDNA) in sodium acetate–acetic acid buffer (pH=4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV–vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that l-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA–NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of l-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode. [Copyright &y& Elsevier]

• Ordered structures of myosin were converted to disordered structures by L-Arg. • The effect was more obvious on rod-rod cross-linking than head-head aggregation. • Increasing pH induced by L-Arg increased the electrostatic repulsion of myosin. • The cationic L-Arg interacted with anionic groups on the myosin at neutral pH. • Heat-induced phase separation of myosin was ameliorated by L-Arg. This investigation aimed to study the potential mechanism of L-arginine (L-Arg) on the heat-induced phase separation phenomenon of myosin from the perspective of conformational changes of myosin. L-Arg ameliorated the phase separation of myosin after a two-step heating procedure via suppression of heat-induced aggregation of myosin. The effect of L-Arg on the heating of myosin at high temperatures (75–85 °C) was more pronounced than that in the setting stage (35–45 °C), suggesting that the ameliorative effects of L-Arg on the heat-induced phase separation of myosin are mainly attributed to the inhibition of rod-rod cross-linking between denatured myosin molecules. Additionally, L-Arg without pH modification exhibited an increased ability to suppress the gelation of myosin compared with pH modification, indicating that both pH effects and the particular structure of L-Arg play noticeable roles in the suppression of myosin gelation. Far-UV circular dichroism, intrinsic fluorescence spectroscopy and differential scanning calorimetry demonstrated that L-Arg induced the absence of ordered secondary structures of myosin molecules, especially β-sheets, and thus generated a looser protein structure, which may represent the dominant suppression mechanisms of L-Arg on the heat-induced aggregation of myosin. This work provided support for the use of L-Arg as a food additive, and the results of this study will be attractive to the meat and beverage products. [ABSTRACT FROM AUTHOR]