## 4 Discussion  
Cold-adapted organisms have developed networks of defense mechanisms to protect themselves from the harsh aquatic environment (S  
the first line of defense against microbial invasions [20]. Hepcidin is one of the most widespread antimicrobial amphipathic peptides involved in fish innate immunity [20]. It was first identified and isolated in human urine, and denominated as LEAP-1 or hepcidin [1], followed by its detection in other mammal and fish species [10; 21; 29; 36; 37; 38; 39]. Previous studies had mostly focused on the antimicrobial activities of the hepcidin with eight cysteine residues [2; 5; 8; 40; 41; 42], whereas little information is available on the antimicrobial activity of novel, four-cysteine LEAPs isolated from Antarctic fish. In the present study, we synthesized novel LEAPs derived from Antarctic notothenioid _D.__mawsoni_ and eelout _L.__debarboni_, with four and eight cysteine residues. The secondary structure of peptides was analyzed by circular dichroism (CD) spectroscopy. Analysis of the CD spectra indicated that the general conformational characteristics were preserved in all the structures of all the LEAPs (Figure 2). All the spectra were typical of \(\beta\)-sheet peptides, stabilized with disulfide bonds that were connected by turn-loop segments [43; 44]. In spite of alterations in the disulfide-bonding pattern of trout hepcidin, the disulfide bonds variants show a typical CD spectrum of hairpin structure and maintain the antibacterial activity [5]. Similarly, the CD spectra of our LEAPs at 5, 15, 25, and 35 \({}^{\circ}\)C showed the characteristics of a typical beta-sheet structure. Cationic antimicrobial peptides are defined as peptides with a net positive charge of +2 to +8, and an excess of arginine, lysine, and histidine amino acid residues [45]. These cationic antimicrobial peptides bind specifically to the membrane. Therefore, gram-positive and gram-negative bacteria, viruses, and fungi are the targets of such peptides [46]. nLEAP-1 and 2 have a net positive charge of +7 and +8, with an excess of arginine and lysine amino acids, which likely gives them the ability to bind to the bacterial membrane and efficiently inhibit their growth. The most potent cationic peptides have a charged hydrophilic portion that is segregated from the hydrophobic portion, for example, an amphipathic structure with a hydrophobic core separating two charged segments, which are well suited for undergoing transitions between hydrophilic and hydrophobic environments [46]. nLEAP-1 and nLEAP-2 have an amphipathic structure, which could be the reasoning behind its potent antibacterial activity against gram-positive and gram-negative bacteria. Generally speaking, more than 50% of the amino acids are hydrophobic, a fact reflected in the interaction of such peptides with the membranes as a part of their antibacterial mechanism. The key factors responsible for the antibacterial activity mechanism of action and selectivity toward bacterial cells are the composition of amino acids in the peptide which decides its flexibility, amphipachity, net charge, hydrogen-bonding capacity, and hydrophobicity [47]. eLEAP-1 has a net +4.1 charge, and thus the peptide bind through the electrostatic interactions between positively charged amino acids on the peptide and negatively charged phospholipids headgroups on bacterial membrane and enter it [48]. eLEAP-2 has a net 0 charge. So, it may have weak electrostatic interaction such as hydrogen bonding with functional group present on the bacterial membrane and the hydrophobic region may help them to cross the outer bacterial membrane. In the present study, we observed that the LEAPs with four cysteine showed an effective antibacterial activity against gram-positive and gram-negative bacteria, compared with the eight-cysteine peptides. As shown in previous studies, synthetic EC-hepcidin1 (four-cysteine), at a concentration of 18.6 \(\upmu\)M or above, inhibits the growth of the gram-negative bacterium _V.__vulniktivas_ and gram-positive bacterium _S. aureus_[21].