## 4 Discussion
Both cecropin A2 and buforin II show a concentration-dependent decrease in the intensity of the DNA band [57; 58]. Human lactoferrin, an 11-amino-acid-containing peptide with 24% \(\beta\)-sheet and 41% \(\alpha\)-helix structure, isolated from human salivary proteins, inhibits DNA migration at a DNA: peptide ratio of 1:100 [59]. P7 and APP, \(\alpha\)-helix peptides with 20 amino acid residues, are analogs of the cell-penetrating peptide (CPP) ppTG2 [60; 61; 65]. P7 and APP inhibit the migration of DNA at a DNA: peptide ratio of 1:4 and 1:0.08, respectively [61]. Competitive EB displacement assays have shown that P7 and APP bind to DNA in an intercalative manner. Human \(\alpha\)-defensin 5 [62], a \(\beta\)-sheet peptide with 32 amino acid residues, localized in the small intestinal Paneth cells in humans, is known to show DNA binding, with a dissociation constant value of 3 \(\upmu\)M. Human neutrophil peptide-I [63], a \(\beta\)-sheet peptide, stored in azurophilic granules of polymorphonuclear neutrophils, inhibits DNA migration at a DNA: peptide ratio of 1:50. CP-1, a \(\beta\)-sheet peptide with 9 amino acid residues, obtained from T-cell antigen receptor (TCR) \(\alpha\)-chain, inhibits DNA migration at a DNA: peptide ratio of 1:150 [64]. These DNA-binding peptides have varying net charges (+3 to +10) and structures (\(\alpha\)-helix, \(\beta\) sheet, and random coil). Furthermore, the DNA-binding property of these peptides is not the outcome of electrostatic interactions between the cationic peptide and the polyanionic DNA. For example, lactoferrin B, a 25-amino-acid-residue peptide with one disulfide bridge and +8 net charge, does not bind to DNA even at a DNA: peptide ratio of 1:200 [66] Human \(\beta\) defensin 4, a \(\beta\)-sheet peptide with 48 amino acid residues shows the considerable damage to _E. coli_ membrane [52]. nLEAP-2 with weak DNA binding affinity has high fluorescence intensity of DNA-bound SYTOX green signal, where nLEAP-1, eLEAP-1 and eLEAP-2 with strong DNA binding affinity have low fluorescence intensity. Taken together, these results strongly support our hypothesis that decrease in the fluorescence intensity of DNA-bound SYCOX green signal is due to the competitive displacement of SYCOX green by LEAPs. In this study, LEAPs with 4 cys residues (nLEAP-2 and eLEAP-2) showed significant antibacterial activity as compared to their 8 cys counterpart (nLEAP-1 and eLEAP-1) against both gram-positive and gram-negative bacteria. It has been suggested that, the LEAP-1 is involved in both iron regulation and antibacterial activity and LEAP-2 is involved only in antibacterial activity [22; 23]. In teleost fish, due to the existence of multiple LEAP genes, functional separation was suggested. In which, the cellular and molecular evidence indicated that, some fish LEAPs may serve only as antimicrobial peptides, other fish and amphibian hepcidins may function as iron regulators [67]. Studies of synthetic human LEAP-1 mutants with 1 disulfide bond removed by pairwise substitution of cysteines with alanine showed only negligible decrease in ferroportin-binding activity and over half of the activity persisted even when 3 of the 4 disulfide bonds were removed leaving only the most terminal disulfide bond intact [44]. If the structure-function relationships of human LEAPs are applicable to Antarctic fish LEAPs, the peptide with 4 cys with 2 disulfide bonds intact may have complete bacterial membrane binding ability. To investigate whether LEAPs had distinct characteristics arising from their origin in fish that inhabit in a cold environment, we investigated the effect of temperature on their structure and antibacterial activity. We analyzed the CD spectra of LEAPs from 5 to 35 \({}^{\circ}\)C. The CD analysis of LEAP-2 showed a distinct change in the CD spectrum, among others, indicating a modification of the peptide structure. Thus, the structure of LEAPs are temperature sensitive [4; 68].