When the ACP was heated with 1 mmol and 2 mmol β-mercaptoethaol at 80°C for 10 min to ensure thiol residues existed in the Selleck EPZ015938 reduced state, no particular change in antimycotic activity was observed. This indicates that the oxidation state of the cysteine residues may not be important for the antimycotic activity [50]. When the dialysed ACP was treated with the reducing agent DTT, no decrease in inhibitory activity was observed, indicating that disulphide bonds are not responsible for biological
activity. It was also observed that storage of ACP at −80°C for 1 year did not significantly affect biological activity. Ammonium sulfate salt as well as sodium phosphate buffer did not inhibit ACP activity at the concentration used and did not modify the result of
the assay. The dialysed concentrate of ACP, dissolved in 20 mmol sodium phosphate buffer, weakly bound with the DEAE Sepharose matrix, indicating that the ACP bears negative charges. Being weakly negative, it was separated https://www.selleckchem.com/products/LY2603618-IC-83.html easily in native polyacrylamide gel electrophoresis. After purification by ammonium sulfate fractionation, dialysis, anion exchange chromatography and gel filtration, the final amount of recovered protein (0.45%) was found very low. This could be increased by using protein engineering and optimization methods. Comparing the partial amino acid sequence of the purified antimycotic protein to other antimicrobial peptides and bacteriocins by using protein-protein BLAST in NCBI revealed no complete homology with other known bacteriocins or AMPs. The combined N-terminal and de novo sequence GPGGPG…WLPPAGLLGRCGRWFRPWLLWLQSGAQYKWLGNLFGLGPK
had high amounts of glycine, proline, leucine and tryptophan. This has been observed in many antimicrobial peptides Romidepsin solubility dmso including Meloxicam bacteriocins like enterocin and acidocin. It was reported earlier that the glycine-rich antifungal peptide tenacin-3 enters the C. albicans cytoplasm [51], although tenacin-3 seems not to induce membrane permeabilisation. Linear peptides with an extended structure were characterised by an unusual proportion of one or more amino acids (most often proline, tryptophan, or glycine) [52, 53]. Penaedins characterised from shrimps and prawns had a high content of Pro/Arg/Gly residues in the extended N-terminal domain [54]. Oxypinin 2 has a GVG motif, and ponericin G has glycine residues flanking the central proline, resulting in a GPG motif with calculated grand average of hydropathicity (GRAVY) of −0.683.20. The presence of Gly-Pro hinges in antimicrobial peptides like oxypinins, ponericins, and cecropins supports the antimicrobial potential of ACP, wherein a similar sequence was observed. The regional flexibility provided by proline was sometimes enhanced by the presence of glycine residues [55]. In another recent report, a penaedin homologue, hyastatin from spider crab [56], was shown to possess a Pro/Gly domain similar to the N-terminal domain of penaedins that bind chitin tightly.