370
P-10 column. Next, ion exchange, gel filtration and reverse-phase high-performance
liquid chromatography (RP-HPLC) led to the extraction of pure HNP-1, HNP-2 and
HNP-3 peptides. These peptides were reduced and alkylated and subjected to hydrolysis in order to determine the amino acid composition. The reduced peptides were
digested with proteases, and the resulting amino acids were quantitated as phenylthiocarbamyl (PTC) derivatives.
HBD-2 a, β-defensin, has been found to augment the antimycobacterial activity
of first-line anti-TB drugs isoniazid and rifampicin (Kalita et al. 2004; Sharma et al.
2001). Kisich et al. (2001) showed that on treatment with HBD-2 mRNA (MIC of
approximately 2 μg/mL), MTB-infected macrophages show a concentrationdependent inhibition of mycobacterial growth, and with a single HBD-2 mRNA
treatment, the antimycobacterial effect lasted for at least 7 days. Administration of
l-isoleucine in drug-sensitive H37Rv strain as well as in MDR-TB-infected mice
showed an increase in the induction of beta-defensins 3 and 4 (Pazgier et al. 2006)
and a significant decrease in bacillary load.
Sequence determination: The S-carboxamidomethylated HNP-1, HNP-2 and
HNP-3 were subjected to gas-phase Edman degradation. Phenylthiohydantoin
(PTH) amino acids were identified by RP-HPLC. The carboxyl-terminal segment of
each defensin was labeled with tritium at its carboxyl terminus and then digested
with trypsin. The digestion fragments were purified by RP-HPLC, and the tritiumcontaining fragments were characterized by amino acid analysis. The human peptides are composed of 29 or 30 amino acids. The sequence of HNP-1 was determined
as “ACYCRIPACIAGERRYGTCIYQGRLWAFCC.”
The human defensins possess a net positive charge at pH 7.0. Six cysteine residues are conserved among human defensins, and it has been proposed that the
molecular conformation afforded by these residues plays a major role in immunity
against microbes.
15.2.6 PR39
PR39 is a neutrophil-derived peptide with antimicrobial activity requiring the 26
residues of amino-terminus (Shi et al. 1996). Its mechanism of action was proposed to be the inhibition of DNA and protein synthesis (6). It was first isolated
from porcine intestine and later from human neutrophils (Agerberth et al. 1991;
Shi et al. 1994). PR39 peptide with amino acid sequence
“RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP” has been successfully
synthesized, purified and tested for its antibacterial activity against E. coli and
Salmonella sp. (Shi et al. 1996). Linde et al. (2001) demonstrated that the proline–
arginine-rich porcine peptide PR39 inhibited M. tuberculosis H37Rv to significant
levels in a concentration-dependent manner in vitro.
S.M. Thayil and A.K. Kesavan
P-10 column. Next, ion exchange, gel filtration and reverse-phase high-performance
liquid chromatography (RP-HPLC) led to the extraction of pure HNP-1, HNP-2 and
HNP-3 peptides. These peptides were reduced and alkylated and subjected to hydrolysis in order to determine the amino acid composition. The reduced peptides were
digested with proteases, and the resulting amino acids were quantitated as phenylthiocarbamyl (PTC) derivatives.
HBD-2 a, β-defensin, has been found to augment the antimycobacterial activity
of first-line anti-TB drugs isoniazid and rifampicin (Kalita et al. 2004; Sharma et al.
2001). Kisich et al. (2001) showed that on treatment with HBD-2 mRNA (MIC of
approximately 2 μg/mL), MTB-infected macrophages show a concentrationdependent inhibition of mycobacterial growth, and with a single HBD-2 mRNA
treatment, the antimycobacterial effect lasted for at least 7 days. Administration of
l-isoleucine in drug-sensitive H37Rv strain as well as in MDR-TB-infected mice
showed an increase in the induction of beta-defensins 3 and 4 (Pazgier et al. 2006)
and a significant decrease in bacillary load.
Sequence determination: The S-carboxamidomethylated HNP-1, HNP-2 and
HNP-3 were subjected to gas-phase Edman degradation. Phenylthiohydantoin
(PTH) amino acids were identified by RP-HPLC. The carboxyl-terminal segment of
each defensin was labeled with tritium at its carboxyl terminus and then digested
with trypsin. The digestion fragments were purified by RP-HPLC, and the tritiumcontaining fragments were characterized by amino acid analysis. The human peptides are composed of 29 or 30 amino acids. The sequence of HNP-1 was determined
as “ACYCRIPACIAGERRYGTCIYQGRLWAFCC.”
The human defensins possess a net positive charge at pH 7.0. Six cysteine residues are conserved among human defensins, and it has been proposed that the
molecular conformation afforded by these residues plays a major role in immunity
against microbes.
15.2.6 PR39
PR39 is a neutrophil-derived peptide with antimicrobial activity requiring the 26
residues of amino-terminus (Shi et al. 1996). Its mechanism of action was proposed to be the inhibition of DNA and protein synthesis (6). It was first isolated
from porcine intestine and later from human neutrophils (Agerberth et al. 1991;
Shi et al. 1994). PR39 peptide with amino acid sequence
“RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP” has been successfully
synthesized, purified and tested for its antibacterial activity against E. coli and
Salmonella sp. (Shi et al. 1996). Linde et al. (2001) demonstrated that the proline–
arginine-rich porcine peptide PR39 inhibited M. tuberculosis H37Rv to significant
levels in a concentration-dependent manner in vitro.
S.M. Thayil and A.K. Kesavan