The Pharmacology of Defensin

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Defensin (DF) is a newer cytolytic antimicrobial peptide having molecular weight of 4 kDa having antimicrobial activity against wide range of gram positive and gram negative bacteria. The present work describes method development of recombinant Defensin protein using internal standard Bovine serum albumin (BSA). A simple, precise and accurate reverse phase high performance liquid chromatography method has been developed and validated for quantification of Defensin protein using BSA as an internal standard using Enable Q C18 column (250mm x 4.6 mm I.D, 5μm particle size 300ºA) as column, water: acetonitrile (60:40 v/v) with Trifluoroacetic acid 0.1% as mobile phase, flow rate of 1ml/min and detection was carried out at 280 nm. The retention time of BSA and Defensin was 2.342 and 5.279 min respectively. The linearity range was found to be 10-50 μg/ml, co-relation coefficient was found to be 0.999. The limits of detection and quantitation of the method were 1.010 and 3.062 μg/ml respectively. So, the developed method was accurate, precise and robust.

Key words: Recombinant Defensin, BSA, RP-HPLC, validation.

Human Defensins (1-5) are small, cationically charged, cysteine-rich endogenous antibiotic peptides with antimicrobial and cytotoxic properties that contain 29-35 amino acid residues, including six invariant disulphide linked cysteines moiety having a molecular weight of 4-5 kDa. The novel trend in drug development is the design and development of biopharmaceuticals, thus, the pharmaceutical industry has focused on bio molecules method development and validation (8-9). HPLC is a widely used technique for proteins and peptides because of its ease of usage, higher selectivity and faster analysis. In this study one of the antim...

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...Table 2). The results for intraday (0.30 – 0.80%) & interday precision (0.34 – 0.62%) study performed on 3 concentrations (20, 30 & 40 μg/ml) of standard solution of BSA & Defensin indicate that the purported method being precise (Table 2). The values for LOD & LOQ were found to be 1.010 & 3.062 μg/ ml respectively (Table 2). These data suggested that the proposed method was sensitive for the estimation of both the proteins.
The recovery study was performed by the standard addition method. The amount of Defensin was calculated by putting value of ratio of Defensin to BSA (IS) in regression equation. And % recovery was calculated for Defensin. The mean recovery obtained was 99.82 – 101.85% which was within the limit of 98 – 102% which proves that the method was accurate (Table 3). To evaluate robustness of the developed method, few parameters were deliberately varied

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