Utilization of gel electrophoreses for the quantitative estimation of digestive enzyme papain.
SDS-PAGE densitometric method for analysis of papain in pharmaceutical formulations was developed and validated for the first time. Standard and samples were mixed with SDS sample buffer and denatured at 95 °C for 5 min and the gel was run at 20 mA and 200 V for 30-40 min in SDS-PAGE buffer. Gels were stained in Coomassie blue solution and distained by 5% methanol and 10% acetic acid. Destained gels were imaged and analyzed using the ChemiDoc™ XRS+ System. Bands of papain appeared atRfvalue 0.78 ± 0.03 corresponding to molecular weight 23406 Da between proteins with molecular weight 31,000 and 21,500 Da of the broad range protein standard. The generated calibration curve was used for quantitative estimation of papain in pharmaceutical formulations. The developed method was validated for precision, accuracy, specificity and robustness as described by the ICH guidelines. The proposed method gives an alternative approach for enzymes and protein analysis.
Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, 11942 Alkharj, Saudi Arabia; Department of Microbiology, College of Science, Al-Azhar University, Nasr City, 11884 Cairo, Egypt.
Magdy M
MM
Muharram
Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, 11942 Alkharj, Saudi Arabia; Department of Pharmacognosy, College of Pharmacy, Alexandria University, Alexandria 21215, Egypt.
Maged S
MS
Abdel-Kader
ICH guidelines; Papain; Quantitative; SDS-PAGE