Development and validation of capillary electrophoresis assay for ribonuclease A.
A quick and reproducible capillary electrophoresis assay was developed and validated for bovine pancreatic Ribonuclease A (Rnase A). Effects of separation buffer pH and concentration as well as separation power were evaluated on Rnase A samples in phosphate buffered saline in the 5-100 micrograms ml-1 concentration range. The separation variables yielded significant influence on peak elution, with phosphate buffer (pH 2.5; 0.2 M) at separation power of 2 W providing an optimum peak at about 4 min. The peak sharpness and detection limit was further improved by a base/sample/acid injection technique. The optimized method showed good linearity with inter- and intraday RSD of peak area in the 2-5% range. Heat, as well as guainidine hydrochloride denatured samples, showed loss of enzymatic activity, changes in tyrosine fluorescence and abnormal electropherograms. The assay was also compared to a bicinchoninic acid assay that is routinely used for protein quantitation, and found to have greater sensitivity and reproducibility.
Nellore
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Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore 21201.
Bhagat
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HR
D002021:Buffers; D004586:Electrophoresis / Q000379:methods*; D011489:Protein Denaturation; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry
D002021:Buffers; D014443:Tyrosine; D012259:Ribonuclease, Pancreatic