Hydrolysis of proteins and peptides in a hermetically sealed microcapillary tube: high recovery of labile amino acids.
A method for the hydrolysis of peptides and proteins in a hermetically sealed microcapillary tube has been developed. The method is based on the concept that oxidative degradation of labile amino acids during acid hydrolysis of proteins and peptides at high temperature can be reduced to a minimum by limiting the ratio of air to liquid (v/v, less than 1:10) in a microcapillary tube. Furthermore, the physical constraints imposed by the capillary tube will restrict the exposure of the protein solution to air at a very limited area at the meniscus of the liquid. This method eliminates the necessity of time-consuming sealing under vacuum and/or flushing with nitrogen to remove oxygen in the hydrolysis tube. High recovery of labile amino acids can be obtained in a reproducible manner. Because of the simplicity and high reproducibility of the method described, it could be the method of choice for the hydrolysis of protein and peptide intended for quantitative amino acid analysis. Performic acid oxidation is performed at 50 degrees C for 10 min instead of 4 to 20 h at 0 degrees C to achieve an equally good yield of cysteic acid and methionine sulfone from peptides and proteins.
Liu
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Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, Maryland 20892.
Boykins
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D005561:Formates / Q000378:metabolism; D006868:Hydrolysis; D008832:Microchemistry; D009113:Muramidase; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic; D014362:Trypsinogen
D000596:Amino Acids; D005561:Formates; D011506:Proteins; C008817:peroxyformic acid; D014362:Trypsinogen; D012259:Ribonuclease, Pancreatic; D009113:Muramidase