Probing the structure and activity of trypsin with amidination.
Trypsin reacts with S-methylisothiourea for 1 to 2 h and the number of primary amine sites at which covalent labeling occurs is determined by mass spectrometry. By digesting the amidinated trypsin and mass analyzing the proteolytic peptides the sites of reaction are determined. The addition of cytochrome c to a solution of amidinated trypsin enables the proteolytic activity and autolytic properties of the enzyme to be studied.
Liu
Xiaohui
X
Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, IN 47405-7102, USA.
Broshears
William C
WC
Reilly
James P
JP
D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D001665:Binding Sites; D002417:Cattle; D045304:Cytochromes c / Q000378:metabolism; D066298:In Vitro Techniques; D007550:Isothiuronium / Q000031:analogs & derivatives / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014108:Tosylphenylalanyl Chloromethyl Ketone; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*
D000588:Amines; D010446:Peptide Fragments; D007550:Isothiuronium; D014108:Tosylphenylalanyl Chloromethyl Ketone; D045304:Cytochromes c; D014357:Trypsin; D010434:Pepsin A; C027744:S-methylisothiopseudouronium