Qualitative determination of N-terminal amino acids of peptides and proteins with cobalt(3) chelates.
1. A method of N-terminal peptide-bond hydrolysis with the cis-beta-hydroxyaquo(triethylenetetramine)cobalt(III) ion, i.e. beta-[Co(trien)(OH)(OH(2))](2+), is reported. The method has been demonstrated with 22 small peptides and ten proteins. 2. The procedure is rapid (an N-terminal amino acid determination can be made easily in one day), it involves no acid hydrolysis step and thus no destruction of labile amino acids, and it involves the use of easily prepared inexpensive reagents. 3. The released N-terminal amino acids can be identified as their cobalt(III) derivatives, or directly as the amino acid or as their dansylated derivatives. 4. The method is to treat 1 mumol of peptide or protein with beta-[Co(trien)(OH)(OH(2))](2+) reagent at pH8.0, 45 degrees C for 3h. Addition of 0.5m-phosphate buffer, pH10.5 at 45 degrees C for 10min cleaves the N-terminal bidentate amino acid-cobalt complex, which can be identified directly. For greater sensitivity with 10nmol of peptide) the free amino acid is prepared from the complex by treatment (with NaCN (0.1m, 40 degrees C, 30min), or H(2)S or NaBH(4) (25 degrees C, 5min), dried, dansylated and the dansyl-amino acid identified by high-voltage electrophoresis. The method is unaffected by the presence of 4-8m-urea, but will not cleave blocked N-terminal acids.
D000596:Amino Acids / Q000032:analysis*; D001894:Borohydrides; D002021:Buffers; D002614:Chelating Agents; D003035:Cobalt; D003486:Cyanides; D003619:Dansyl Compounds; D004586:Electrophoresis; D006358:Hot Temperature; D006862:Hydrogen Sulfide; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008722:Methods; D010443:Peptide Chain Termination, Translational; D010455:Peptides / Q000032:analysis*; D010710:Phosphates; D011506:Proteins / Q000032:analysis*; D014508:Urea
D000596:Amino Acids; D001894:Borohydrides; D002021:Buffers; D002614:Chelating Agents; D003486:Cyanides; D003619:Dansyl Compounds; D010455:Peptides; D010710:Phosphates; D011506:Proteins; D003035:Cobalt; D014508:Urea; D006862:Hydrogen Sulfide