Single-strand conformation polymorphism analysis for the detection of point mutations in the mitochondrial DNA

Kim, Y.L.; Brown, M.D.; Wallace, D.C.

Analytical Biochemistry 224(2): 608-611

1995


ISSN/ISBN: 0003-2697
PMID: 7733466
DOI: 10.1006/abio.1995.1096
Accession: 033401865

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Abstract
Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vaporphase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-terminal proline.

Single-strand conformation polymorphism analysis for the detection of point mutations in the mitochondrial DNA