Slag Kumpf mining

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A process for C -terminal sequencing of peptides , developed by Paul Schlack and W. Kumpf in 1926, is known as Schlack-Kumpf degradation .

principle

The process must take place in the absence of oxygen in order to prevent undesired oxidations, e.g. B. from cysteines to cysteine ​​sulfonic acids or disulfide bridges . The slag kump breakdown can be divided into the following three steps:

  1. Activation : The C-terminal amino acid is activated under conditions that are harsh for the peptide: At temperatures of 60 to 80 ° C, the terminal carboxy group is acetylated using acetic anhydride in glacial acetic acid, resulting in a mixed peptide anhydride. This can also lead to an esterification of the side chains of lysine, serine and tyrosine, as well as the acidic amino acids. Some of the labile bonds in the peptide backbone can also be hydrolytically cleaved as a side reaction in this step.
  2. Coupling : With the participation of the highly reactive thiocyanic acid , a hydantoin ring is formed. Due to the instability of thiocyanic acid, the latter is obtained immediately before the reaction by acidifying ammonium thiocyanate . Before the subsequent cleavage, the reagents used in step 1 and 2 must be separated from the sample using inert gas and similar methods.
  3. Cleavage : By acting on NaOH or KOH for a few minutes at room temperature, the thiohydantoin amino acid can be cleaved from the residual peptide. The subsequent removal of the base represents an experimental difficulty because washing out is potentially associated with a loss of peptide.

Analysis of the thiohydantoin amino acid

Reverse phase chromatography ( reverse phase HPLC) can be used to separate residual peptide and thiohydantoin amino acid and identify the latter. Compared to the hydrophobicity of the PTH derivative resulting from Edman degradation , the thiohydantoin amino acid is much less hydrophobic, which makes chromatographic analysis considerably more difficult. Improvements result from increasing the number of theoretical plates and from increasing the hydrophobicity of the stationary phase . Even under optimal conditions, only a few amino acids at the C-terminal end of a protein can be analyzed with the Schlack-Kumpf breakdown. Degradation with carboxypeptidases has established itself as a biochemical alternative to C -terminal degradation .

Alternatively to the thiocyanic Triphenylgermanylisothiocyanat (TPG-ITC) can be used with peptides N -Terminal of 1,4-phenylene diisothiocyanate (DITC) glass beads coupled to.

literature

  • F. Lottspeich, JW Engels: bioanalytics . 3. Edition. Springer Spectrum, Berlin / Heidelberg 2012, ISBN 978-3-8274-2942-1 .
  • Gerhard Richter: Practical Biochemistry: Basics and Techniques . Georg Thieme, 2003. ISBN 9783131323811 , pp. 209f.

Individual evidence

  1. ↑ P. Schlack, W. Kumpf: About a new method for determining the constitution of peptides . In: Hoppe-Seyler's journal for physiological chemistry. Volume 154, Issue 1-3, pp. 125-172, doi : 10.1515 / bchm2.1926.154.1-3.125 .
  2. ↑ John M. Walker: The Protein Protocols Handbook. Springer Science & Business Media, 1996, ISBN 978-0-896-03338-2 , pp. 557-568.
  3. ↑ J. Li, S. Liang: C-terminal sequence analysis of peptides using triphenylgermanyl isothiocyanate. In: Analytical biochemistry. Volume 302, number 1, March 2002, ISSN  0003-2697 , pp. 108-113, doi : 10.1006 / abio.2001.5505 , PMID 11846383 .