C-terminal analysis

Another class of exopeptidases, the carboxypeptidases, can be used for the C-terminal analysis of polypeptides. Carboxypeptidases, in direct analogy to 

Alternatively, chemical methods can be employed, for example hydrazinolysis. The polypeptide is treated with anhydrous hydrazine at 90 °C for 20-100 h under mild acidic conditions. This reaction produces aminoacyl hydrazine derivatives of all amino acid residues, except for the C-terminal residue, which is released as a free amino acid (Fig. 7.4). After chromatographic separation of the reaction mixture, this free amino acid can be identified. Hydrazinolysis also leads to a great number of side products. 

There is no reliable process comparable to the Edman degradation for sequential C-terminal analysis. None of the described methods can be employed in a cyclic fashion to extract one amino acid after the other and identify them in an automated fashion. 

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Figure 6. HSA (1 nmol), applied to a Zitex membrane, was Edman-sequenced on the HP G1005A N-terminal sequencer for 5 cycles and then transferred to the HP G1009A C-terminal sequencer. Cycles 1 and 2 of the Edman cycles are shown above the cycles 1 and 2 of the C-terminal analysis. Residues are unambiguously assigned in both cases.

C-terminal racemisation of a peptide and specific deuteration of the C-terminal residue can be achieved by cyclisation of the peptide to the peptide oxazolone and quenching in 2H20. This specific reactivity of the C-terminal amino-acid residue has formed the basis of a C-terminal analysis of peptides the C-terminal residue is the only one to be racemised in this way and the identity of the C-terminal residue is revealed by analytical methods for determining d l ratios of amino-acid mixtures (Section 4.18.2 Sih and Gu, 1995). 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Alike any other G-protein coupled receptors (GPCRs), mGlu receptors have seven transmembrane helices, also known as the heptahelical domain (Fig. 2). As observed for all GPCRs, the intracellular loops 2 and 3 as well as the C-terminal tail are the key determinants for the interaction with and activation of G-proteins. However, sequence similarity analysis as well as specific structural features make these mGlu receptors different from many other... 

Microheterogeneity below specified level Polyacrylamide gel electrophoresis C- and N-terminal analysis. Amino acid composition... 

Figure 2 Depiction of the active ( open ) and inactive ( closed ) conformations of Src kinase based on the analysis of x-ray structures of c-Src tyrosine kinase crystallized in its inactive state [7]. The stabilization of the inactive conformation is influenced by multiple events including intramolecular binding of the tyrosine-phosphorylated C-terminus tail to the SH2 domain as well as interactions between the SH3 domain and the SH2-kinase linker. CT, C-terminal NT, N-terminal. 

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