Terminal Residue and Sequence

The presence of C-terminal arginine in several protamines was confirmed by means of modern techniques using DFP-treated carboxypeptidase B, hydrazinolysis, and thiohydantoin procedures [Ando et al. 1958 (1) Tobita et aL 1968 Akabori et al. 1952, 1956 Kawanishi et aL 1964 Tristram, 1947, 1949]. These results are also shown in Table VI-1. 

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The use of the method of dinitrophenylation (DNP) (Sanger, 1945) enabled Porter and Sanger (1948) to confirm the presence of proline at the N-terminus of salmine. Since then, many workers have studied N-terminal residues and sequences... 

Ando, T., Abukumagawa, E., Nagai, Y., Yamasaki, M. (2) On the N-terminal residues and sequence of clupeine from Clupea pallasii The occurrence of proline in addition to alanine as the N-terminus. J. Biochem. (Tokyo) 44, 191—194 (1957). 

If the phenylisothiocyanate method is used, the cyclization and release of the N-terminal derivative occurs under mild conditions that leave the rest of the chain intact. It is therefore possible to take the protein chain, now without its original N-terminal residue, and repeat the procedure to determine the second residue in the sequence, and so on. Unfortunately, at each step, there is a finite chance of additional peptide hydrolysis or incomplete reaction, and uncertainty tends to accumulate after 10 to 20 cycles. 

Better results are obtained for chain C, which corresponds to the chymotrypsinogen COOH-terminal sequence, when aqueous extracts of oxidized a-chymotrypsin (A4) are chromatographed on Sephadex G-50 equilibrated with 0.05 M HCl (79). Figure 8 shows that three well-separated peaks emerge. The first one is an ill-defined mixture containing chains B and C. The second is chain C in an apparently pure form. The third is the small chain A which does not absorb at 280 m/t. Chromatographically purified chain C can be obtained in an over-all yield of 30%. It contains alanine as the single NH2-terminal residue and all amino acids except histidine and phenylalanine. 

The dry membrane is inserted into a C-terminal sequencer column (inert Kel-F columns fitted with inert endfiits) and installed in any one of the four sample positions on the HP G1009A sequencer. The sequencer column reactions that occur on the Zitex membrane include the chemical coupling and cyclization of the C-terminal residue and the cleavage and extraction that releases the thiohydantoin-amino acid derivative. The thiohydantoin-amino acids are extracted off the Zitex... 

Mouse immimoglobulin G (ISOkDa) was applied as a phosphate buffer solution directly on a Zitex reaction membrane and subjected to C-terminal sequence analysis (Figure 2). The C-terminal cycle-1 identified the extent of protein processing at the C-terminus of the heavy chain by the detection and relative recoveries of the heavy chain Lys (K, expected full-length sequence C-terminal residue) and Gly (G) residues. The C-terminal residue of the light chain was identified as the expected Cys (C) residue. 

A series of amino acids joined by peptide bonds form a polypeptide chain, and each amino acid unit in a polypeptide is called a residue. A polypeptide chain has polarity because its ends are different, with an a -amino group at one end and an a -carboxyl group at the other. By convention, the amino end is taken to be the beginning of a polypeptide chain, and so the sequence of amino acids in a polypeptide chain is written starting with the aminoterminal residue. Thus, in the pentapeptide Tyr-Gly-Gly-Phe-Leu (YGGFL), phenylalanine is the amino-terminal (N-terminal) residue and leucine is the carboxyl-terminal (C-terminal) residue (Figure 3.19). Leu-Phe-Gly-Gly-Tyr (LFGGY) is a different pentapeptide, with different chemical properties. 

It is many years since Schlack and Kumpf showed that a simple A-acyl peptide treated with ammonium thiocyanate and acetic anhydride (Scheme 5.7) underwent cyclisation at the C-terminus to yield l-acyl-2-thiohydantoins (5.29). Mild alkaline hydrolysis then yielded the 2-thiohydantoin (5.30) corresponding to the C-terminal terminal residue and an A-acylpeptide containing one amino acid fewer. This reaction sequence should lead to a cyclic procedure at the C-terminus analogous to the Edman procedure at the A-terminus. Despite several attempts to avoid side reac-... 

The synthesis of a dipeptide, NH3 CHR CONI ICIIICCOO, from the constituent amino acids involves forming the peptide bond so that the amino-acid sequence is correct and enantiomerisation (Section 7.7) at the chiral a-carbon atoms is avoided. The latter point does not arise, of course, with glycine. In order to produce the correct sequence and to prevent the formation of a mixture of higher peptides, the amino group of the intended TV-terminal residue and the carboxy group of the intended C-terminal residue are normally protected. 

Fig. 1. Amino acid sequence of GMF-beta. The sequence was established for bovine brain GMF-beta by automated Edman degradation (microsequencing) and tandem mass spectrometry. Identical sequence was obtained for recombinant hGMF-beta, which was deduced from nucleotide sequence of the cDNA and verified by microsequencing of the first ten NH2-terminal residues and by carboxylpeptidase de adation of the first four COOH-terminal residues. The one-letter abbreviations for the amino acids are A, Ala C, Cys D, Asp E, Glu F, Phe G, Gly H, His I, He K, Lys L, Leu M, Met N, Asn P, Pro Q, Gin R, Arg S, oer T, Thr V, Val W, Trp and Y, Tyr. The three cysteine residues (at positions 7, 86 and 95) are underlined. (Adapted from ref. 13)...

Finally, synthesis of oligonucleotides by degradation Homologues of oligouridylate have been prepared by successive digestion of RNA with RNase Ti and RNase U2, followed by deamination, to afford oligouridylate sequences with 3 -terminal purine nucleotides, which were subjected to enzymic dephosphorylation, periodate oxidation and P-elimination of the terminal residue, and a second dephosphorylation step.325 xhis afforded (Up)nU species (n = 1 - 7), which were separated chromatographically. 

Specific enzymes, cofactors, and other components for removai of initiating residues and signal sequences, additional proteolytic processing, modification of terminal residues, and attachment of phosphate, methyl, carboxyl, carbohydrate, or prosthetic groups... 

Extensive studies have been performed to disclose the biosynthetic pathway of microcystin and their lower mass analogs, the nodularins (53,85). One of the major questions was the origin of the Adda residue. The methyl substimtion pattern was indicative of incorporation of either propionate or acetate followed by methylation via S-adenosylmethionine. Although both propionate and methionine were found to be incorporated, the pattern of labelled metabolites was clearly indicative of an acetate-plus-methionine sequence for Cl through C8. The remainder of Adda presumably derives from phenylalanine via phenylacetic acid. The other subunits are for the most part derived from predictable pathways. According to the biosynthetic intermediates isolated the assembly of the linear penta- and octapeptides occurs with the Adda unit as N-terminal residue and Arg as C-terminus. Cyclization apparently represents the last step, since conversion of N-methyl-serine and -threonine to Mdha and Mdhb, respectively, occurs in earlier steps. 

What treatments could you apply to the following hemoglobin fragment to determine the amino-terminal residue and to obtain two sets of peptides with overlaps so that the complete amino acid sequence can be established Give the sequences of the peptides obtained. 

In order to determine the sequence of amino acids in a peptide, initially it was subjected to partial hydrolysis. Thereby fragments were formed, smaller peptides the sequence of which could be more easily established. After micropreparative separation, e.g. by paper chromatography, the fragments were hydrolyzed and analyzed for amino acid composition both before and after treatment with nitrous acid. The amino add missing in the treated, desaminated, sample, was its N-terminal residue. The sequence of the whole peptide was then reconstructed through the appropriate combination of the structurally identified peptide fragments. A typical example, the eluddation of the structure of gramiddin S, is described on p. 205. 

Methods for sequence determination by sequential d radation from the C-terminal end have in neral not been well developed. In an attempt to improve this situation. Louden and co-workers have investigated various chemical methods for determining C-terminal residues and degrading peptides from the C-terminal end. - ... 

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