Met-containing peptides

Sulfur-bound L-Met, as opposed to S,N-chelated L-Met, is more reactive as a ligand on Pt(II) and can be slowly replaced by N7 of G (95, 96). Transfer of Pt onto DNA via Met-containing peptides or proteins may therefore be possible. Monofunctional adducts of the type [Pt(en)(G)(L-Met-S)] appear to be very stable (97) and so methionine may play a role in trapping these adducts. Also, the high trans influence of S as a Pt(II) ligand can lead to the facile labilization of trans-am(m)ine ligands and this allows cisplatin to react with GMP faster in the presence of L-Met then in its absence (98), which introduces another route to DNA platination. 

The H2/Pd hydrogenolysis system for the removal of the Z group from peptides is limited to sulfur-free peptides. In order to overcome this limitation, Meienhofer successfully conducted hydrogenolysis in liquid ammonia as a solvent to remove the Z group from Cys- or Met-containing peptides.As shown in Scheme 23, protected somatostatin was prepared in a stepwise manner starting from H-Cys(tBu)-OtBu. 

Figure 2. Tryptic jj tide maps of native KGF (A) and the individual oxidized KGF species (B-F) from Figure 1. Met containing peptides were labelled gl, hi, il, jl, kl, and 11, while the peptides containing the free sulfhydryl Cys 40 were labelled ml and nl. Oxidized forms of the same peptides were labelled g2, h2, i2, j2, k2,12, m2, and n2, respectively. Peptide i2 coelutes with the peptide T-V-A-V-G-l-V-A-I-K, while peptide 12 co-elutes with the peptide E-L-l-L-E-N-H-Y-N-T-Y-A-S-A-K.

In another study, related to the E. coli K12 proteome, by another group [37], the tryptic digest was first fractionated in 40 fractions by RPLC. Next, Met-containing peptides in these fractions were oxidized by HjOj. These modified proteins were separated from the bulk unmodified proteins by RPLC and fractionated in 8 fractions. These fractions were subsequently analysed by nano-LC-MS-MS on a Q-TOF instrument operated under DDA. After the first RPLC run, an exclusion hst was generated from peptides identified in a MASCOT search of the data, and the sample was then remn. Following this procedure, 754 proteins were identified, which is about 34% of the expressed E. coli K12 proteome. 

K. Gevaert, J. Van Damme, M. Goethals, G.R. Thomas, B. Hoorelbeke, H. Demol, L. Martens, M. Puype, A. Staes, J. Vandekerckhove, Chromatographic isolation of Met-containing peptides for gel-free proteome analysis Identif cation of more than 800 Escherichia coliproteins. Mol Cell Proteomics 1 (2002) 896. 

Oxidation. Can occur during all manipulations involving Met-containing peptides... 

Following the same principle, Severin has developed assays for His- and Met-containing peptides in water at neutral pH, for concentrations of analytes as low as 0.3 iM. The organometallic host was a Cp Rh(III) complex (52) combined with the indicator azophloxine. With the same Rh... 

FIGURE 5. Assay for Met(0)-peptide reductase. The assay for Met(OJ-peptide reductase is carried out in a two-step incubation. The first incubation mixture contains Met(OJ-L12, DTT and the Met(OJ-peptide reductase. At the end of this incubation, purified L12 transacetylase and [3H]acetylCoA are added and the amount of radioactivity incorporated into L7 is determined by its ability to be retained on a nitrocellulose filter. The latter represents the amount of Met(0)-L12 reduced since the oxidized protein is not a substrate for the transacetylase. 

FIGURE 6. Gel electrophoresis of purified E. coli Met(0)-peptide reductase. (A) 15% polyacrylamide containing 0.1% NaDodSO. (B) 13% polyacrylamide (pH8.8) nondenaturing. Reproduced by permission of Academic Press from Brot and coworkers112. 

The incubation mixture is described in the legend to Table 5 and contained partially purified E. coli Met(0)-peptide reductase and 10.2 pmol oxidized a-1-PI. The inhibition ofelastase, due to reactivation of oxidized a-l-PI, was assayed after a 60-min incubation. 

FIGURE 8. The reduction of fMet(O)-Leu-Phe by a human neutrophil and purified E. coli Met(O) peptide reductase. Each assay contained in a final volume of 30 /j1 25 mM Tris-HCl (pH 7.4), lOmM MgCl, 15 mm dithiothreitol 540pmole fMet(O)-[ H]Phe-Leu and Met(0)-peptide reductase. After incubating at 37 °C for 60 min, the incubation mixture is acidified and extracted with ethyl acetate. After centrifugation, an aliquot of the organic phase is removed and assayed for radioactivity. Reproduced by permission of Academic Press from Fliss and coworkers ... 

A type I one-electron photo-oxidation of methionine-methionine-containing peptides by triplet carboxybenzophenone in air-saturated aqueous solution has been reported the S+ radical cation that is formed then reacts with the other Met-S to form an S-S three-electron complex which reacts with superoxide radical anion before hydrolysis to Met(=0)-Met(=0) bis-sulfoxide. Alternatively, cyclization of the A-terminal NH2 on to the S can occur to give a three-electron S-N complex which can react with superoxide radical anion to give a cyclic sulfonium intermediate. 

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