Specificity of trypsin digestion and conformational flexibility at different sites of unfolded lysozyme

Abstract

Fourteen tryptic peptides and nine intermediates were identified as products of trypsin digestion of reduced and S-3-(trimethylated amino) propylated lysozyme. Kinetics of the appearance and disappearance of these products were observed by monitoring the peak areas on the chromatogram. In spite of the complicated reaction pathways, kinetics of the digestion of proteins and several intermediate products show simple decay curves with a single rate constant. In this paper, the trypsin susceptibility of the individual cleavage site is defined as a hydrolytic rate constant of the susceptible peptide bond in the presence of 10 nM trypsin. The cleavage sites of unfolded lysozyme are classified into two groups in terms of the trypsin susceptibility: one has a high susceptibility (10-20 h-1) and the other a low susceptibility (1.0-2.0 h-1). In the unfolded state of lysozyme, in conclusion, the region from residues 15 to 61 has a strong resistance to trypsin digestion; on the other hand, the C-terminal half of the polypeptide chain is flexible enough to fit into the active site of trypsin. In addition, six kinds of pentapeptides were synthesized as analogues of lysozyme fragments including Arg 14, Arg 21, Lys 33, Arg 45, Arg 61, and Arg 73. Kinetics of tryptic digestion of them were observed. Both kcat and KM were determined for these synthetic pentapeptides. The susceptibility of each cleavage site in pentapeptides is determined and compared with that corresponding in proteins. The susceptibility is usually higher when the susceptible peptide bond is included in proteins than in pentapeptides, so long as the conformation of peptide chain is flexible. However, susceptibilities of a few sites in proteins are lower than those in pentapeptides. This means that the peptide chains tend to fold locally to prevent trypsin from binding to the sites. It was found that the sites of Arg 21 and Arg 45 are indeed resistant to trypsin, but the site of Lys 33 is not so much, although the hydrolytic rate at Lys 33 itself is extremely slow.

Trypsin Sheds Light on the Singular Case of Seminal RNase, a Dimer with Two Quaternary Conformations

“…The latter interpretation appears as the only realistic one, based on the following considerations. (i) The rates measured for MxM and MϭM are 2-3 orders of magnitude lower than those reported in the literature for tryptic hydrolysis of proteins, folded or unfolded (21,22). Furthermore, when a preparation of S protein dissociated from S peptide was treated with trypsin under the same conditions employed for MxM and MϭM, a value of k hydrolysis ϭ 1,240 ϫ 10 Ϫ4 min Ϫ1 was measured.…”

Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis

“…MALDI-TOF MS analysis of region 1 identified the protein as AGP or orsomucoid (Table 1). Due to the preferential action of trypsin [21] and less ionization efficiency of glycopeptides [22], the sequence coverage of some of the proteins were found to be less. In the present study it was observed that potential N-linked glycopeptides were not covered by PMF spectra and this may be reflected in low sequence coverage.…”

Novel Baculovirus DNA Elements Strongly Stimulate Activities of Exogenous and Endogenous Promoters

“…Therefore, the early PHCm promoter is probably stronger than most, if not all, of the currently known early type promoters that can be expressed in recombinant baculoviruses. It has been shown that the unfolding of a protein may result in protease degradation (35,36), and improper protein modifications occur at a late stage of infection (37)(38)(39). These previous observations imply that the production of relatively lower quantity of protein with better activity may be the result of a relatively better folding or modifications on proteins generated by the early type PHCm promoter than those generated by the very late p10 promoter.…”