Specific Isolation of Biologically-Active Peptides by Means of Immobilized Anhydrotrypsin and Anhydrochymotrypsin

Abstract

Anbydrotrypsin, a derivative of bovine trypsin, immobilized on Sepharose tightly adsorbs various peptides containing L-arginine at the carboxyl termini, such as bradykinin and tuftsin. These peptides correspond to the specific products of the action of trypsin-like enzymes. Native trypsin immobilized on Sepharose does not show such strong affinity. Fragment 2, a peptide with 41 amino acid residues, which has been released together with bradykinin from bovine high-molecular-weight kininogen by the action of plasma kallikrein, is also adsorbed on the immobilized anhydrotrypsin. When only the carboxyl-terminal arginine is removed with carboxy-peptidase B, however, the peptide loses its adsorptive ability. Immobilized anhydrochymotrypsin, on the other hand, exerts specific affinity for the peptides which correspond to the products of chymotrypsin. These results suggest that the anhydro-derivatives of serine-pr0 teases in general may be of great use in the affinity chromatography of respective series of various naturally occurring peptides.

Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom‐up shotgun proteomics strategy

“…Ishii et al [18] confirmed the loss of affinity to AHT after CPB treatment of a peptide sample containing C-terminal arginine [17]. In our work, a soluble form of CPY at a concentration of 0.5 mg/50 lL was used to treat the neurotensin trypsin digest.…”

“…Method described in Section 2.2 has been used for AHT preparation with a recovery of around 80%, and results in the presence of residual unmodified active trypsin, which should be eliminated before the main bioaffinity carrier preparation. Yokosawa and Ishii (1976) introduced a method of carrier preparation for the affinity purification of AHT, based on the immobilization of tryptic peptides of Protamine sulfate (a protein isolated from Salmon) with an affinity for active trypsin onto a convenient support [18]. In our work, the affinity carrier was prepared by utilizing macroporous bead cellulose Perloza MT 500 after sodium periodate activation (see Section 2.3) and used as stationary phase (ST-Perlose) for low pressure LC in a BioLogic LP system (BioRad, USA) (Fig.…”

“…AHT has no affinity for free amino acids and/or peptides with Arg, Lys, and AECys presented elsewhere in the peptide molecule than at the C-terminus [22]. According to the literature the affinity ligand AHT, immobilized either on sepharose (AT-Sepharose) or agarose, was successfully used to trap peptides that corresponded to the products generated by the action of various trypsin-like proteases [16,18], biologically active peptides [21,23], trypsin inhibitors [24,25], or for recombinant proteins with low biological activity where commonly used affinity chromatography is not convenient [19].…”

Anhydroelastase: Enhanced affinity toward product‐type ligands revealed by affinity chromatography

“…Stronger affinity was observed at slightly acidic pH (e.g., K i = 2.4 mM at pH 5 and K, = 9.5 mM at pH 8.2). Such pH dependence has also been observed with other serine proteases such as trypsin (Kasai and Ishii, 1978) and chymotrypsin (Johnson and Knowles, 1966;Ishii et al, 1979). Therefore, the affinity chromatography of product-type peptides was mainly carried out at pH 5, as was the case of anhydrotrypsin-Sepharose (Kumazaki et al, 1986).…”

“…These data coincide with those previously reported on anhydrotrypsin and anhydrochymotrypsin (Ako et al, 1974;Yokosawa and Ishii, 1977). Ishii et al demonstrated that trypsin and chymotrypsin have remarkably enhanced affinities toward Bz-Arg-OH (Yokosawa and Ishii, 1977) and Ac-Trp-OH (Ishii et al, 1979), respectively, on dehydration at serine residues in their active sites. Since then this affinity enhancement in these serine proteases has been generally observed toward product-type ligands that correspond to the peptides formed as products of the respective enzyme reaction (Kumazaki et al, 1986(Kumazaki et al, , 1987(Kumazaki et al, , 1988.…”