Figure 3
From: Enzymatization of mouse monoclonal antibodies to the corresponding catalytic antibodies
FRET-HA peptide and cleavage reaction. (A) The chemical structure of the FRET-HA peptide. The peptide GLFGAIAGFIE (aa 372–382; part of the fusion peptide) was added before GMVDGWYG (aa 387–394; immunogen peptide) owing to GMVDGWYG being hydrophobic. The fluorescent reagent 7-MCA and quenching reagent DNP were bound to Lys. These reagents were introduced into the N- or C-terminus of the GLFGAIAGFIE-GMVDGWYG peptide. Additionally, six D-arginine residues (D-Arg)6 [rrrrrr] were added to enhance peptide hydrophilicity. (B) Time course of the cleavage reaction of InfA-3L/wt and InfA-3L/P95(−). FRET-HA peptide, 25 μM; InfA-3L/wt (open circle), 5 μM; InfA-3L/P95(−) (closed circle), 5 μM. InfA-3L/wt did not exhibit any catalytic activity for cleaving the FRET-HA peptide. Contrastingly, InfA-3L/P95(−) clearly decomposed the FRET-HA peptide in a time-dependent manner. (C) Cleaved peptide bond. The reaction products obtained in the above experiment were analyzed using HPLC and MS. Some peaks were observed, as shown in the chromatogram. Two of these peaks were detected. The first was obtained from E-GMVDGWYGK(DNP)rrrrrr-NH2 and the other was H–K(7-MCA)-GLFG. InfA-3L/P95(−) cleaved the peptide bond between G–A and I–E. (D) The time course of the cleavage reaction of InfA-6L/wt and InfA-3L/P95(−). FRET-HA peptide, 25 μM; InfA-6L/wt (open circle), 5 μM; InfA-6L/P95(−) (closed circle), 5 μM. InfA-6L/wt did not exhibit any catalytic activity for cleaving the FRET-HA peptide. Contrastingly, InfA-6L/P95(−) clearly decomposed the FRET-HA peptide in a time-dependent manner. (E) The time course of the cleavage reaction of InfA-9L/wt and InfA-9L/P95(−). FRET-HA peptide, 25 μM; InfA-9L/wt (open circle), 5 μM; InfA-9L/P95(−) (closed circle), 5 μM. InfA-9L/wt did not exhibit any catalytic activity for cleaving the FRET-HA peptide. Contrastingly, InfA-9L/P95(−) clearly decomposed the FRET-HA peptide in a time-dependent manner. (F–H) represent the results of the kinetic analysis under the following conditions: The concentration of mutant such as InfA-3L/P95(−), InfA-6L/P95(−), and InfA-9L/P95(−) was fixed at 5 μM and that of the FRET-HA peptide varied from 5 to 600 μM at 37 °C. [S], FRET-HA peptide; [v], initial rate. (F) InfA-3L/P95(−). The Lineweaver–Burk plot demonstrated that the cleavage reaction by the InfA-3L/P95(−) mutant fits the Michaelis–Menten kinetics equation, indicating that the reaction is enzymatic. (G) InfA-6L/P95(−). InfA-6L/P95(−) exhibited a linear relationship between 1/[v] vs. 1/[S], indicating that degradation by the mutant obeyed the Michaelis–Menten kinetics. (H) InfA-9L/P95(−). The degradation reaction obeyed Michaelis–Menten kinetics, indicating that the degradation was enzymatic.
