Journal: bioRxiv

Article Title: Serpin-Driven Green Camouflage and NIR Fluorescence in Frogs

doi: 10.64898/2026.02.11.704363

Figure Lengend Snippet: (A) Sequence alignment of RCL sequences of known neutrophil elastase inhibitors and TpBBS. The scissile bond of TpBBS is shifted one amino acid relative to the other canonical inhibitors (red arrow). (B) Cleavage of rTpBBS by (human) neutrophil elastase monitored with western blotting using anti-his antibody. The cleaved rTpBBS (black arrow) exhibited lower molecular weight than the uncleaved protein (red arrow). (C) Normalized MALDI-TOF of the relaxed rTpBBS (blue) compared to untreated rTpBBS (black). The peak at ∼4.1 kDa confirms the cleavage of rTpBBS at the scissile bond, in the same position as the endogenous TpBBS. Inset, shows the cleaved protein (43kDa) as analyzed by MALDI-TOF. (D) Absorbance spectra of relaxed rTpBBS bound to biliverdin (blue) normalized to the Soret band of free biliverdin at the same concentration, and fluorescence (dotted lines, excitation 390 nm, emission 650 nm-850 nm), endogenous TpBBS (black), and free biliverdin (red). (E) Ligand binding curves (%bound vs concentration) of relaxed rTpBBS after 4 h of equilibration. A fixed concentration of biliverdin (15 nM) was titrated with increasing concentrations of recombinant apoprotein (0.02 nM-30 nM). Apparent K d of 3.5 nM for the relaxed rTpBBS.

Article Snippet: The relaxed TpBBS was prepared by incubating the recombinant TpBBS with human neutrophil elastase (Innovative Research, Inc.) at 1:3 ratio at 25 °C for 6 h in 1X PBS.

Techniques: Sequencing, Western Blot, Molecular Weight, Concentration Assay, Fluorescence, Ligand Binding Assay, Recombinant