(6) Assay Card

General Information
Summary Kalata B1 and B2 and acyclic permutants were found to be resistant to chemical denaturation in the oxidised state but not when reduced.
Condition Fluorescence emission spectra were measured between 280 and 500 nm on a Perkin-Elmer luminescence spectrometer using an excitation wavelength of 280 nm and quartz cuvettes with an optical path length of 4 mm. The excitation and emission slit widths were set to 4 nm with a photomultiplier voltage of 650 V. Experiments were performed at 25 C with the temperature maintained using a Grant LTD6 temperature control unit. All peptide emission spectra were corrected by background subtraction of the spectrum from the equivalent buffer at the given GdHCl concentration and were the average of three scans. Using mellitin as a positive control and kB1 as a test compound, the optimum sample concentration for fluorescence measurement was determined. Solutions containing 25 uM peptide and DTT were prepared and incubated at 37 C for 30 min; GdHCl was then added and the mixture incubated at room temperature prior to analysis. The final concentration of DTT in the solutions was 5 mM, and the GdHCl concentration ranged between 0 and 6 M.
Result The fluorescence spectrum of kB1 alone was low in intensity, with a maximum emission wavelength (max) of 335 nm. Upon addition of 6 M GdHCl, there was no significant change in either the fluorescence intensity or emission wavelength, although the former decreased slightly. The addition of DTT to kB1 followed by incubation at 37 C for 30 min resulted in a large and significant increase in the fluorescence intensity (~5-fold), and the wavelength of maximum emission was red-shifted to ~345 nm. Addition of GdHCl to a solution of the DTT-reduced peptide caused a further increase in the intensity, but no change in the emission maximum. Kalata B2 behaved in a manner similar to that of kB1. Upon reduction, an increase in fluorescence intensity was observed, but the increase was ~2-fold compared to an increase of 5-fold for kB1. Unlike the case for kB1, the subsequent addition of GdHCl caused no further change in the fluorescence spectrum. The acyclic permutant des(24-28)kB1 and the two-disulfide analogue in solutions of phosphate buffer, 6 M GdHCl, 5 mM DTT, or both the reducing agent and denaturant. The two species gave similar low-intensity spectra as seen for native kB1, with maximum fluorescence emission occurring at ~335 nm. Upon addition of DTT, large increases in fluorescence intensity were observed, ca. 5-fold for the acyclic permutant and ca. 6-fold for the two-disulfide permutant, and red shifts in the maximum fluorescence to 341 and 346 nm, respectively, were observed. This is consistent with the 5-fold intensity increase and 6 nm red shift observed for kB1 upon reduction. Incubation of des(24-28)kB1 with GdHCl resulted in no significant changes to the fluorescence spectrum, as was also the case for native kB1, and the max did not change significantly. Incubation of the acyclic permutant with both GdHCl and DTT resulted in an intensity increased compared to that of the reduced species, without a discernible change in the wavelength of maximum fluorescence (344 nm). The two-disulfide mutant was the only species to show an increased fluorescence intensity upon addition of 6 M GdHCl to the oxidized form (3-fold increase). The wavelength of maximum emission was red-shifted (6 nm shift) compared to the spectrum in buffer alone. Addition of 6 M GdHCl to the reduced [A1,15] mutant caused an increase in fluorescence intensity comparable to those observed for both reduced kB1 and reduced des(24-28)kB1.
AssayType Chemical stability

References
Colgrave ML, Craik DJ (2004) Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 43:5965-75

Cross-references
Proteins Assayed kalata b1-5
kalata B1
des(24-28)kB1
[Ala1,15]kB1