Sub-Nanosecond Pulsed LEDs

PLS 265 to 600

Apomyoglobin in buffer

Full length apomyoglobin has 154 amino acids including two tryptophans and three tyrosines. A 5 µM solution of this protein in acetate buffer was excited with the PLS 280, driven by the PDL 800-B driver at 2.5 MHz repetition rate. The FluoTime 200 spectrometer was equipped with Glan-Taylor polarisers in order to select the vertically polarized component of the excitation light and the magic-angle polarized component of the fluorescence. The collected emission passed a monochromator set to 350 nm with 30 nm bandpass. The pulses from PMA-185 detector were processed by the TimeHarp 200 TCSPC board. The measured instrument response function (IRF) has a FWHM of 700 ps and 1.2 million counts were collected within 4 minutes measurement time. The measurement results were analysed using FluoFit - in this case the decay is best described by a triple exponential decay funciton. The picture below shows the IRF (red), the measured decay (blue) and the result of the fit (black) along with the three recovered lifetimes and their fractional intensities.

Further information about the protein dynamics can be obtained by a quick anisotropy decay analysis. Two more histograms were collected, the so called parallel and perpendicular polarized decays. The simplest anisotropy analysis involves a direct calculation of the anisotropy decay curve R(t) from those polarized decays. The necessary G-factor was determined in an independent decay measurement with horizontally polarized excitation.The picture below shows the time evolution of the anisotropy (blue) during the first 20 ns and a fitted single exponential decay curve (black). The time resolved anisotropy reveals an average rotational correlation time of 1.6 ns and a residual anisotropy of 0.05. This residual anisotropy is a clear indication of a slower process, which cannot be resolved using the fluorescence of tyrosine and tryptophane as an indicator. In fact, the expected rotational correlation time of apomyoglobin is in the order of 7-8 ns and this process is most likely the reason for the residual anisotropy.