2.3. Intrinsic Tryptophan Fluorescence (ITF)

Changes in fibrinogen structure induced by the RSNOs, SNAP and NAP as well as the non-RSNO, SNP, were determined using an assay that measured the fluorescence intensity of intrinsic tyrptophan in fibrinogen which fluoresces around 350 nm ^87–92. This assay has been described previously ²¹. Briefly, ITF spectra of fibrinogen/RSNO solutions in HBSS were measured with the use of the Cytation 3 microplate reader in fluorescence mode. The samples in a 96-well plate were excited at 290 nm. The emission spectrum between 300 and 460 nm was monitored at each concentration of RSNO (1–1000 μM). To determine the fluorescence quenching of the internal tryptophan moieties in fibrinogen, Stern-Volmer plots were used to analyze the quenching effects of the RSNOs on fibrinogen. The Stern-Volmer equation was used to describe the fluorescence quenching ⁸⁹,

Where I_o and I represent the fluorescence intensity in the absence and presence of each RSNO concentration, respectively, and K_SV is the Stern-Volmer constant. As changes in the Stern-Volmer relationship occur then it is hypothesized that changes in the tertiary conformational state of fibrinogen is occurring.

To determine the effects of 100 or 1000 ppm NO gas bubbled or infused through silicone tubing on the ITF, the fibrinogen solutions were incubated for 1 h at 37°C. The measured ITF between 300 and 460 nm was monitored as described above.