The interaction of collagen with the hydrophobic fluorescent probe-2-p-toluidinylnaphthalene-6-sulfonate.

Three kinds of fluorescence enhancement result from the interaction of 2-p-toluidinylnaphthalene-6-sulfonate and calf-skin collagen. They are negatively cooperative, independent, and highly cooperative fluorescence enhancement. In the independent region at pH 3.7, the binding number is about 36 moles of 2-p-toluidinylnaphthalene-6-sulfonate per mole of tropocollagen with a binding constant of 2.0 × 10⁴ M^?1; with ΔG = ?5.7 kcal/mole, ΔH = ?4.0 kcal/mole, and ΔS = 6 e.u. The pH dependence of fluorescence of native collagen shows that the deprotonated forms of the β and γ carboxyl groups of aspartic and glutamic acid decrease the intensity, possibly by charge repulsion of the negatively charged sulfonate group of 2-p-toluidinylnaphthalene-6-sulfonate. The positive charge of lysine is found to be unimportant in the interaction of 2-p-toluidinylnaphthalene-6-sulfonate with collagen. Fluorescence enhancement is caused mainly by the hydrophobic interactions of 2-p-toluidinylnaphthalene-6-sulfonate and collagen. Salt bridge formation between basic and acidic side chains in very low salt concentration may be detectable by 2-p-toluidinylnaphthalene-6-sulfonate fluorescence.