Fluorescence polarization of Limulus hemocyanin

Abstract

The polarization of the native fluorescence of Limulus hemocyanin (Hc) as a function of pH (4–10) has been measured. At pH 7, the polarization decreases in the order oxyHc, deoxyHc, stripped Hc, apoHc. The observed changes in polarization are explained in terms of conformational changes and the association-dissociation behavior of the 48-mer (60S), 24-mer (37S), and 12-mer (24S) aggregates and the monomer (5S) subunits. For oxyHc (pH 5.0–7.5), polarization as function of pH is reversible and independent of time, whereas at higher pH (7.8–8.4) the polarization decreases as a function of time. The polarization-time plots show a first rapid fall in the polarization followed by a phase of much slower change, with the rate of initial depolarization increasing with increasing pH. At pH 8.4, the depolarization is ascribed to dissociation from the 48-mer and 24-mer to the monomer species. In the pH range 4.5–6.5, the reaction scheme is proposed as follows: deprotonation of P 12(H +) 2i to form P 12H +, aggregation of two P 12H + into P 24(H +) 2, deprotonation of two amino acid residues of P 24(H +) 2 to form P 24, and aggregation of two P 24 into P 48 (60S).