P1 contrast analysis in the presence of iohexol. Square roots of the forward-scattered intensities by P1, I(0)1/2, measured at various iohexol concentrations are indicated by colored circles and are identical to those shown in the inset in Fig. 3(a). The experimental CMP is extrapolated from a phenomenological quadratic fit and is below that expected for P1 from its sequence (shaded gray area). I(0)1/2 does not display the linear behavior expected for an isolated protein (continuous gray line). The slope at the origin (linear fit through red and orange data points, broken black line) would yield an `apparent' contrast-match point (CMP*) well above that expected from the P1 sequence. We explain the complex behavior of I(0)1/2 by two concomitant phenomena: (1) specific binding of 2–3 iohexol molecules to the P1 surface at low concentrations (and reaching saturation at intermediate concentrations) and (2) global exclusion of iohexol molecules from a hydration layer/shell around the P1 surface, in particular at higher concentrations.