Fluctuation of the lysozyme structure

Abstract

The kinetics of hydrogen-deuterium exchange in hen egg-white lysozyme (muramidase) has been followed in aqueous solutions of various pH values and in solutions with various concentrations of lithium chloride, by an infrared absorption measurement. It was found that, in every case, 34% of the total peptide hydrogen atoms exchange relatively slowly with a rate of a first-order reaction. This amount corresponds to 44 peptide groups per molecule, and this is equal to the number of peptide NH-groups which are found to be involved in hydrogen bonds with the carbonyls of other peptide groups in the lysozyme molecule in the crystalline state. Each rate constant determined is in good agreement with the value expected from two simple assumptions. (1) The scheme of the isotope exchange reaction is N ⇌ D → D ∗ (⇌ N ∗ ), where N is the native form of the molecule, D a denatured (unfolded) form, and ∗ indicates the deuterated products. (2) The N ⇌ D fluctuation rate is much higher than the rate of the isotope exchange reaction D → D ∗ . It has been shown that the N ⇌ D transition postulated here is the same as that which can be followed by circular dichroism measurement and by some other physical measurements. The effect of lithium chloride on the exchange reaction rate is solely attributable to the change in the N ⇌ D equilibrium caused by the salt, whereas the effect of pH (in the 5 to 8 range) is wholly ascribed to the catalytic action of the OH − anion on the D → D ∗ reaction rate. From the deuterium exchange rate observed, an effective value of the mole fraction of the D form is estimated to be 3 × 10 −6in the solution with no lithium chloride at 20 °C and of pH = 5 to 8.