Pressure acceleration of proteolysis: A general mechanism

Abstract

Remarkable acceleration of enzymatic proteolysis by pressure at kbar range is reported with ubiquitin as substrate and α-chymotrypsin as enzyme. The acceleration is interpreted in terms of the shift of conformational equilibrium in ubiquitin from the non-degradable folded conformer to the enzyme-degradable unfolded conformer by pressure because of the lower volume of the latter, while the enzymatic activity of α-chymotrypsin is still largely retained. This mechanism is considered generally applicable to most globular proteins and the method of pressure-accelerated proteolysis will have an enormous potential utility in systems wherever efficient removal of proteins is needed.

Keywords: pressure-enhanced proteolysis; shift of conformational equilibrium; ubiquitin; unfolded conformer; α-chymotrypsin.

Figure 1.

SDS-PAGE patterns for ubiquitin subjected to enzymatic hydrolysis with α-chymotrypsin at 37°C for 50 min at following pressures. a. 1bar, b. 500 bar, c. 1000 bar, d. 1500 bar, e. 2000 bar, f. 2500 bar, g. 3500 bar. The solution contained ubiquitin (31 μM) and α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer, pH 7.0. Lanes for ubiquitin alone and α-chymotrypsin alone are also shown for comparison.

Figure 2.

Fluorescence spectra of α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer, pH 7.0 at 37°C, measured at various pressures from 1 bar up to 3800 bar. The maximum emission wavelength λ_max at 332.2 nm gradually shifts to red with increasing pressure, finally to 350.5 nm at 3.8 kbar.

Figure 3.

General mechanism for pressure enhancement of enzymatic proteolysis of a globular protein. Pressure shifts the population from N to U according to eq. 1 and U is subsequently degraded by an proteolytic enzyme, e.g. α-chymotrypsin under pressure.