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Pressure acceleration of proteolysis: A general mechanism

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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.

No MeSH data available.


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 λmax at 332.2 nm gradually shifts to red with increasing pressure, finally to 350.5 nm at 3.8 kbar.
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f2-4_29: 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 λmax at 332.2 nm gradually shifts to red with increasing pressure, finally to 350.5 nm at 3.8 kbar.

Mentions: As a convenient measure of stability of the native conformation of α-chymotrypsin against pressure, we monitored its Trp fluorescence as a function of pressure in 10 mM Tris-HCl buffer (pH 7.0) at 37°C (Fig. 2). The maximum emission wavelength (λmax) was initially at 332.2 nm, indicating that Trp residues are buried in the folded conformation of α-chymotrypsin. It gradually increased with increasing pressure, but stayed within 332∼336 nm below 3 kbar, which strongly suggests that the original native Trp environment is largely unchanged. Finally at 3.8 kbar, λmax shifts to 350.5 nm, indicating that at this pressure the protein is largely denatured. The above results strongly suggest that, under the present experimental condition for the proteolytic reaction under pressure, the native environment of α-chymotrypsin is largely retained at least below 3 kbar and therefore that its catalytic activity would be retained, if not at the same level as that at 1 bar.


Pressure acceleration of proteolysis: A general mechanism
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 λmax at 332.2 nm gradually shifts to red with increasing pressure, finally to 350.5 nm at 3.8 kbar.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036607&req=5

f2-4_29: 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 λmax at 332.2 nm gradually shifts to red with increasing pressure, finally to 350.5 nm at 3.8 kbar.
Mentions: As a convenient measure of stability of the native conformation of α-chymotrypsin against pressure, we monitored its Trp fluorescence as a function of pressure in 10 mM Tris-HCl buffer (pH 7.0) at 37°C (Fig. 2). The maximum emission wavelength (λmax) was initially at 332.2 nm, indicating that Trp residues are buried in the folded conformation of α-chymotrypsin. It gradually increased with increasing pressure, but stayed within 332∼336 nm below 3 kbar, which strongly suggests that the original native Trp environment is largely unchanged. Finally at 3.8 kbar, λmax shifts to 350.5 nm, indicating that at this pressure the protein is largely denatured. The above results strongly suggest that, under the present experimental condition for the proteolytic reaction under pressure, the native environment of α-chymotrypsin is largely retained at least below 3 kbar and therefore that its catalytic activity would be retained, if not at the same level as that at 1 bar.

View Article: PubMed Central - PubMed

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.

No MeSH data available.