The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

Abstract

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

Figure 1

Electrophoretic study of binase. (a) Polyacrylamide gel electrophoresis (PAGE) with 0.1% SDS at nanoconcentrations of the enzyme (M, molecular weight markers). (b) Zymogram of binase at nanoconcentrations. (c) PAGE of binase at microconcentration (10 µg) in the presence of strong denaturing agents: 1 - 1.5 % SDS; 2 - 2 % SDS.

Figure 2

CD spectra of binase in units of molar (on a residue) ellipticity (a) and relative content of secondary structure elements of binase (b) for enzyme concentrations of 0.05 mg/ml (dark) and 1 mg/ml (light symbols), pH 7.

Figure 3

Changes in 216 nm absorbance of binase solution at pH 6.1 (light symbols) and pH 3 (dark symbols) with respect to protein concentration.

Figure 4

The model of binase dimer [8] and experimental hydrodynamic size (R = 2.1 nm) of protein determined by means of NMR techniques.