Technique employing the Protein Assay Kit (Bio-Rad, Moscow, Russia) and bovine serum albumin because the regular. Molar concen-Biology 2021, 10,4 oftration of enzyme options was determined by titration on the enzyme active websites with p -guanidinobenzoic acid p-nitrophenyl ester as ��-Carotene Purity & Documentation described in [28]. Buffer exchange was performed utilizing a 30 kDa cutoff centrifugal filter device (Millipore, MA, USA). To identify the oligomeric state of wild-type and modified PSP, the protein ( 2 mg/mL concentration) was applied to a Superdex 200 10/30 GL column (GE Healthcare, Chicago, IL, USA) equilibrated with 20 mM Tris-HCl, pH 8.0 and 200 mM NaCl. 2.3. Enzymatic Study Kinetic parameters of substrate hydrolysis by wild-type and modified PSP variants have been determined as described in [28,29]. Briefly, hydrolysis of N-benzoyl-D,L-arginine-pnitroanilide (BAPNA) (Sigma-Aldrich, St. Louis, MI, USA) and two other p-nitroanilide (pNA) substrates, Z-RR-pNA and Z-KR-pNA (Z = benzyloxycarbonyl) (Bachem AG, Budendorf, Switzerland), was monitored as an increase in the absorption at 405 nm (25 C) due to the formation of free p-nitroaniline (405 = ten.400 M-1 cm-1 ). The initial hydrolysis prices have been determined from the initial linear a part of the kinetic curve (extent of hydrolysis didn’t exceed ten ) by monitoring the increase in the absorbance at 405 nm in 0.1 M Tris-HCl, pH eight.0, 2 DMSO, at 25 C. At the least ten concentration points (in duplicate or triplicate with various concentrations of your enzyme) of every single substrate had been made use of to determine kinetic constants, generally in the array of 0.02.4 mM. The variance of v/[E] values at identical substrate concentrations didn’t exceed 50 . Kinetic parameters (Kcat and Km) had been calculated from the Michaelis enten equation utilizing nonlinear regression. The normal error did not exceed 10 . For evaluation of the impact of spermine around the initial hydrolysis rates, 14 nM of either PSP or PSPmod and 0.1 mM BAPNA have been used. The reactions have been carried out in triplicate for every concentration of spermine. 2.4. Far-UV Circular Dichroism Spectroscopy CD spectra and absorption spectra of wild-type and modified PSP variants were recorded in wavelength range 18020 nm on Chirascan spectrometer (Applied Photophysics, Leatherhead, Surrey, UK) with 1 nm slit width and 1 nm step at 20 C. SharedAccess Equipment Centre “Industrial Biotechnology” of Federal Study Center “Dodecyl gallate In Vitro Fundamentals of Biotechnology” Russian Academy of Sciences supplied the gear. Protein samples (1 mg/mL) were prepared inside a ten mM Na-phosphate buffer, pH 8.0, supplemented with 40 mM NaF. Optical path length was ten mm. Protein concentrations had been verified employing extinction coefficients of peptide bond at 205 nm. All measurements were repeated twice for every sample. two.five. Differential Scanning Calorimetry Protein samples (two mg/mL) were prepared in a 25 mM Na-phosphate buffer, pH 7.83, in duplicate either supplemented or not with 2 mM spermine. The excess heat capacity from the denaturation was measured with DASM-4M differential adiabatic scanning microcalorimeter with 467 capillary cells. The experiment was performed beneath a constant pressure of 2.2 atm at a heating price of 1 K/min. 2.6. Protein Crystallization, Data Collection, Processing, Structure Refinement and Analysis Crystallization of oligopeptidase B from S. proteomaculans with modified hinge region and its E125A and S532A mutants are described in [34,35]. Diffraction data in the crystals had been collected at the Kurchatov sy.
