Cryostructuring of polymer systems. Proteinaceous wide-pore cryogels generated by the action of denaturant/reductant mixtures on bovine serum albumin in moderately frozen aqueous media.

Freeze-thaw processing of bovine serum albumin (BSA) aqueous solutions, which contain also the additives of denaturants (urea in this case) and thiol-bearing reductants [cysteine (Cys) in this case] leads to the formation of wide-pore cryogels. The properties and porous morphology of these spongy gel matrices were demonstrated to depend on the initial concentration of all precursors and on the freezing/frozen storage temperature. The optimum conditions for preparing such BSA-based cryogels were found to be as follows: [BSA] = 3-5 g dL(-1), [urea] = 0.5-2.0 mol L(-1), [Cys] = 0.01 mol L(-1), and freezing temperatures in the range of -15 to -20 °C. The size of gross pores in thus prepared cryogels is ∼50-150 µm. The spatial network of BSA-cryogels was shown to be cross-linked chemically via interchain disulfide bridges. The significant role of hydrophobic interactions in the stabilization of 3D networks of these cryogels is inferred, as well as the supposition about the relay-race sequence mechanism of the intermolecular disulfide cross-link formation is made.

Cloning, sequencing, expression, and characterization of thermostability of oligopeptidase B from Serratia proteamaculans, a novel psychrophilic protease.

Protease from Serratia proteamaculans (PSP) is the first known psychrophilic oligopeptidase B. The gene of S. proteamaculans 94 oligopeptidase B was cloned, sequenced and expressed in Escherichia coli. The unfolding of PSP molecule following heat treatment at 37°C by measuring fluorescence spectra was examined in parallel with the residual activity determination. The effect of PSP thermostabilization by glycerol at 37-50 °Ð¡ was revealed. Calcium ions and buffer solution of low molarity cause the opposite effect - the acceleration of PSP inactivation at 37°C. The thermal stability of PSP molecule in the presence of 0-100mM CaCl2 was also investigated by means of high-sensitivity differential scanning calorimetry. The artificial reconstruction of the natural complex PSP-chaperonin from S. рroteamaculans was carried out: the stable complex (1:1) of chaperonin E. сoli GroEL with active recombinant enzyme PSP was obtained. It was shown that complex formation with chaperonin promotes PSP thermostability at 37°C.

Biomembrane sensitivity to structural changes in bound polymers.

Anionic liposomes containing a 4:1 molar ratio of neutral to anionic phospholipids were treated with an excess of five zwitterionic polymers differing only in the spacer length separating their cationic and anionic moieties. Although the polymers do not disrupt the structural integrity of the liposomes, they can induce spacer-dependent molecular rearrangements within the liposomes. Thus, the following were observed: spacer length = 1, no binding to the liposomes; spacer length = 2, adsorption to the liposomes, but no molecular rearrangement; spacer length = 3, lateral lipid segregation but little or no flip-flop; spacer length = 4 or 5, lateral lipid segregation and flip-flop. These diverse behaviors are relevant to the use of biomedical formulations where polyelectrolytes play a role.