Inclusion of proteins into polyelectrolyte microparticles by alternative adsorption of polyelectrolytes on protein aggregates.

A new method of protein immobilization into polyelectrolyte microparticles by alternative adsorption of the oppositely charged polyelectrolytes on the aggregates obtained by salting out of protein is proposed. The model protein alpha-chymotrypsin (ChT) was included in the polyelectrolyte microparticles obtained by various number of polyelectrolyte adsorption steps (from 1 to 11). The main parameters of ChT inclusion into microparticles were calculated. Scanning electron and optical microscopy were used for characterization of morphology and determination of particle size which was from 1 to 10 micro m in most cases. It was shown that the size and shape of protein-containing particles and protein aggregates used as a matrix were similar. Change in ChT enzymatic activity during entrapment into polyelectrolyte particles and activity of released protein were studied. The effect of pH on release of incorporated proteins was investigated; it was shown that change in pH and the number of polyelectrolyte adsorption steps allows protein release to be manipulated.

Encapsulation of proteins by layer-by-layer adsorption of polyelectrolytes onto protein aggregates: factors regulating the protein release.

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer-by-layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility.

Effect of hydrophobization of basic pancreatic proteinase inhibitor on the inhibition of bovine trypsin and human leukocyte elastase.

The effect of modification of basic pancreatic trypsin inhibitor (BPTI) by derivatives of fatty acids (oleic, stearic) on the inhibition of bovine trypsin and human leukocyte elastase (HLE) was studied. Kinetic constants of interaction with trypsin and inhibition constants of both enzymes were determined. Hydrophobization of BPTI had virtually no effect on its high affinity for trypsin. The coupling of cis-unsaturated oleoyl radicals to the inhibitor molecule significantly increased the efficiency of HLE inhibition, whereas the coupling of saturated stearoyl radicals completely canceled the anti-elastase activity and in some cases promoted the substrate hydrolysis.

Interaction of human leukocyte elastase with plasma fibronectin and its inhibition by soybean Bowman-Birk protease inhibitor.

Binding of human leukocyte elastase (HLE) to human plasma fibronectin (Fn) was determined by its competition with an oligopeptide chromogenic substrate. Kinetic curves of accumulation of the substrate hydrolysis product in the presence of Fn were specific for slow-binding one-step inhibitors of the enzyme. Values of rate constants for HLE association with Fn and for the dissociation of the complex were, respectively, 2.2.10(3) M(-1).sec(-1) and 1.4.10(-3) sec(-1) at pH 7.5 and 25 degreesC. The dissociation constant of the HLE--Fn complex determined independently by titration of the enzyme with the protein substrate and neglecting the hydrolysis was 3.9.10(-7) M. The resulting values were suggested to describe a high-affinity site in the Fn molecule which was subjected to the primary attack by HLE. The soybean Bowman--Birk protease inhibitor (BBI) efficiently inhibited the HLE-induced degradation of Fn under conditions of both inhibitor preincubation with the protease and its addition into the reaction mixture of the protease with Fn.