[New derivatives of azobenzene for the directed modification of proteins].

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

A comparative study of functional properties of calf chymosin and its recombinant forms.

The action of calf chymosin obtained from transgenic sheep milk and the recombinant protein expressed in yeast Kluyveromyces lactis (Maxiren) on fluorogenic peptide substrates, namely Abz-A-A-F-F-A-A-Ded, Abz-A-A-F-F-A-A-pNA, Abz-A-F-F-A-A-Ded, Abz-A-A-F-F-A-Ded, Abz-A-A-F-F-Ded, Abz-A-A-F-F-pNA, and heptapeptide L-S-F-M-A-I-P-NH2, a fragment of kappa-casein (the native chymosin substrate), was investigated. It has been established that transgenic chymosin and recombinant chymosin (Maxiren) differ from the native enzyme in their action on low molecular weight substrates, whereas there was no difference in enzymatic action on protein substrates. Pepstatin, a specific inhibitor of aspartic proteinases, inhibits the recombinant chymosin forms less efficiently than the native enzyme.

Activity and stability of native and modified subtilisins in various media.

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.

Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions.

Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly.