Structural and functional features of protein-polysaccharide complexes based on cysteine proteases and hydrophilicly modified chitosan

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Abstract

In this work, complexes of cysteine proteases, namely bromelain, papain and ficin, with a graft copolymer of chitosan and poly(N,N-dimethyl amino ethyl methacrylate) were obtained. It was determined that the enzyme catalytic activity in the complexes is reduced compared to their native forms. The results of molecular docking showed that modified polysaccharide located in the catalytic pocket of cysteine proteases globules. The resulting complexes have increased stability when stored under physiological conditions, which makes them promising candidates for use in the development of treatments for wounds.

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About the authors

M. S. Lavlinskaya

Voronezh State University

Email: holyavka@rambler.ru
Russian Federation, Universitetskaya Ploshchad 1, Voronezh, 394018

A. V. Sorokin

Voronezh State University

Email: holyavka@rambler.ru
Russian Federation, Universitetskaya Ploshchad 1, Voronezh, 394018

S. S. Goncharova

Voronezh State University

Email: holyavka@rambler.ru
Russian Federation, Universitetskaya Ploshchad 1, Voronezh, 394018

M. G. Holyavka

Voronezh State University; Sevastopol State University

Author for correspondence.
Email: holyavka@rambler.ru
Russian Federation, Universitetskaya Ploshchad 1, Voronezh, 394018; Universitetskaya ul. 33, Sevastopol, 299053

M. S. Kondratyev

Voronezh State University; Institute of Cell Biophysics of the RAS

Email: holyavka@rambler.ru
Russian Federation, Universitetskaya Ploshchad 1, Voronezh, 394018; Institutskaya ul. 3, Pushchino, 142290

V. G. Artyukhov

Voronezh State University

Email: holyavka@rambler.ru
Russian Federation, Universitetskaya Ploshchad 1, Voronezh, 394018

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Supplementary files

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2. Scheme 1. The process of formation of the target product – a graft copolymer of chitosan and poly-N,N-dimethylaminoethyl methacrylate.

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3. Fig. 1. Protein content (mg/g carrier) in complexes of bromelain, papain and ficin with grafted copolymer of chitosan and poly-N,N-dimethylaminoethyl methacrylate. The yield of complex formation for protein is indicated, expressed as a percentage of the sorbed enzyme from its amount in the solution during the interaction, taken as 100%.

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4. Fig. 2. Total catalytic activity (units/ml of solution) of bromelain, papain and ficin complexes with a grafted copolymer of chitosan and poly-N,N-dimethylaminoethyl methacrylate. The efficiency of enzyme complexation (by total catalytic activity) is also indicated, expressed as a percentage of the retention of the proteolytic activity of the enzyme after immobilization in relation to the enzyme activity in solution, taken as 100%.

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5. Fig. 3. Topology of the complexes of bromelain (a), papain (b), and ficin (c) with a grafted copolymer of chitosan and poly-N,N-dimethylaminoethyl methacrylate calculated by the flexible molecular docking method. A fragment of the grafted copolymer of chitosan and poly-N,N-dimethylaminoethyl methacrylate molecule is shown in pink. The α-helical fragments of the enzyme molecule, which are predominantly part of the L-domain, are shown in turquoise. The β-pleated regions, which predominate in the R-domain, are shown in red. A depression (catalytic pocket) containing the active center of the enzyme is formed at the junction of the L- and R-domains.

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6. Fig. 4. Bonds and interactions between bromelain (a), papain (b), and ficin (c) and the graft copolymer of chitosan and poly-N,N-dimethylaminoethyl methacrylate (dashed lines indicate hydrogen bonds, bond lengths are given in angstroms). Catalytically significant residues are shown in bold.

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7. Fig. 5. Residual catalytic activity of enzymes after incubation of samples at 37°C: in units/ml of solution (a) and as a percentage of the initial level (b). The legend for both diagrams is given in Fig. 5a.

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