Solid-state conjugation of proteins with hydrophobic compounds in non-denaturing conditions. I. Acylation of proteins by dansyl proline using a polymeric N-hydroxysuccinimide ester.

A method of conjugating protein molecules under native conditions with water-insoluble hydrophobic compounds is developed. It permits very water-insoluble acids to be gently coupled to the primary amines on proteins or other biopolymers. For this purpose we synthesized a polymer (co-polymer of N-hydroxymaleimide and N-vinylpyrrolidone cross-linked by benzidine) which swells equally well in water and in organic solvents. Hydrophobic substances are first activated by esterification to this polymer in organic solvent and then conjugated to protein by acylation in aqueous medium at pH 8.0-8.5. Thus, the contact of native protein with organic co-solvent may be completely avoided. The application of this approach is demonstrated by labeling trypsinogen and plasminogen with dansyl proline.

[Natural and synthetic inhibitors of thrombin. I. Natural inhibitors]. / Prirodnye i sinteticheskie ingibitory trombina. I. Prirodnye ingibitory.

Structures and properties of main physiological (heparin, antithrombin III, heparin cofactor II) and nonphysiological (hirudin, thrombin-binding aptamers, cyclotheonamides) natural thrombin inhibitors and its fragments and synthetic analogs (hirugen, hirulogs, hirunorms, pentasaccharides, macrocyclic alpha-keto amides) were reviewed. The molecular mechanisms of interaction of these compounds with thrombin and their anticoagulant activity at preclinical and clinical trials are discussed. The examined of natural thrombin inhibitors and their synthetic fragments and analogs are perspective for prophylaxis and treatment of different thrombo-embolic diseases.

[Natural and synthetic inhibitors of thrombin. II. Synthetic low-molecular-weight inhibitors]. / Prirodnye i sinteticheskie ingibitory trombina. II. Sinteticheskie nizkovmolekuliarnye ingibitory.

The up-to-date problems, concerning the structure and properties of two types of inhibitors are reviewed. It is particularly considered properties of low-molecular weight thrombin inhibitors that have electrophilic groups capable to react with Ser-195 of thrombin (peptidyl-chloromethyl ketones, aldehydes, ketomethylene derivatives and derivatives of boric and phosphoric acids) and the competitive reversible thrombin inhibitors. The review focuses on methods of modification of the structure in the natural inhibitors and design of new peptidomimetics. The prospects for prophylaxis and treatment of diverse thromboembolic diseases are discussed.

[New donor-acceptor pairs for fluorogenic substrates with intramolecular fluorescence energy transfer for thrombin and trypsin]. / Novye donorno-aktseptornye pary dlia fluorogennykh substratov s vnutrimolekuliarnym perenosom énergii fluorestsetsii dlia trombina i tripsina.

New substrates for thrombin and trypsin are described: a fluorogenic substrate Abz-Pro-Arg-Gly-Nph (I), whose action is based on intramolecular fluorescence energy transfer, and H-D-Trp-Pro-Arg-pNA (II), which can be used both as a chromogenic substrate and as a substrate with the intramolecular fluorescence energy transfer. In substrate (I), a 4-nitrophenylhydrazide group was first used as an acceptor of excitation energy of the 2-aminobenzoyl group. The substrate is poorly hydrolyzed by thrombin (kcat/K(m) = 1.4 x 10(3) M-1 s-1) and is efficiently cleaved by trypsin (kcat/K(m) = 3.15 x 10(6) M-1 s-1). The hydrolysis of (II) can be monitored both spectrophotometrically, by absorbance at 405 nm, and from the increase in fluorescence at 340 nm. In the efficiency of hydrolysis with thrombin (kcat/K(m) = 3.0 x 10(6) M-1 s-1), compound (II) is comparable with the known chromogenic substrates for this enzyme. The proposed donor-acceptor pairs are promising in designing substrates with the intramolecular fluorescence energy transfer for a variety of proteolytic enzymes.

Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS).

A prospective class of intramolecular fluorescence energy transfer substrates (IFETS) is described. In contrast to the known chromogenic and fluorogenic substrates that are used widely in the scientific and medical research, IFETS allow to control the enzymatic cleavage at any point of the peptide chain and thus permit simultaneous studies of enzymes of different specificity. We discuss the main types of donor and acceptor, their advantages and drawbacks and the effectiveness of exited-state energy transfer between them. High sensitivity and selectivity of IFETS in the assay of proteinases was demonstrated. They prove to be a very useful and very promising instrument for enzymology and medicine.

[Inducing conformational changes in thrombin by ligands as a way of regulating its activity]. / Indutsirovanie ligandami konformatsionnykh izmenenii v trombine kak sposob reguliatsii ego aktivnosti.

This review summarizes data on changes in thrombin conformation induced by various ligands including hirudin, hirugen, similar acidic peptides, fibrinogen, thrombomodulin, heparin, antithrombin, prothrombin, and platelet receptor. Studies of enzyme kinetics, spectroscopy, X-ray structure, and thermodynamics of thrombin complexes with various ligands indicate that the thrombin molecule undergoes conformational changes. Possible mechanisms of allosteric regulation of thrombin activity by high molecular weight ligands are discussed which can be important during physiologic or pathophysiologic reactions of the organism.

Synthesis and study on a novel inhibitor of thrombin containing no side chain at the P1 position of the polypeptide chain.

The presented work allows one to speculate that the hydrophobic contacts of the residues located at the P2 and P3 positions with the corresponding subsites of thrombin (S2 and S3) allow the synthesis of compounds that can react with thrombin specifically and probably may not interact with other trypsin-like serine proteases. Substitution of proline by m-Abz-residue may result in the development of novel substrates and inhibitors for thrombin.

[Thrombin substrates containing aminobenzoic acids]. / Sybstraty trombina, soderzhashchie aminobenzoinye kisloty.

In order to study the interactions of synthetic substrates with the active site of thrombin the compounds of R-CO-Abz-Arg-OMe were obtained which contained, o-, m- and p-amino benzoic acids (Abz) at P2-subsite. R-CO-moiety was butyric, perfluorobutyric, benzoyl, 2-fluorobenzoyl and perfluorobenzoyl groups. Kinetic parameters of their hydrolysis by thrombin were measured and antithrombin activity was studied. Introduction of fluorine atoms into N-acyl groups caused insignificant alterations of the substrate or inhibitor properties of the compounds containing N-butyric group, but significantly influenced the substrate properties, when peptides contained N-benzoyl group. The interaction mechanism of the obtained substrates with thrombin was proposed.

[Fluorescent substrates of proteolytic enzymes with internal quenching of fluorescence]. / Fluorestsentnye substraty proteoliticheskikh fermentov s vnutrennim tusheniem fluorestsentsii.

The design and application of a successfully developed type of fluorogenic substrates for proteolytic enzymes are discussed. Proteolytic enzymes play a key role not only in the regulation of cellular protein turnover but also in the control of many other physiological functions. Substrates consisting of the appropriate peptides with chromophore or fluorophore at the C-termini are often employed to detect protease activity. Proteases exhibit the primary specificity to the particular amino acid residue and hydrolyze the following peptide bond. However, it is impossible to investigate the specific recognition of the other side of a scissible bond. Intramolecularly quenched fluorescent substrates are very useful instruments for research and medical purpose due to their high sensitivity and selectivity to proteases.

[Use of water-soluble 2-nitro-4-sulfophenyl ester of adipic acid for preparation of peptide protein conjugates]. / Primenenie vodorastvorimogo 2-nitro-4-sul'fofenilovogo efira adipinovoi kisloty dlia polucheniia kon"iugatov peptid-belok.

Two synthetic peptides were conjugated with bovine serum albumin by means of 2-nitro-4-sulfophenyl ester of adipic acid. The amino acid analysis of the conjugates has shown that 14-15 molecules of the peptide are coupled per 1 molecule of the albumin during 10 min. The number of coupled molecules is the same when the reaction time increases to 24 hours. So, 2-nitro-4-sulfophenyl ester of adipic acid may be used parallel with the known bifunctional reagents to obtain peptide-protein conjugates.

[Chromogenic and fluorogenic peptide substrates of proteolytic enzymes]. / Khromogennye i fluorogennye peptidnye substraty proteoliticheskikh fermentov.

The review describes approaches to designing chromogenic and fluorogenic substrates for proteolytic enzymes, mainly for assay of serine proteinases. Principles of substrate polypeptide chain construction and some methods for detection of chromogenic and fluorogenic products of their hydrolysis are considered. The use of these substrates for the study of blood clotting enzymes and for clinical diagnostics is briefly treated. Methodology of chemical synthesis of principal chromogenic and fluorogenic substrates is also discussed.

[Analysis of the relationship between the reactivity of synthetic substrates and thrombin inhibitors and their structure]. / Issledovanie zavisimosti reaktsionnoi sposobnosti sinteticheskikh substratov i ingibitorov trombina ot ikh stroeniia.

Kinetics of thrombin- and trypsin-catalyzed hydrolysis of diphenylacetyl-L-arginine esters was studied at pH 8.5 and 25 degrees C, and the antithrombin activity of in vitro synthesized compounds was examined. The anticlotting activity of arylsulphonyl-L-arginine methyl esters appeared to be higher than that of the derivatives of diphenyl arginine. Relations were found connecting polar (delta) and steric (Es) characteristics of substituent (R) in R-C6H4-SO2-Arg-OCH3 esters with their antithrombin activity in vitro or with efficiency of their thrombin-catalyzed hydrolysis. This gives supplementary possibilities for synthesis of new substrates and more potent thrombin inhibitors.

[Therapeutic effect of myelin basic protein and synthetic encephalitogenic peptide in guinea pigs with experimental allergic encephalomyelitis]. / Izuchenie lechebnogo deistviia osnovnogo belka mielina i sinteticheskogo éntsefalitogennogo peptida u morskikh svinok s éksperimental'nym allergicheskim éntsefalomielitom.

Effect of antigenic factors of experimental allergic encephalomyelitis (alkaline protein of myelin and synthetic encephalitogenic peptide) was studied in guinea pigs with distinct manifestations of the experimental allergic encephalomyelitis. The animals recovered after prolonged administration of alkaline protein of myelin within 14 days, if they were sensibilized either by bovine alkaline protein of myelin or by synthetic encephalitogenic peptide. Synthetic encephalitogenic peptide was only effective in treatment of the disease caused by the peptide.