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The rate of the H-D exchange of the peptide NH atoms of the isolated alpha and beta subunits of human Hb were studied at the pH range 5.5-9.0 and 20 degrees C by the IR spectroscopy. The factor retardation of the exchange rate of subunits -P in the range -10(2)-10(7). In comparison with tetramer Hb the probability of local fluctuations (1/P) is increased to a slightly greater extent for the monomeric alpha subunits then for the tetramer beta subunits. Unlike Hb oxygenation of subunits does not influence on the probability of the local fluctuations and subunits have no the pH-dependent change of the value 1/P observable for the ligand Hb. The possible mechanisms of the overall intensification of the local fluctuations upon the splitting of the Hb tetrameric contacts between subunits are discussed with the inviting of the structural crystallographic data.

The rate of the H-D exchange of the peptide NH atoms of the different forms of human Hb was studied at the range of pH 5-10 and temperature 10-63 degrees C by the IR spectroscopy. The pH-dependence of the H-D exchange rate is accordance with the EX2 mechanism. Two pH-dependent conformers of ligand forms of Hb existes at 10-30 degrees C with lower probability of local fluctuations of the alkaline conformer. The difference between two conformers vanishes at 40 degrees C with the appearance of the third conformer with higher probability of local fluctuations. The deoxyHb at 20 degrees C and pH range 6-9 has no pH-dependent conformers and the probability of local fluctuations is considerably reduced in comparison to the acid conformer of ligand Hb. Upon the destabilization of the ligand Hb structure by the pH decreasing to 5.0 at 20 degrees C or the temperature increasing up to 50-60 degrees C at pH 7.1 the global fluctuations of the native structure are intensified providing the H-D exchange of the slowest exchanging NH atoms. The nature of the local and global fluctuations and possible similarity between the two pH-dependent conformers of ligand Hb and its functional R and R2 states revealed by the X-ray analysis and NMR spectroscopy were discussed.

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water-magnesium complex involved in their catalytic acts. In the enzyme-substrate complexes, the Mg2+ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3'-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3'-leaving group. A strained metalloenzyme-substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction. The English version of the paper.

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 A to R factor of 12.4% and Rfree factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O delta 1 atom of Asn119 were shown to be within a range of 2.01-2.11 A. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 A2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 A2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.

A comparative research of individual peptide structures obtained after hydrolysing of Bacillus circulans and B. amyloliquefaciens RNases by the Glu-specific staphylococcal protease was carried out by means of mass-spectrometry and Edman degradation methods. A complete amino acid sequence of B. circulans RNase was determined. Gln15, Gly65 and Gln104 residues in B. amyloliquefaciens RNase were found to be substituted by Leu, Ala and Lys residues in B. circulans RNase, respectively. Catalytic properties of the B. circulans RNase in transesterification reactions with poly- and oligonucleotides as substrates were investigated.

Two enzyme forms were isolated from the commercial preparation of extracellular endonuclease of Serratia marcescens strain B10 M1. The chromatographic and electrophoretic properties, isoelectric points and N-terminal amino acid residues are different for both enzymes. At the final step of the purification procedure including ion-exchange chromatography on phospho- and DEAE-cellulose columns the yields of nucleases Sm1 and Sm2 were 13% and 25%, respectively. No significant differences were found in the specific activities of nucleases Sm1 and Sm2 (3.6 x 10(6) and 4.0 x 10(6) un. act./mg of protein). A comparative analysis of tryptic nuclease hydrolysate peptides was carried out. The amino acid sequences of some polypeptide segments of the proteins were determined. The structural similarity of the enzyme was established and the amino terminal regions of the proteins were identified. The localization of the disulfide bonds in the molecules of the both nucleases was determined. The similarity of nucleases Sm1 and Sm2 strain B10 M1 to S. marcescens endonucleases obtained from other strains was demonstrated.

Some variants of the quantitative dansyl method in combination with thin-layer chromatography on polyamide plates have been compared, and principles of its optimization have been demonstrated. Sensitivity of the method is about 10(-11) mole, the error does not exceed 10%. Data are given on the analysis by the dansyl method of peptides of the tryptic hydrolysate of bovine pancreatic ribonuclease after their separation by peptide mapping procedure.

We have analysed reassociated Ig molecules, containing heavy (H) or light (L) chains of Ig-B2 monoclonal antibody with human fibronectin binding activity and L of H chains of normal mouse serum immunoglobulin (Ig-NM). Examination of Ig-B2 idiotype expression in reassociated Ig indicated that 0.4% L-NM and 0.8% H-NM were able to restore Ig-B2 idiotype. The analysis of antigen binding capacity of reassociated Ig demonstrated, that only 4% H-NM created antigen binding site in complex with L-B2. We have determined the leading role of L-chain in creation of idiotype and binding site of Ig-B2. Selectivity of interaction between H and L chains is discussed. The results indicate, that not more than 4-6% of random H--L combinations produce functional Ig.

Extracellular RNase Fl1 has been purified from the culture filtrate of Fusarium lateritium. The enzyme has been obtained in the electrophoretically homogeneous state with the yield about 90% and 300 fdd degree of purification. RNase Fl1 is a guanyl specific enzyme (EC 3.1.27.3) with the specific activity on RNA 1420 units/mg of protein. The total primary structure of the RNase has been determined by the automated Edman degradation of two non-fractionated peptide hydrolysates produced by trypsin and Staphylococcus aureus protease and of the hydroxylamine cleavage products of the protein. It was shown that hydroxylamine converts the RNase Fl1 N-terminal residue, pyroglutamic acid, into the hydroxyamic acid derivative sensitive to Edman degradation. RNase Fl1 consists of 105 amino acid residues (Mr 10,852) and is a structural homologue of the Fus. moniliforme RNase F1, differing from the latter by 15 amino acid substitutions outside the enzyme active site.

The complete amino acid sequence of an extracellular guanyl-specific RNase from Aspergillus pallidus fungi has been established. The RNase contains 104 amino acid residues (Mr 11,029). Its primary structure was analyzed basing on the automated Edman degradation of the carboxymethylated RNase followed by tryptic digestion and sequencing of the resultant hydrolysate. An additional structural information was obtained by means of the automatic sequencing of the cyanogen bromide peptide mixture and by studying the kinetics of the RNase's digestion with carboxypeptidase Y.

Extracellular guanyl-specific RNAase of the fungus Aspergillus pallidus (RNAase ApI) was isolated in preparative amounts with a 40% yield and purified to homogeneity (938-fold). The complete amino acid sequence of the protein (104 amino acid residues) was determined: 6 Asp, 4 Asn, 4 Thr, 14 Ser, 3 Glu, 4 Gln, 4 Pro, 15 Gly, 9 Ala, 4 Cys, 6 Val, 2 Ile, 4 Leu, 10 Tyr, 4 Phe, 3 His, 4 Arg, 1 Trp. RNAase ApI has a molecular mass of 11,029 Da and is homologous to the family of fungal extracellular guanyl-specific RNAases. The primary structure of the protein is close to that of RNAase C2 from Asp. clavatus and differs from it by only 4 substitutions of amino acid residues. Monocrystals of RNAase ApI were grown which can be used for the X-ray analysis of proteins.

The theoretical conformational analysis of the pyridoxal-5'-phosphate aldimine complex with a substrate analogue--threo-alpha-cycloglutamic acid has been carried out. The calculation method used takes into account the possibility of hydrogen bonding between a carboxyl group of the substrate and the phosphate moiety of the cofactor. The conformational properties and geometrical parameters of the lowest energy structures are discussed. It is shown that of the three conformations permitted for the productive enzyme--substrate complex formations only one is electrostatically favourable.

The paper describes a method for producing gram-scale quantities of beef pancreatic ribonuclease A (RNase A) (EC 3.1.4.22) from commercial, amorphous and crystalline RNases. The method involves enzyme purification by chromatography on SP-Sephadex G-25, rechromatography on Bio-Rax 70 followed by concentration, storage and desalting in optimal conditions. In their properties the resultant preparations of RNase A are not inferior to the best commercial samples of the enzyme.

The physico-chemical properties have been studied of RNase A selectively modified at the E-NH2-group of Lys-7 and Lys-41 with pyridoxal-P. Modification did not affect conformational stability of the protein globule, thus all changes in the molecule of the modified RNase A were localised around the alkylated Lys residue. In the both cases pyridoxyl-P. The residue was shown to be localized in the active site region of the (P-Pxy)-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. (P-Pxy) E-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. In the Lys-41 derivative, pyridoxamine-P was situated exactly in the active site and is partially hidden in the protein grobule. The pH-dependence of absorption spectra indicates that the chromophore of pyridoxyl-P in modified proteins is quite sensible to the ionic state of its surrounding. The usefulness of pyridoxyl-P as a reporter group was proved in the study with (P-Pxy)-Lys-7-RNase A. Some conformational changes involving His-119 were shown to take place in the course of the enzyme-nucleotide complex formation.

The complete amino acid sequence of a guanyl-specific RNAse from Streptomyces aureofaciens has been established using a rapid method of primary structure analysis which eliminates the peptide fractionation. The automated Edman degradation of the carboxymethylated RNAse Sa and of non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the modified protein were used. The RNAse contains 96 amino acid residues, Mr 10,566. The secondary structures of RNAse Sa and microbial RNAses have been calculated using a modified Chou--Fasman procedure. A comparison of the primary and secondary structures of the RNAses revealed different degrees of sequence homology and a similar distribution of predicted structural regions (alpha-helices, beta-structure and beta-turn). The predicted secondary structure patterns are discussed in the light of the RNAse X-ray analysis date.

The kinetics of nitration of tyrosine residues in histones F1 and F2a1 by tetranitromethane has been investigated. At low ionic strength and 30-fold molar excess of nitrating agent the nitration reaction results in fast modification of all tyrosine residues in both histones. At the same time the rates of modification of different tyrosine residues in histone F2a1 are not identical and markedly exceed the rate of N-Ac-OEt-Tyr nitration in a model system. The increase of reaction mixture ionic strength causes an increase of modification rates. The differential UV-absorption spectra of histone F1 obtained by temperature perturbation show an abnormal positive characteristic maximum at 286.8 nm. Analysis of the dependence of nitration rates of tyrosine residues in histones in saline solutions upon the ionic strength and of difference UV-absorption spectra of histones leads to a conclusion that there are specific interactions of definite parts of histone polypeptide chains. These interactions may arise from aggregation of histone molecules.

The phosphorylation of lysine-rich histones F1, F2a2 and F2b of calf thymus has been investigated using homogeneous histone kinase from pig brain. 32P-labelled phosphopeptides from tryptic digests of corresponding histones were obtained. According to N-terminal analysis and the quantitative determination of amino acid composition of the obtained radioactive peptides the sites of phosphorylation were identified in the primary structure of lysine-rich histones, namely, Ser-38 for the polypeptide chain of histone F1, Ser-19 or 18 for histone F2a2 and Ser-14 and 36 for histone F2b. Thus, the high specificity of brain histone kinase in vitro was demonstrated.