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The paper deals with investigation of silver ion interaction with sulfur-bearing amino acids and cysteine-bearing peptides using an electrospray ionisation orthogonal ion introduction time-of-flight (ESI-o-TOF) mass spectrometer. It has been shown that Cys and Hcy demonstrate the largest affinity for silver, both in relation to methionine and cysteine residues in peptides. The studies have for the first time revealed the effect of predominant forming of ions containing two silver atoms per a sulfhydryl group of cysteine-bearing peptides if the nearest microenvironment does not hinder this.

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Potentially biologically-active nanostructures can be created from single chains of unmodified peptides by cross-linking different regions of the chain by disulfide bonds and cleaving the chain at specified sites to obtain the final configuration. The availability of techniques for assembly and characterization of such structures was tested on a two-loop structure created from a 21-residue linear peptide. Directed intra-molecular disulfide bond formation was performed by inserting partial sequences favoring intra-molecular SS bond formation ("loops") separated by partial sequences disfavoring such a process ("spacers") into the precursor sequence. Peptide bond cleavage by partial acid hydrolysis at specific sites (GG, NP/DP) inside the loops opened them; the same process in the spacer separated the loops. Synthesis, oxidation and bond cleavage were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). The hydrolysis fragments of the produced nanostructures were characterized by tandem electrospray ionization Fourier transform mass spectrometry (ESI FT-MS) with collisional and electron capture dissociations. The latter technique was especially useful as it cleaves SS bonds preferentially. The feasibility of the proposed synthesis approach and the adequacy of the analysis techniques for the test structure were demonstrated.

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A comparative study of substrate specificity of bovine duodenal proteinases--chymotrypsin-like duodenase (ChlD) and dual-specificity duodenase (dsD)--was carried out using oligopeptide substrates (human proinsulin, glucagon, melittin, angiotensinogen fragment 1-14). ChlD displayed mainly chymotrypsin-like properties towards these substrates, hydrolyzing peptide bonds carboxy-terminally to bulky aliphatic or aromatic residues. In melittin, ChlD additionally cleaved peptide bonds after Thr and Ser residues. Dual-specificity duodenase (dsD) significantly restricted its specificity to only trypsin-like or only chymotrypsin-like or displayed full activity, combining both specificities, depending on substrate. Both ChlD and dsD efficiently hydrolyzed a single peptide bond (Phe8--His9) in angiotensinogen fragment 1-14. The kinetic parameters of angiotensinogen fragment 1-14 cleavage by ChlD and dsD were determined (k(cat)/K(m) = 80,500 M(-1) x sec(-1) for ChlD and 103,000 M(-1) x sec(-1) for dsD).

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A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for the tested sample by enzymatic hydrolysis of the same sample (with known concentration) in (18)O-water. A mathematical algorithm was developed which uses the isotopic patterns of the substance, the internal standard, and the substance/internal standard mixture for accurate quantitation of the substance. A great advantages of the proposed method is the absence of molecular weight limitation for the protein quantitation and the possibility of quantitation without previous fractionation of proteins and peptides. Using this strategy, the peptide angiotensinogen and two proteins, RNase and its protein inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.

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Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.

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Three forms of the extracellular alkaline ribonuclease of B. polymyxa (RNases Bpo I, Bpo II and Bpo III) were obtained in the homogeneous states. Relative molecular weights, N- and C-terminal amino acid sequences of the proteins were determined. The two higher-molecular weight forms of the enzyme (RNases Bpo I and Bpo III) were shown to be precursor molecules which differed from the mature B. polymyxa RNase (RNase Bpo II) by the presence of a extra twelve and six amino acid residues at the amino terminus, respectively. The study of catalytic properties of the enzyme elucidated that the extent of specificity of B. polymyxa RNase in respect to highpolymeric substrates is different from that of other natural Bacillus RNases. Amino acid composition of B. polymyxa RNase was established and structural similarity within family of Bacillus RNases is discussed. B. polymyxa RNase was shown to be inhibited by intracellular protein inhibitor of B. amyloliquefaciens RNase with the dissociation constant of 2.7 x 10(-12) M.

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The kinetics of tryptic digestion of melittin was studied by combined electrospray ionization time-of-flight mass spectrometry and high-performance liquid chromatography. The ratios of the kinetic constants for cleavage of the peptide bonds that are susceptible to trypsin action were determined. It is shown that trypsin does not manifest affinity for the hydrolysis of the peptide bonds inside the Arg,Lys cluster series as efficiently as it cleaves the peptide at the separately localized Lys residue. This feature demonstrates clearly the advantage of the kinetic approach to tryptic mapping of proteins. The kinetic approach allows the determination of not only discrete structural segments in protein structure but also their relative locations and their amino acid sequences. Using the melittin digests and some artificially prepared amino acids and dipeptides mixtures as models, it is shown that the presence and nature of basic amino acids predetermines the charge states of the molecules analyzed by electrospray but not the yields of their ions. The aliphatic parts of the molecules seem to be more important in determining the actual ion yields.

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The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.

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The kinetics of proteolysis of bradykinin and its metabolites in human blood plasma was studied, the limiting stages were determined and each stage of the degradation was correlated with the appropriate plasma enzyme. A scheme of the pathways of major and minor degradation processes has been proposed.

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