Ultra-rapid preparation of milligram quantities of the purified polypeptide chains of human fibrinogen

In order to study the epitopes in fibrin towards which monoclonal antibodies are directed we needed the pure individual polypeptide chains of human fibrinogen in reasonable quantity. We report here a simplified, rapid method of separation of high-purity human fibrinogen chains. Following reduction and S-carboxymethylation of human fibrinogen, the sample was injected directly onto a column of the polymeric reversed-phase perfusion packing POROS 20-R2, and the chains were completely resolved in less than 3 min at a flow-rate of 10 ml/min. The capacity was equivalent to that of a similar sized conventional silica-based column. However the throughput was approximately five to ten times as high. The column was durable and robust in day-to-day use.     

Optical anisotropy of polypeptide chains

Optical anisotropies γ² of N-t-butylacetamide (tBA), N-Methylacetamide (MA), and N, N-dimethylacetamide (DMA) have been determined from the Rayleigh ratios for depolarzed scattering by dilute solutions of the amides in p-dioxane. Traceless optical polarizability tensors \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document} for the amides are derived from these results in conjunction with the Kerr constant for tBA determined by LeGèvre and co-workers. It is shown that the tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document}_i for the glycyle unit in a polypeptide chain may be identified with \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document}MA . Methods for deriving corresponding tensors for other peptide units are indicated and the traceless polarizability tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document} for a polypeptide chain in any specified configuration is formulated.     

Comparisons of the polypeptide chains of globins

Summary Vertebrate myoglobins and hemoglobins each consist of units containing a heme group and an associated polypeptide chain. The polypeptide chains form an homologous series and can be compared with each other to measure amino acid differences and minimum base differences per codon. These differences are the result of mutations which have been incorporated during divergent evolution from a common ancestral gene. Each such base replacement is termed an evolutionary event. Each amino acid replacement is the result of one or more evolutionary events. However there can be only one amino acid difference between two sites. The minimum mutational difference between two sites is perceptible only as one, two or three base differences per codon, but there may be more evolutionary events than base differences, because of revertants and multiplicity of base replacements at the same site. When all three bases in a codon are changed, the result is recognizable in only about five per cent of cases. Therefore such recognizable three-base changes indicate a large number of evolutionary events.     

Oxidation-induced modification of the fibrinogen polypeptide chains

By using the mass-spectrometry method, the oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains induced by its oxidation have been studied. The αC-region has been proven to be the most vulnerable target for the oxidizer (ozone) as compared with the other structural elements of the Aα chain. The Bβ chain mapping shows that the oxidative sites are localized within all the structural elements of the chain in which the β-nodule exhibits high susceptibility to oxidation. The γ chains are the least vulnerable to the oxidizer action. The results obtained demonstrate convincingly that the self-assembly centers dealing with interactions of knob “A”: hole “a” are not involved in oxidative modification. It is concluded that the numerous oxidative sites revealed are mainly responsible for inhibiting lateral aggregation of protofibrils. The part of amino acid residues subjected to oxidation is supposed to carry out the antioxidant function.     

Different asparagine-linked sugar chains on the two polypeptide chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) purified from placenta, like urinary hCG, is shown to have the sialylated forms of three neutral oligosaccharides: Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4(Fucα1→6)GlcNAc (N-1), Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-2) and Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-3). Gel permeation chromatographic analysis of oligosaccharides released from α- and β-subunits of placental hCG has revealed that the α-subunit has one each of sialylated N-2 and N-3, while the β-subunit has one each of sialylated N-1 and N-2.     

Dimerization of the polypeptide chains of skeletal muscle tropomyosin

The composition of alpha and beta chains in tropomyosin dimers present in fetal and adult skeletal muscle of cow has been analysed by SDS-polyacrylamide gel electrophoresis after cross-linking of the chains by disulphide bridges. The results indicate that in vivo alpha beta heterodimers of tropomyosin are assembled preferentially and only the excess of particular chains forms homodimers, i.e., alpha alpha dimers in adult and beta beta ones in fetal muscle. The original dimers of tropomyosin were dissociated with urea in the presence of dithiothreitol. Subsequent reassembly of the tropomyosin dimers from the mixture of alpha and beta chains approaches the random model.     

The lattice energetics of some polypeptide chains

The interchain energetics of alpha, beta, and PGII conformations of polyglycine, the PPII and left-handed 3.30 fold helical conformations of trans poly-L -proline, and the Yonath and Traub triple helix and left-handed three fold helices of poly(gly-pro-pro) were investigated. Intra- and inter-chain stabilization energies appear to be inversely related, and the interchain stabilization energy can be as large its the intrachain energy. The minimization of the interchain energy can be described by the simultaneous optimization of interchain hydrogen bonding and intermolecular-sidechain digitation. The stability of the poly(gly-pro-pro) triple helix can be readily explained in terms of these two factors. In all cases the experimentally observed lattice packing is predicted, although the calculated lattice constants are slightly larger than those observed. The small differences between observed and predicted lattice constants probably reflect small errors in present conformational potential functions. Homopolymers are probably the best systems to use in the refinement of conformational potential functions because solvent effects arc minimized and the experimentally observed lattice constants provide a check on the configurational calculations.     

Statistical studies of flexible nonhomogeneous polypeptide chains

Unfolded proteins attract increasing attention nowadays because of the accumulation of experimental evidence that they play an important role in different biological processes. Therefore, studies of various statistical properties of flexible protein-like polypeptide chains are becoming increasingly important as well. This paper presents distributions (histograms) of distances between atoms of titratable residues for flexible polypeptide chains with various residue compositions and with the hard-spheres potential taken into consideration. The factors influencing the parameters of the obtained histograms have been identified and analyzed. It was found that the sensitivity of the distributions with respect to the internal structure of intermediate residues increases with the number of residues between the considered charged residues. It was shown that branching at C(beta) atoms of the side chains of the intermediate residues is among the most considerable factors influencing the shape of the distance distribution and the average distance between atoms in flexible chains. Despite the model simplicity, the results of the calculations can be applied for systems with other types of interactions presented, and this was demonstrated for the charge-charge interactions. In particular, it was shown that those interactions have a significant effect on distances between the unlike charges, while such an effect for the like charges is much less pronounced. The comparison of predictions made on the basis of the presented calculations to some experimental data is also given, and possible applications of the theoretical concept described in the paper are discussed.     

Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains

Protein aggregation is associated with a variety of pathological conditions, including Alzheimer's and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.     

Circular dichroism calculation for random-coil polypeptide chains

A method of calculation of the circular dichroism (CD) of random-coil polypeptides has been developed by means of a Monte-Carlo approach to the treatment of statistical systems and exciton theory of optical activity of polymers. The contribution of π–π^* and n–π^* amide transitions to the CD has been taken into account. The π–π^* transition gives rise to two CD bands: a negative short-wavelength band and a weak positive long-wavelength one. The n –π^* transition gives rise to one negative CD band.     

Theoretical calculation of the circular dichroism of unordered polypeptide chains

A calculation has been done of the circular dichroism (CD) spectrum of an unordered polypeptide chain. This has been based on a Boltzmann averaging over a dipeptide conformational CD map. This is shown to be valid by comparing the CD spectra of 28-mer oligopeptides with those generated by summing dipeptide CD spectra. The calculated CD spectrum of an unordered polypeptide chain is found to agree with the assignment proposed by Tiffany and Krimm from experimental studies.     

Characterization of the major polypeptide chains of reduced bovine thyroglobulin

The metabolism of bioreactive lipid mediators was studied in two types of activated macrophages (M phi). We compared the capacity of resident and activated M phi to release, upon a zymosan challenge, cyclooxygenase and lipoxygenase products as well as PAF-acether (platelet-activating factor) and its 2-lyso precursor. Activated M phi were obtained from mice injected intraperitoneally either with nonviable C74 streptococci (St-M phi) or with trehalose dimycolate, a defined immunostimulant isolated from Mycobacterium tuberculosis (TDM-M phi). Both activated populations exhibited common features: conversion of endogenous [14C]arachidonic acid into prostaglandin E2 and thromboxane A2 rather than into prostaglandin I2 and low biosynthesis of PAF-acether, probably due to an impairment of the acetylation step. However, contrary to St-M phi, TDM-M phi did not display a marked overall reduction of arachidonate metabolism. In addition, as compared to resident M phi, TDM-M phi presented a ratio of thromboxane B2/6-ketoprostaglandin F1 alpha 30-fold higher, a better conversion of leukotriene C to leukotriene D and a higher capacity to release the PAF-acether they synthesize. These macrophages thus seem to be valuable tools for studying the formation of mediators and for determining specific markers of an activated state.