Cleavage and deprotection of peptides on MBHA-resin with hydrogen bromide.

Dilute hydrogen bromide in trifluoroacetic acid containing pentamethylbenzene and thioanisole was used in the cleavage and deprotection of peptides on MBHA-resin. Particular attention was paid to potential applicability of the method to kilogram scale synthesis of thymosin alpha 1. In the HPLC purification of the peptides, acetonitrile was replaced by relatively nontoxic isopropanol. The change should be economically and environmentally very attractive.     

An autocatalytic reaction as a model for the kinetics of the aggregation of beta-amyloid

Alzheimer's disease is the commonest form of senile dementia, affecting almost 20 million people worldwide. This neurodegenerative disorder is characterized by amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, beta-amyloid. Fibrillation of beta-amyloid is a nucleation-dependent polymerization process, which is controlled by two kinetics parameters: the nucleation rate and the elongation or growth rate. As the kinetics of fibrillation is strongly dependent on the presence of trace amounts of fibrils, we suggest that the aggregation of beta-amyloid is a model of autocatalytic reaction. A mathematical analysis, permitting quantitative monitoring of the kinetics of fibrillogenesis of beta-amyloid, nucleation, and elongation constants, is presented. The model was checked by applying it to the aggregation of the fragment 1-40 of the beta-amyloid. Understanding of these rate constants may facilitate the study of the effect of substances used for controlling fibril creation and growth. The disaggregating effect of dodecyl trimethylammonium bromide, a cationic surfactant, was easily quantified by means of the model.     

Fluorescence imaging of the interaction of amyloid beta 40 peptides with live cells and model membrane

Amyloid beta peptides (Aβ) found in plaques in the brain have been widely recognised as a hallmark of Alzheimer's disease although the underlying mechanism is still unknown. Aβ40 and Aβ40(A2T) peptides were synthesized and their effects on neuronal cells are reported together with the effect of tetramer forms of the peptides. ThT assay revealed that mutation affected the lag time and aggregation and the presence of lipid vesicles changed the fibril formation profile for both peptides. The A2T mutation appeared to reduce cytotoxicity and lessen binding of Aβ40 peptides to neuronal cells. Fluorescence microscopy of the interaction between Aβ40 peptides and giant unilamellar vesicles revealed that both peptides led to formation of smaller vesicles although the tetramer of Aβ(A2T) appeared to promote vesicle aggregation.This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

The interaction of calmodulin with fluorescent and photoreactive model peptides: evidence for a short interdomain separation

Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2(+)-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, alpha-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal domain was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long alpha-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met-124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthesized. After photocross-linking the Bpa residue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein.     

Resolution of racemic gossypol and interaction of individual enantiomers with serum albumins and model peptides

Racemic gossypol has been resolved by HPLC separation of diastereomeric (-) norepinephrine adducts on a reverse-phase column. The binding constants for the interaction of the three gossypol forms (+, - and +/-) with human and bovine serum albumins have been determined by fluorescence quenching studies. The KD values demonstrate that all three forms bind equally effectively to the two proteins, suggesting an absence of chiral discrimination in albumin-gossypol interactions. Circular dichroism studies of (+)-gossypol binding to the model dibasic peptides, Boc-Lys-Pro-Aib-Lys-NHMe and gramicidin S, suggest that distortions of binaphthyl geometry may occur only for specific orientations of interacting residues at the receptor site.     

A reaction of glucose with peptides

Valylhistidine (Val-His) reacts with glucose (Glc) in a mixture of pyridine and acetic acid to form glucosylvalylhistidine (Glc-Val-His). The pK of the alpha-amino group is thereby lowered to about 5.6 as judged by electrophoretic mobility. The reaction: [Formula: see text] also occurs in an aqueous solution of pyridine and acetic acid of pH6.2 at 50 degrees C, in which it exhibits a half-time of about 30h and a dissociation constant of about 0.3m. Isoleucyltyrosine and glucose react similarly in aqueous solution. The Glc-Val-His has the chromatographic, electrophoretic and chemical properties reported by Holmquist & Schroeder (1966a) for the substance released by proteolysis from the N-terminus of the beta-chains of haemoglobin A(Ic); the value of the dissociation constant means that the concentration of haemoglobin A(Ic) found naturally could be explained by reaction of haemoglobin A with the blood glucose.     

A general model of reaction kinetics in biological systems

Dynamic mathematical models in biotechnology require, besides the information about the stoichiometry of the biological reaction system, knowledge about the reaction kinetics. Modulation phenomena like limitation, inhibition and activation occur in different forms of competition with the key enzymes responsible for the respective metabolic reaction steps. The identification of a priori unknown reaction kinetics is often a critical task due to the non-linearity and (over-) parameterization of the model equations introduced to account for all the possible modulation phenomena. The contribution of this paper is to propose a general formulation of reaction kinetics, as an extension of the Michaelis-Menten kinetics, which allows limitation/activation and inhibition effects to be described with a reduced number of parameters. The versatility of the new model structure is demonstrated with application examples.     

Cell selectivity and interaction with model membranes of Val/Arg‐rich peptides

Antimicrobial peptides are major components of the innate self‐defence system and a large number of peptides have been designed to study the mechanism of action. In the present study, a small combinatorial library was designed to study whether the biological activity of Val/Arg‐rich peptides is associated with targeted cell membranes. The peptides were produced by segregating hydrophilic residues on the polar side and hydrophobic residues on the opposite side. The peptides displayed strong antimicrobial activity against Gram‐negative and Gram‐positive bacteria, but weak haemolysis even at a concentration of 256 µM. CD spectra showed that the peptides formed α‐helical‐rich structure in the presence of negatively charged membranes. The tryptophan fluorescence and quenching experiments indicated that the peptides bound preferentially to negatively charged phospholipids over zwitterionic phospholipids, which corresponds well with the biological activity data. In the in vivo experiment, the peptide G6 decreased the bacterial counts in the mouse peritoneum and increased survival after 7 days. Overall, a high binding affinity with negatively charged phospholipids correlated closely with the cell selectivity of the peptides and some peptides in this study may be likely candidates for the development of antibacterial agents. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A kinetic study on the interaction of deprotonated purine radical cations with amino acids and model peptides

By use of pulse radiolysis techniques, the radical cations of purine nucleotides have been successfully produced by the SO4- ion oxidation. Time-resolved spectroscopic evidence is provided that the one-electron-oxidized radicals of dAMP and dGMP can be efficiently repaired by aromatic amino acids (including tyrosine and tryptophan) via electron transfer reaction. As a model peptide, Arg-Tyr-AcOH was also investigated with regard to its interaction with deprotonated purine radical cations. The rate constants of the electron transfer reactions were determined to be (1 approximately 5) x 10(8) dm(3) mol(-1) s(-1). These results suggest that the aromatic amino acids in DNA-associated proteins may play some role in electron transfer reactions through DNA.     

The interaction of calmodulin with amphiphilic peptides

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.     

On the interaction of esters and peptides with carboxypeptidase B.

The specificity of porcine carboxypeptidase B towards basic and non-basic substrates was studied by employing several esters of phenyllactate. The structure of these depsipeptides complement exactly those of the corresponding phenylalanyl oligopeptide substrates. These non-basic ester-peptide pairs as well as the basic ester-peptide pair of arginyl derivatives, permits the direct comparison of the pH dependencies of the kinetic constants for the hydrolysis of those substrates by carboxypeptidase B. The data is interpreted in terms of three specific ionizing groups located at the active site of the enzyme. The mode and extent of inhibition of the hydrolysis of a specific substrate by another substrate was characterized kinetically. These results are discussed in relation to a proposed model for esterolytic and proteolytic action of carboxypeptidase B.     

A mathematical model of tumor oxygen and glucose mass transport and metabolism with complex reaction kinetics

Hypoxia imparts radioresistance to tumors, and various approaches have been developed to enhance oxygenation, thereby improving radiosensitivity. This study explores the influence of kinetic and physical factors on substrate metabolism in a tumor model, based on a Krogh cylinder. In tissue, aerobic metabolism is assumed to depend on glucose and oxygen, represented by the product of Michaelis-Menten expressions. For the base case, an inlet pO(2) of 40 mmHg, a hypoxic limit of 5 mmHg, and a tissue/capillary radius ratio of 10 are used. For purely aerobic metabolism, a hypoxic fraction of 0.16 and volume-average pO(2) of 8 mmHg are calculated. Reducing the maximum oxygen rate constant by 9%, decreasing the tissue cylinder radius by 5%, or increasing the capillary radius by 8% abolishes the hypoxic fraction. When a glycolytic term is added, concentration profiles are similar to the base case. Using a distribution of tissue/capillary radius ratios increases the hypoxic fraction and reduces sensitivity to the oxygen consumption rate, compared to the case with a single tissue/capillary radius ratio. This model demonstrates that hypoxia is quite sensitive to metabolic rate and geometric factors. It also predicts quantitatively the effects of inhibited oxygen metabolism and enhanced mass transfer on tumor oxygenation.     

Interpreting the aggregation kinetics of amyloid peptides

Amyloid fibrils are insoluble mainly beta-sheet aggregates of proteins or peptides. The multi-step process of amyloid aggregation is one of the major research topics in structural biology and biophysics because of its relevance in protein misfolding diseases like Alzheimer's, Parkinson's, Creutzfeld-Jacob's, and type II diabetes. Yet, the detailed mechanism of oligomer formation and the influence of protein stability on the aggregation kinetics are still matters of debate. Here a coarse-grained model of an amphipathic polypeptide, characterized by a free energy profile with distinct amyloid-competent (i.e. beta-prone) and amyloid-protected states, is used to investigate the kinetics of aggregation and the pathways of fibril formation. The simulation results suggest that by simply increasing the relative stability of the beta-prone state of the polypeptide, disordered aggregation changes into fibrillogenesis with the presence of oligomeric on-pathway intermediates, and finally without intermediates in the case of a very stable beta-prone state. The minimal-size aggregate able to form a fibril is generated by collisions of oligomers or monomers for polypeptides with unstable or stable beta-prone state, respectively. The simulation results provide a basis for understanding the wide range of amyloid-aggregation mechanisms observed in peptides and proteins. Moreover, they allow us to interpret at a molecular level the much faster kinetics of assembly of a recently discovered functional amyloid with respect to the very slow pathological aggregation.     

Developing the reaction kinetics for a biodiesel reactor

The aim of this paper was to investigate the kinetics of the biodiesel reaction in order to find out how best to reach 96.5% methyl ester. The purity of the biodiesel product was examined using gas chromatography to the EN14214 FAME standard and real-time optical microscopy was used to observe the reaction. The problem was the reaction does not reach completion and the mechanism is not understood. It was observed that droplet size had a major influence on reaction end point and that the reaction was mass-transfer limited. This observation was confirmed by developing a mass-transfer based reaction model using the data from the batch reactor which agreed with results from other researchers. The model predicted better conversion with more mixing intensity. The results show that significant improvements could be made to the conventional FAME process.     

Comparison of the interaction of methionine and norleucine-containing peptides with phospholipid bilayers

Norleucine is a structural analog of methionine with a methylene group replacing the thio ether. Despite the close structural similarity of these two amino acids, norleucine-containing peptides have markedly different behaviour with phospholipids compared with methionine-containing peptides. For example, HCO-L-Ahx-L-Leu-L-Phe-OMe behaves as a hydrophobic peptide when mixed with dimyristoylphosphatidylcholine. This peptide lowers the enthalpy of the lipid phase transition. The effect is independent of the rate of heating. With the homologous peptide, HCO-L-Met-L-Leu-L-Phe-OMe, the results are markedly dependent on scan rate with a higher enthalpy observed at faster scan rates. Only at a scan rate of 0.2 K min-1 do the two peptides approach similar behaviour. The higher enthalpy observed for samples with the methionine peptide at higher scan rates can be explained assuming that the peptide aggregates at low temperature. As the phase transition temperature is approached, the more hydrophilic methionine peptide partitions more slowly into the membrane than the norleucine peptide. Partitioning of the peptides between aqueous and lipid phases was measured at 37 degrees by centrifuging down the lipid-bound fraction. At a peptide concentration of 15 microM and a lipid concentration of 1.4 mM, 89% of the HCO-L-Ahx-L-Leu-L-PheOMe and 97% of the HCO-L-Met-L-Leu-L-PheOMe remained in the supernate; indicating a greater tendency of the norleucine-containing peptide to partition into the lipid phase. The peptides Ac-L-Phe-L-Met-L-Arg-L-Phe-NH2 and Ac-L-Phe-L-Ahx-L-Arg-L-Phe-NH2 are readily soluble in water.(ABSTRACT TRUNCATED AT 250 WORDS)