The measurement of amino groups in proteins and peptides.
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ABSTRACT:
SUBMITTER: Fields R
PROVIDER: S-EPMC1177228 | biostudies-other | 1971 Sep
REPOSITORIES: biostudies-other
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Project description:We report a method for expressing the solvent accessibility of histidine imidazole groups in proteins. The method is based on measuring the rate of the hydrogen exchange (HX) reaction of the imidazole C(?1)-hydrogen. The rate profile of the HX reaction as a function of pH gives a sigmoidal curve, which reaches the maximal rate constant (k(max)) on the alkaline side of the sigmoidal curve. To quantitatively describe the solvent accessibility of imidazole groups in proteins, it is necessary to compare the k(max) of the imidazole groups with their intrinsic k(max) ((i)k(max)), the maximal rate constants for the given imidazole groups when they are fully exposed to the bulk solvent. However, the mechanism of the HX reaction suggests that the (i)k(max) of an imidazole group differs depending on its pK(a), and no systematic study has been conducted to clarify how the (i)k(max) is affected by pK(a). We therefore investigated the relationship between (i)k(max) and pK(a) using four imidazole derivatives at three different temperatures. The experimentally determined pK(a)-specific (i)k(max) values allowed us to derive a general formula to estimate the (i)k(max) value of any given imidazole group exhibiting a specific pK(a) at a specific temperature. Using the formula, the protection factors (PF), the ratio of (i)k(max) to k(max), of five imidazole groups in dihydrofolate reductase were obtained and used to express the magnitude of their solvent accessibility. In this definition, the smaller the PF value, the higher the solvent accessibility, and a value of 1 indicates full exposure to the bulk solvent. The solvent accessibility expressed by the PF values agreed well with the solvent accessible surface areas obtained from the X-ray diffraction data.
| S-EPMC3462353 | biostudies-literature
Project description:1. The reaction of several peptides and proteins with diborane was studied under different conditions to determine those most suitable for the specific reduction of carboxyl groups. 2. In the reaction of model peptides and the cyclic peptides bacitracin and tyrocidin, reduction at 0 degrees was entirely specific for the carboxyl groups without affecting the peptide bonds. Acid amide residues were not reduced. Some tripeptides showed anomalous results in that the C-terminal residue was quite resistant to reduction. 3. Specific reduction of carboxyl groups was achieved in each of the following proteins: human serum albumin, egg albumin, adult human haemoglobin, sperm-whale apomyoglobin, horse heart cytochrome c and egg-white lysozyme. The C-terminal amino acid was usually reduced. 4. Conditions for specific reduction of all available carboxyl groups are not easily found and may vary from one substance to another. Specific reduction of a limited number of available carboxyl groups may be generally accomplished by reactions at -10 degrees . 5. It is suggested that this chemical modification, which has the advantage of permanence, may be useful in studying the role of carboxyl groups in the conformation of proteins and in the biological properties of peptides and proteins.
| S-EPMC1187580 | biostudies-other
Project description:Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new class involving modifications installed by a cytochrome P450, a multi-nuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes encoded by the BGC were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization with 2D-NMR and Marfey's method on the resulting RiPP demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C crosslink between two Tyr residues with the B12-rSAM generating β-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid while the methyltransferase acted on the β-carbon of the α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configurations of the atropisomer that formed upon biaryl crosslinking. The conserved Cys residue in the precursor peptide was not modified as in all other characterized MNIO-containing BGCs; However, mutational analyses demonstrated that it was essential for the MNIO activity on the C-terminal Asp. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to discover new macrocyclic RiPPs and that RiPPs remain a significant source of previously undiscovered enzyme chemistry.
| S-EPMC10635010 | biostudies-literature
Project description:Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 × 10-9 M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10-8 M for OP-peptides and 1 × 10-12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 μg/mL of depY was 0.025 μg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.
| S-EPMC6006506 | biostudies-literature
Project description:Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.
| S-EPMC5352754 | biostudies-literature
Project description:Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.
| S-EPMC1163376 | biostudies-other
Project description:The ability of 18-crown-6 (18C6) to form noncovalent complexes with cationic groups in the gas phase has been leveraged in numerous, largely orthogonal mass spectrometry-based applications. Although the fundamental interaction between 18C6 and a charged group in the gas phase is quite strong, the strength of attachment of 18C6 to large molecules is more difficult to predict because intramolecular binding of the cation can be competitive. Herein, we demonstrate in experiments with model peptides that 18C6 adducts are not strongly attached to flexible molecules with numerous potential hydrogen bonding sites. 18C6 adduct stability is increased if intramolecular charge complexation is inhibited by sterics or competitive binding. It is demonstrated with molecular mechanics that significant structural changes occur upon loss of 18C6 in model peptides. Examination of the loss of 18C6 adducts from proteins following collisional activation reveals that lower charge states lose the most 18C6. The degree of 18C6 adduct stability may reflect the degree of structural reorganization that occurs following collisional activation, suggesting that lower charge states represent structures that are not similar to gas phase idealized states. In this regard, 18C6 may serve the function of protecting solution phase protein structure. Collisional activation of holomyoglobin with 18C6 adducts attached reveals that heme loss occurs primarily after 18C6 loss, further supporting the notion that 18C6 protects native structure by solvating charged sites.
| S-EPMC3806280 | biostudies-literature
Project description:A method is described for determining carbonyl groups that is especially suitable for use with proteins and peptides. It involves the determination of the extinction at 370nm of a sample solution after adding 2,4-dinitrophenylhydrazine. The reaction of 2,4-dinitrophenylhydrazine with pyruvoylglycine and with transaminated ribonuclease T(1) is presented; the isolation of protein hydrazones is discussed.
| S-EPMC1176632 | biostudies-other
Project description:1. The kinetics of the reaction of 2,4,6-trinitrobenzenesulphonic acid with various amino acids, peptides and proteins were studied by spectrophotometry. 2. The reaction of the alpha- and in-amino groups in simple amino acids was found to be second-order, and the unprotonated amino group was shown to be the reactive species. 3. By allowing for the concentration of unreactive -NH(3) (+) group, intrinsic reactivities for the free amino groups were derived and shown to be correlated with the basicities. 4. The SH group of N-acetylcysteine was found to be more reactive to 2,4,6-trinitrobenzenesulphonic acid than most amino groups. 5. The reactions of insulin, chymotrypsinogen and ribonuclease with 2,4,6-trinitrobenzenesulphonic acid were analysed in terms of three exponential rate curves, each referring to one or more amino groups of the proteins. 6. The reaction of lysozyme with 2,4,6-trinitrobenzenesulphonic acid was found to display an acceleration effect. 7. From the reaction of 2,4,6-trinitrobenzenesulphonic acid with glutamate dehydrogenase at several enzyme concentrations, it was possible to discern two sets of amino groups of different reactivity, and to show that the number of groups in each set was decreased by aggregation of the enzyme.
| S-EPMC1198823 | biostudies-other
Project description:1. A method is described for determining the ionization constants and reactivities of individual amino groups in proteins. The principle is that in the presence of a trace amount of radioactive label, the various reactive groups in a protein molecule will compete for the label and the amount incorporated into any one group will be determined by its nucleophilicity, pK and micro-environment. The relative amounts of label incorporated into various groups will be proportional to their second-order rate constants and by comparing these rate constants with those expected on the basis of a linear free-energy relationship obtained with a series of standard compounds, the micro-environment can be defined for a particular amino group. 2. The method consists of treating a protein and an internal standard with a limiting amount of radioactive reagent and then with an excess of unlabelled reagent to yield a chemically homogeneous but heterogeneously labelled compound. After appropriate enzymic digestion peptides containing each labelled group are isolated and their rates of reaction, relative to the internal standard, are determined from their specific radioactivities. The entire procedure is repeated at several pH values. 3. When the method was applied to the amino groups of porcine elastase by using tritiated acetic anhydride as the labelling reagent, the N-terminus was found to have pK(a) 9.7 and a much lower than normal reactivity. Lysine-87 and lysine-224 were found to have pK(a) 10.3 and normal reactivities. At pH values greater than 10.5 there are discontinuities in all the titration curves, indicating that the entire molecule is undergoing a structural reorganization.
| S-EPMC1177144 | biostudies-other