Blockage of lysine by Mallard's reaction. II. Chemical properties of derivatives N-(deoxy-1-D-fructosyl-1) and N-(deoxy-1-D-lactulosyl-1) lysine

Lysine is often inactivated by ‘Maillard-type’ reactions in food proteins; the values obtained for it by different methods cannot be compared because the chemical behaviour of inactivated lysine is unknown. In this paper, the behaviour of an important form of inactivated lysine, namely its 1-deoxyketosyl derivatives, during acid hydrolysis as well as on the different assays for available lysine (F-DNB, TNBS, pipsylchloride and guanidination) is studied. The relative proportions of regenerated lysine and of formed furosine and pyridosine are established as a function of the conditions of hydrolysis. The validity of the methods used to estimate available lysine from its 1-deoxyketosyl derivatives is discussed.     

Absorption of chemically unmodified lysine from proteins in foods that have sustained damage during processing or storage

When a food is processed or stored, amino acids can react with a number of chemical entities to produce new compounds that are often nutritionally unavailable to the consumer. During acid hydrolysis used in amino acid analysis, some of these compounds revert back to the parent amino acid, leading to errors in estimates of both the amino acid content of foods and amino acid digestibility. This is a particular concern for the amino acid lysine in damaged food proteins. Chemical assays have thus been developed to allow determination of unaltered or reactive lysine. However, there is evidence that, in damaged food proteins, not all of the reactive lysine is released during digestion and absorbed. The development and application of an assay for absorbed (ileal digestible) reactive lysine is discussed.     

Liquid chromatographic determination of the total available free and intrachain lysine in various foods

A method for the quantitative determination of the total available lysine in various foods is developed. The method is based on the reaction of the amino groups on the lysine molecule with fluorodinitrobenzene and is capable of furnishing simultaneous determination of the available intrachain lysine (known as N-epsilon-[2,4-dinitrophenyl]-L-lysine) or the available free and/or N-terminal lysine (known as N,N'-di-[2,4-dinitrophenyl]-L-lysine). Optimum conditions for separation and quantitation are studied. The results show the proposed method to be both accurate and precise and suitable for food samples containing hydrolyzed proteins.     

Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355?nm

We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, Hde, and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight tandem mass spectrometry (TOF-TOF-MS/MS) and post-source decay (PSD). We also reported the absence of adduct formation when using ??-cyano-4-hydroxycinnamic acid (CHCA) matrix. Further mass spectrometric analysis of disulfide-intact and disulfide-reduced over-expressed HdeA and HdeB proteins from lysates of gene-inserted E. coli plasmids suggests covalent attachment of SA occurs not at cysteine residues but at lysine residues. In this revised hypothesis, the attachment of SA is preceded by formation of a solid phase ammonium carboxylate salt between SA and accessible lysine residues of the protein during sample preparation under acidic conditions. Laser irradiation at 355?nm of the dried sample spot results in equilibrium retrogradation followed by nucleophilic attack by the amine group of lysine at the carbonyl group of SA and subsequent amide bond formation and loss of water. The absence of CHCA adducts suggests that the electron-withdrawing effect of the ??-cyano group of this matrix may inhibit salt formation and/or amide bond formation. This revised hypothesis is supported by dissociative loss of SA (?224?Da) and the amide-bound SA (?206?Da) from SA-adducted HdeA and HdeB ions by MS/MS (PSD). It is proposed that cleavage of the amide-bound SA from the lysine side-chain occurs via rearrangement involving a pentacyclic transition state followed by hydrogen abstraction/migration and loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal (?206?Da).     

Integrative Chemical Biology Approaches for Identification and Characterization of “Erasers” for Fatty‐Acid‐Acylated Lysine Residues within Proteins

Acylation of proteins with fatty acids is important for the regulation of membrane association, trafficking, subcellular localization, and activity of many cellular proteins. While significant progress has been made in our understanding of the two major forms of protein acylation with fatty acids, N‐myristoylation and S‐palmitoylation, studies of the acylation of lysine residues, within proteins, with fatty acids have lagged behind. Demonstrated here is the use of integrative chemical biology approaches to examine human sirtuins as de‐fatty‐acid acylases in vitro and in cells. Photo‐crosslinking chemistry is used to investigate enzymes which recognize fatty‐acid acylated lysine. Human Sirt2 was identified as a robust lysine de‐fatty‐acid acylase in vitro. The results also show that Sirt2 can regulate the acylation of lysine residues, of proteins, with fatty acids within cells.     

ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

-Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acid analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm² total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid or in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. The incubation of lens proteins with dehydroascorbic acid or l -threose, but not fructose, produced equivalent glycation, protein crosslinking and sensitizer activity. The relative sensitizer activity of the 4 week glycated sample was quantitatively very similar to that of a water-insoluble fraction from aged human lenses. These data are consistent with the hypothesis that the protein-bound brunescence in the lens may be advanced glycation endproducts, which are formed in large part by the oxidation products of ascorbic acid, and that these compounds may contribute significantly to the UVA sensitizer activity present in aged human lenses.     

Photochemical addition of amino acids and peptides to homopolyribonucleotides of the major DNA bases

Abstract— The photochemical quantum yields for addition of glycine and the L-amino acids commonly occurring in proteins (excluding proline) to polyadenylic acid, polycytidylic acid, polyguanylic acid and polyribothymidylic acid have been determined in deoxygenated phosphate buffer at Λ 254 nm and pH 7, using a fluorescamine assay technique. Polyadenylic acid was reactive with eleven of the twenty amino acids tested, with phenylalanine, tyrosine, glutamine, lysine and asparagine having the highest quantum yields. Polyguanylic acid reacted with sixteen amino acids; phenylalanine, arginine, cysteine, tyrosine, and lysine displayed the largest quantum yields. Polycytidylic acid showed reactivity with fifteen amino acids with lysine, phenylalanine, cysteine, tyrosine and arginine having the greatest quantum yields. Polyribothymidylic acid, reactive with fifteen of nineteen amino acids surveyed, showed the highest quantum yields for cysteine, phenylalanine, tyrosine, lysine and asparagine. None of the polynucleotides were reactive with aspartic acid or glutamic acid.
The quantum yields for photoaddition of eighteen dipeptides of the form glycyl X (X being one of the amino acids commonly occurring in proteins, including proline), and of L-alanyl-L-tryptophan, L-seryl-L-seryl-L-serine, L-threonyl-L-threonyl-L-threonine, L-cystine- bis -glycine, and N_α-acetyllysine to polyadenylic acid, polycytidylic acid and polyguanylic acid were measured. All of these were found to add photochemically to each of these polymers. Polyribothymidylic acid, tested with eleven of these peptides and with N_α-acetyllysine, was found to be reactive with all.     

Available lysine in foods: a brief historical overview

A brief historical overview is presented on the development of the science addressing lysine bioavailability in foods. Early observations that dietary protein utilization did not always correlate with gross amino acid composition led to an understanding that the amino acid lysine, in particular, can be easily damaged during food processing. Conventional amino acid analysis, involving a strong acid hydrolysis step, can lead to a significant degree of overestimation of lysine in processed foods. More recently, it has been found that not only food lysine content values but also estimates of lysine digestibility and digestible lysine contents may be erroneous. Estimates of absorbed (true ileal digestible) reactive lysine are accurate measures of available lysine. Technically, bioassays such as the slope-ratio assay determine utilized rather than available lysine.     

Covalent binding of human serum albumin and ovalbumin by chloramine-T and chemical modification of the proteins

The covalent binding of ³⁵S-chloramine-T to human resum albumin (HSA) and ovalbumin is described. At pH 6.5, up to 24 chloramine-T molecules were found to be covalently bound per molecule of HSA; with ovalbumin the binding was only 5–7 molecule per protein molecule. Binding was accompanied by extensive modification of methionine, cysteine, histidine, tyrosine and lysine. Three new peaks appeared in the amino acid profiles of the modified proteins; two were identified as 1-aminoadipic acid (oxidation of lysine) and 3-chlorotyrosine. The most sites for covalent binding are lysine residues.     

Helical peptide models for protein glycation: proximity effects in catalysis of the Amadori rearrangement.

INTRODUCTION: Non-enzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. The observation that only a fraction of all lysine residues undergo glycation indicates the role of the immediate chemical environment in the glycation reaction. Here we have constructed helical peptide models, which juxtapose lysine with potentially catalytic residues in order to probe their roles in the individual steps of the glycation reaction. RESULTS: The peptides investigated in this study are constrained to adopt helical conformations allowing residues in the i and i+4 positions to come into spatial proximity, while residues i and i+2 are far apart. The placing of aspartic acid and histidine residues at interacting positions with lysine modulates the steps involved in early peptide glycation (reversible Schiff base formation and its subsequent irreversible conversion to a ketoamine product, the Amadori rearrangement). Proximal positioning of aspartic acid or histidine with respect to the reactive lysine residue retards initial Schiff base formation. On the contrary, aspartic acid promotes catalysis of the Amadori rearrangement. Presence of the strongly basic residue arginine proximate to lysine favorably affects the pK(a) of both the lysine epsilon-amino group and the singly glycated lysine, aiding in the formation of doubly glycated species. The Amadori product also formed carboxymethyl lysine, an advanced glycation endproduct (AGE), in a time-dependent manner. CONCLUSIONS: Stereochemically defined peptide scaffolds are convenient tools for studying near neighbor effects on the reactivity of functional amino acid sidechains. The present study utilizes stereochemically defined peptide helices to effectively demonstrate that aspartic acid is an efficient catalytic residue in the Amadori arrangement. The results emphasize the structural determinants of Schiff base and Amadori product formation in the final accumulation of glycated peptides.     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.     

Utility of N-peracetylation of proteins for their structure determination by mass spectrometry

Acetylation of the animo groups (N-terminus and lysine) of proteins before enzymatic or chemical cleavage was explored as an approach to provide additional information in the course of the determination of amino acid sequences. The major advantage is the ability to differentiate glutamine from lysine, because only the latter is acetylated and thus increases in mass by 42 Da. Horse heart cytochrome c could be fully N-actetylated and even on prolonged digestion with chymotrypsin underwent very little tryptic cleavage, in contrast to the native protein where this side reaction is extensive. Sperm whale myoglobin is more difficult to acetylate, but even at 40%–50% average acetylation, all 19 lysines could be identified unambiguously. A proteolytic digest of acetylated protein is thus a useful component of strategies for the determination of the primary structure of proteins by tandem mass spectrometry.     

Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C(18) high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.     

NON-ENZYMIC METHODS FOR THE PHOSPHORYLATION OF PROTEINS1

Abstract

Non-enzymic methods for the phosphorylation of proteins are reviewed with respect to type of reagent, protein source, reactive site (N[sbnd], O[sbnd] or S[sbnd]), extent of reaction, and effect on physical properties, functionality and biological properties. Experimental procedures are given to illustrate the most important methods. Some enzymic methods are included for comparison. The evidence for phosphorylation of specific amino acid residues, such as serine, threonine, histidine or lysine, is critically assessed. Conclusions are drawn regarding the status of phosphorylation as a synthetic method for modifying proteins.     

Hydroxamic Acid‐Piperidine Conjugate is an Activated Catalyst for Lysine Acetylation under Physiological Conditions

Lysine acylation of proteins is an essential chemical reaction for posttranslational modification and as a means of protein modification in various applications. N,N‐Dimethyl‐4‐aminopyridine (DMAP) derivatives are widely‐used catalysts for lysine acylation of proteins; however, the DMAP moiety mostly exists in a protonated, and thus deactivated, form under physiological conditions due to its basicity. An alternative catalytic motif furnishing higher acylation activity would further broaden the possible applications of chemical lysine acylation. We herein report that the hydroxamic acid‐piperidine conjugate Ph‐HXA is a more active catalytic motif for lysine acetylation than DMAP under physiological conditions. In contrast to DMAP, the hydroxamic acid moiety is mostly deprotonated under aqueous neutral pH, resulting in a higher concentration of the activated form. The Ph‐HXA catalyst is also more tolerant of deactivation by a high concentration of glutathione than DMAP. Therefore, Ph‐HXA might be a suitable catalytic motif for target protein‐selective and site‐selective acetylation in cells.     

Potentiometric sensor array for the determination of lysine in feed samples using multivariate calibration methods

A potentiometric sensor array has been developed for the determination of lysine in feed samples. The sensor array consists of a lysine biosensor and seven ion-selective electrodes for NH4+, K+, Na+, Ca2+, Mg2+, Li+, and H+, all based on all-solid-state technology. The potentiometric lysine biosensor comprises a lysine oxidase membrane assembled on an NH4+ electrode. Because the selectivity of the lysine biosensor towards other cation species is not sufficient, there is severe interference with the potentiometric response. This poor selectivity can be circumvented mathematically by analysis of the richer information contained in the multi-sensor data. The sensor array takes advantage of the cross-selectivity of lysine for each electrode, which differs from the other species and quantification of lysine in complex feed sample extracts is accomplished with multivariate calibration methods, such as partial least-squares regression. The results obtained are in a reasonable agreement with those given by the standard method for amino acid analysis.     

Theoretical Study on the Mechanisms of Action of Sulfurand Nitrogen-containing Amino Acid Residues with Monofunctional Guanine Adduct Formed by Antitumor Drugs trans-[PtCl2（Am）（isopropylamine）] （Am = Isopropylamine,Dimethylamine and Propylamine） and Their cis Isomers

The mechanisms of action on monofunctional guanine adducts of analogues of transplatin with aliphatic amine ligands,such as trans-[Pt（Am）（isopropylamine）（G）（H2O）] where Am represents dimethylamine,propylamine or isopropylamine and their cis isomers reacting with sulfur- and nitrogen-containing amino acid residues,were explored. Histidine and lysine residues are chosen as the model ligands of nitrogen-containing amino acid residues of proteins; meanwhile,methionine and cysteine residues are chosen as the model ligands of sulfur-containing amino acid residues of proteins. A dominating preference for sulfur-containing ligand over nitrogen-containing ligand is established. The calculated smallest activation barrier for sulfur-containing ligand is 9.9,and 21.1 kcal/mol for nitrogen-containing ligand in aqueous solution,and both of them have trans configurations. The difference in activation energy is 11.2 kcal/mol,indicating the platination of sulfur-containing amino acid residues is faster by seven to eight orders of magnitude than that of nitrogen-containing amino acid residues.     

Detection and quantitation of additions of soybean proteins in cured-meat products by perfusion reversed-phase high-performance liquid chromatography

Perfusion liquid chromatography has been applied in this work to the determination of soybean proteins in commercially available cured meat products, enabling the detection of additions of soybean proteins in cured meat products to which the addition of these vegetable proteins is forbidden and the quantitation of soybean proteins in cured meat products to which the addition of these proteins is allowed up to a certain limit. The analytical methodology is based on a sample treatment (fat extraction and soybean protein solubilization) prior to chromatographic analysis. Fat extraction with acetone and soybean protein solubilization with a buffer solution at basic pH (pH 10 or 9) were necessary to obtain selective and sensitive conditions. Use of water-acetonitrile-trifluoroacetic acid or water-tetrahydrofuran-trifluoroacetic acid linear binary gradients at a flow rate of 3 mL/min, a temperature of 50 degrees C, and UV detection at 280 nm enabled chromatographic analysis of soybean proteins in cured meat products in less than 3 min.