Solid phase isotope exchange with spillover hydrogen in amino acids,peptides, and proteins

We summarize here information on the theoretical and experimental study of high-temperature (150–200°C) solid phase catalytic isotope exchange (HSCIE) carried out with amino acids, peptides, and proteins under the action of spillover hydrogen. Main specific features of the HSCIE reaction, its mechanism, and its use for studying spatial interactions in polypeptides are discussed. A virtually complete absence of racemization makes this reaction a valuable preparative method. The main regularities of the HSCIE reaction with the participation of spillover tritium have been revealed in the case of peptides and proteins, and the dependence of reactivity of peptide fragments on the spatial organization of their molecules has been studied. An important peculiarity of this reaction is that HSCIE proceeds at 150–200°C with a high degree of chirality retention in amino acids and peptides. This is provided by its reaction mechanism, which consists in a synchronous one-center substitution at the saturated carbon atom characterized by the formation of pentacoordinated carbon and a three-center bond between the carbon and the incoming and outgoing hydrogen atoms.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 3–21.Original Russian Text Copyright © 2005 by Zolotarev, Dadayan, Borisov.     

The solid-state catalytic synthesis of tritium labeled amino acids,peptides and proteins

Summary New catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993     

Peptide design using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins

Incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded conformations: beta-turns in short peptides and 3(10)-helices in larger ones. A few exceptions-namely, alpha-helix or flat beta-bend ribbon structures-have also been reported in a few cases. The most favorable conformation of DeltaPhe residues are (phi,psi) approximately (-60 degrees, -30 degrees ), (-60 degrees, 150 degrees ), (80 degrees, 0 degrees ) or their enantiomers. DeltaPhe is an achiral and planar residue. These features have been exploited in designing DeltaPhe zippers and helix-turn-helix motifs. DeltaPhe can be incorporated in both right and left-handed helices. In fact, consecutive occurrence of three or more DeltaPhe amino acids induce left-handed screw sense in peptides containing L-amino acids. Weak interactions involving the DeltaPhe residue play an important role in molecular association. The C--H.O==C hydrogen bond between the DeltaPhe side-chain and backbone carboxyl moiety, pi-pi stacking interactions between DeltaPhe side chains belonging to enantiomeric helices have shown to stabilize folding. The unusual capability of a DeltaPhe ring to form the hub of multicentered interactions namely, a donor in aromatic C--H.pi and C--H.O==C and an acceptor in a CH(3).pi interaction suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures.     

Hydrolysis technology of biomass waste to produce amino acids in sub-critical water

Hydrolysis of biomass waste (such as fish waste, chicken waste, hair and feather) to produce amino acids was studied in sub-critical water, with reaction temperatures from 180 to 320 degrees C and reaction pressures from 3 to 30 MPa. The product of amino acid was determined by Amino Acid Analyzer (BioLC), and 18 kinds of amino acid were obtained. The results show that the controlling of reaction atmosphere, pressure, temperature and time of hydrolysis is very important to obtain high yield of amino acid; most of amino acids reached maximum yield at reaction temperature range of 200-290 degrees C and reaction time range of 5-20 min. There are obvious changes of amino acids yield at reaction pressures of 6-16 MPa and reaction temperature around 260 degrees C, owing to the homogeneousness of the first two phases of water in the formation of vapor and liquid. There are different yields of the same amino acid in different reaction atmospheres (e.g. air, carbon dioxide and nitrogen).     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria

Based on primary structure comparison between four highly homologous DNA-binding proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the thermophilic HUBst from Bacillus stearothermophilus (Tm = 61.6 degrees C) and the mesophilic HUBsu from Bacillus subtilis (Tm = 39.7 degrees C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the protein (Tm = 45.1 degrees C) at the level of its mesophilic homologue HUBsu. By replacing these amino acids in HUBsu with their thermophilic counterparts, the HUBsu-E15G/D34E/142V mutant was generated with thermostability (Tm = 57.8 degrees C) at the level of thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely thermophilic HUTmar from Thermotoga maritima (Tm = 80.5 degrees C), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V421 (Tm = 35.9 degrees C) converted the extremely thermophilic protein HUTmar to mesophilic. The various forms of HU proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the protein are responsible for the stability of HU proteins by acting cooperatively in a common mechanism for thermostability.     

Peptide Formation Mechanism on Montmorillonite Under Thermal Conditions

The oligomerization of amino acids is an essential process in the chemical evolution of proteins, which are precursors to life on Earth. Although some researchers have observed peptide formation on clay mineral surfaces, the mechanism of peptide bond formation on the clay mineral surface has not been clarified. In this study, the thermal behavior of glycine (Gly) adsorbed on montmorillonite was observed during heating experiments conducted at 150 °C for 336 h under dry, wet, and dry–wet conditions to clarify the mechanism. Approximately 13.9 % of the Gly monomers became peptides on montmorillonite under dry conditions, with diketopiperazine (cyclic dimer) being the main product. On the other hand, peptides were not synthesized in the absence of montmorillonite. Results of IR analysis showed that the Gly monomer was mainly adsorbed via hydrogen bonding between the positively charged amino groups and negatively charged surface sites (i.e., Lewis base sites) on the montmorillonite surface, indicating that the Lewis base site acts as a catalyst for peptide formation. In contrast, peptides were not detected on montmorillonite heated under wet conditions, since excess water shifted the equilibrium towards hydrolysis of the peptides. The presence of water is likely to control thermodynamic peptide production, and clay minerals, especially those with electrophilic defect sites, seem to act as a kinetic catalyst for the peptide formation reaction.     

Hydrogen bonds in crystal structures of amino acids, peptides and related molecules

The results of a survey of 439 hydrogen bonds in 95 recently determined crystal structures of amino acids, peptides and related molecules suggest that the following generalizations hold true for linear (angle X-H---Y greater than 150 degrees) hydrogen bonds. (1) The charge on the acceptor group does not influence the length of a hydrogen bond. (2) For a given acceptor group, the hydrogen bond lengths increase in the order imidazolium N--H less than ammonium N-H less than guanidinium N-H; this order holds true for oxygen anion acceptor groups. Cl-ions and the uncharged oxygen of water molecules. (3) The uncharged imidazole N-H group forms shorter hydrogen than the amide N-H GROUP. (4) The carboxyl O-H groups form shorter hydrogen bonds than other hydroxyl groups. (5) The hydrogen bonds involving a halogen ion are longer than hydrogen bonds with other acceptors when corrected for their longer van der Walls radii. The observed differences between the lengths of hydrogen bonds formed by different donor and acceptor groups in amino acids and peptides, imply differences in the energetics of their formation.     

Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins

Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10-25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at alpha-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.     

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B_1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.     

The measurement of amino groups in proteins and peptides

A technique is examined for determining amino groups with 2,4,6-trinitrobenzenesulphonic acid, in which the extinction at 420nm of sulphite complexes of the trinitrophenylated amino groups is measured. The sensitivity of the method is 5-200nmol of amino group. The method is especially suitable for checking the extent of blocking or unblocking of amino groups in proteins and peptides, owing to the short time required for reaction (5min at room temperature). The reaction of the reagent with thiol groups has been studied and was found to proceed 30-50 times faster than with in-amino groups of model compounds. The in(420) of a trinitrophenylated thiol group was found to be 2250m(-1).cm(-1). The reaction with several amino acids, peptides and proteins is presented. The in(420) of a typical alpha-amino group was found to be 22000m(-1).cm(-1) and that of an in-amino group, 19200m(-1).cm(-1). Difficulties inherent in the analysis of constituent amino group reactions in proteins are discussed.     

Value-added subcritical water hydrolysate from rice bran and soybean meal

New value-added product was derived from agricultural by-products: rice bran and soybean meal by means of subcritical water (SW) hydrolysis. The effect of temperature (200-220 degrees C), reaction time (10-30 min), raw material-to-water weight ratio (1:5 and 2:5), was determined on the yields of protein, total amino acids, and reducing sugars in the soluble products. The suitable hydrolysis time was 30 min and the proper weight ratio of the raw material-to-water was 1:5. The reaction temperature suitable for the production of protein and amino acids was 220 degrees C for raw and deoiled rice bran, 210 degrees C for raw soybean meal, and 200 degrees C for deoiled soybean meal. The products were also found to have antioxidant activity as tested by ABTS(.)(+) scavenging assay. In addition, sensory evaluation of milk added with the hydrolysis product of deoiled rice bran indicated the potential use of the product as a nutritious drink.     

Protein modification by diazotized arsanilic acid: synthesis and characterization of the phenylthiohydantoin derivatives of azobenzene arsonate-coupled tyrosine, histidine, and lysine residues and their sequential allotment in labeled peptides

Analytical procedures are elaborated for the sequential allotment of azobenzene arsonate binding sites in proteins and peptides. The reaction of diazotized arsanilic acid with proteins leads to covalent modification of tyrosine, histidine and, in part, lysine residues. Synthetic peptides containing these amino acids were modified with diazotized arsanilic acid and subjected to N-terminal sequence analysis. The amino acid derivatives phenylthiohydantoin(Pth)-azobenzene-arsonate-tyrosine, Pth-azobenzene-arsonate-histidine, and alpha-Pth-epsilon-hydroxycaproic acid are recovered upon Edman degradation of selected peptides. Phenylthiohydantoins of modified and nonmodified amino acids are fully separated by reverse-phase HPLC on a Zorbax-PTH column. For identification purposes, phenylthiohydantoins of azobenzene arsonate-labeled amino acids have been synthetized. They are characterized with respect to spectral absorption characteristics and retention times on reverse-phase supports.     

A review of adsorption of amino acids on minerals: Was it important for origin of life?

Minerals more readily adsorb amino acids with charged R groups than uncharged R groups, so that the incorporation of amino acids with charged R groups into peptides would be more frequent than for amino acids with uncharged R groups. However, 74% of the amino acids in the proteins of modern organisms contain uncharged R groups. Thus, what could have been the mechanisms that produced peptides/proteins with more amino acids with uncharged R groups than precursors with charged R groups? Should we expect the composition of amino acids adsorbed on minerals to be similar to those of present proteins? Was the adsorption of amino acids on minerals important for the origin of life? The lipid world offers an alternative view of origin of life. Liposomes contributed to elongation of peptides as well as select hydrophobic amino acids and peptides. These experiments could be showing the mechanism, which hydrophobic amino acids have been selected. However, liposomes have no influence on the stereoselectivity in the oligomerization of amino acids. In the present paper, several other mechanisms are also discussed that could produce peptides with a greater proportion of amino acids with uncharged R groups.     

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.     

Hydrolysis of proteins and peptides in a hermetically sealed microcapillary tube: high recovery of labile amino acids

A method for the hydrolysis of peptides and proteins in a hermetically sealed microcapillary tube has been developed. The method is based on the concept that oxidative degradation of labile amino acids during acid hydrolysis of proteins and peptides at high temperature can be reduced to a minimum by limiting the ratio of air to liquid (v/v, less than 1:10) in a microcapillary tube. Furthermore, the physical constraints imposed by the capillary tube will restrict the exposure of the protein solution to air at a very limited area at the meniscus of the liquid. This method eliminates the necessity of time-consuming sealing under vacuum and/or flushing with nitrogen to remove oxygen in the hydrolysis tube. High recovery of labile amino acids can be obtained in a reproducible manner. Because of the simplicity and high reproducibility of the method described, it could be the method of choice for the hydrolysis of protein and peptide intended for quantitative amino acid analysis. Performic acid oxidation is performed at 50 degrees C for 10 min instead of 4 to 20 h at 0 degrees C to achieve an equally good yield of cysteic acid and methionine sulfone from peptides and proteins.     

Recognition of a flipped base in a hairpinloop DNA by a small peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258-259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 x 10(6) variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use pi-pi stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 x 10(6) M(-1) at 25 degrees C and the resulting binding free energy change at 25 degrees C (DeltaG degrees (25)) was -8.2 kcal mol(-1). The binding of the peptide to AP2 was also analyzed and the resulting binding constant and DeltaG degrees (25) were about 4.2 x 10(4) M(-1) and -6.3 kcal mol(-1), respectively. The difference in the binding free energy changes (DeltaDeltaG degrees (25)) of 1.9 kcal mol(-1) was comparable to the values reported in other systems and was considered a consequence of the loss of pi-pi stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Determination of methionine sulfoxide in protein and food by hydrolysis with p-toluenesulfonic acid

Methionine sulfoxide in peptides and proteins was determined by use of 3 N p-toluenesulfonic acid as a hydrolyzing agent. Samples were hydrolyzed at 110 degrees C for 22 h in an evacuated sealed tube and analyzed for amino acid content. Amino acid analysis showed that the recovery of methionine sulfoxide from a synthetic peptide and its mixture with proteins was consistently better than 90%. The recovery of all other amino acids except tryptophan was complete, and was similar to that observed after hydrolysis with 6 N HCl. The presence of carbohydrates had no effect on the yield. Thus, the present procedure can be used for general and simultaneous determination of methionine sulfoxide as well as other amino acids in proteins.     

A non-specific aminopeptidase from Aspergillus

A fermentation broth supernatant of the Aspergillus oryzae strain ATCC20386 contains aminopeptidase activity that releases a wide variety of amino acids from natural peptides. The supernatant was fractionated by anion exchange chromatography. Based on the primary amino acid sequence data obtained from proteins in certain fractions, polymerase chain reaction (PCR) primers were made and a PCR product was generated. This PCR product was used to screen an A. oryzae cDNA library from which the full length gene was then obtained. Fusarium venenatum and A. oryzae were used as hosts for gene expression. Transformed strains of both F. venenatum and A. oryzae over-expressed an active aminopeptidase (E.C. 3.4.11), named aminopeptidase II. The recombinant enzyme from both fungal hosts appeared as smears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After deglycosylation of the N-linked sugars, both samples were a sharp band at approximately 56 kDa and had identical N-terminal amino acid sequences. Aminopeptidase II is a metalloenzyme with, presumably, Zn in the active site. Using various natural peptides and para-nitroanilides (pNAs) of amino acids as substrates, the aminopeptidase was found to be non-specific. Only X-Pro bonds demonstrated resistance to hydrolysis catalyzed by this aminopeptidase. The optimal enzyme activity was observed at pH 9.5 and 55 degrees C. Among amino acid pNAs, Leu-pNA appears to have the highest value of bimolecular constant of 40 min(-1) mM(-1) (k(cat) = 230 min(-1); K(m) = 5.8 mM) at pH 7.5 and 21 degrees C. Among Xaa-Ala-Pro-Tyr-Lys-amide pentapeptides, the velocity of catalytic hydrolysis at pH 7.5 and 21 degrees C was in a decreasing order: Pro, Ala, Leu, Gly and Glu.