Preparation and characterization of N epsilon-(2,3-dihydroxypropyl)-L-lysine and its phenylthiohydantoin derivative

Convenient methods for preparative synthesis of N epsilon-(2,3-dihydroxypropyl)-L-lysine and its phenylthiohydantoin derivative are described. The former compound was characterized by elemental analysis, melting point, and ion-exchange chromatography and the latter by elemental analysis, melting point, UV-spectrum, HPLC and thin-layer chromatography. This study was performed for investigations of lysine residues in proteins.     

Synthesis and characteristics of modified oligodeoxyribonucleotides containing 2'-O-(2,3-dihydroxypropyl)uridine and 2'-O-(2-exoethyl)uridine

A new uridine derivative, 2'-O-(2,3-dihydroxypropyl)uridine, and its 3'-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2'-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2'-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2' position of the carbohydrate moiety was confirmed by the interaction with p-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units is practically indistinguishable from that of the natural analogues.     

The mammalian hypusine-containing protein, eukaryotic initiation factor 4D. Structural homology of this protein from several species

A single cellular protein of Mr approximately 18,000 and pI near 5.1, recently identified as eukaryotic translation initiation factor eIF-4D, contains the unusual amino acid hypusine [N epsilon-(4-amino--2-hydroxybutyl)lysine] formed post-translationally from lysine with a structural contribution from the polyamine spermidine. When the 3H-labeled hypusine-containing protein isolated from Chinese hamster ovary (CHO) cells that were grown with radioactive polyamine is digested with trypsin and the digest is subjected to two-dimensional separation, a single radioactive peptide is seen. A labeled peptide that occupies this same position is found in a digest of the [3H]hypusine protein from human lymphocytes and the single hypusine-containing tryptic peptide from purified rabbit reticulocyte eIF-4D also moves to this identical position. Stepwise Edman degradation of the tryptic digest of CHO cell hypusine-protein releases the radioactivity as a single peak in accordance with our earlier evidence for a single hypusine residue per molecule of eIF-4D. The similar patterns of radioactive peptides obtained from tryptic digests of radioiodinated eIF-4D from CHO cells, human lymphocytes, and rabbit reticulocytes suggest a highly conserved primary structure for this protein.     

A 15N NMR study on D-lysine metabolism in Neurospora crassa

The metabolic relationship of D-lysine, L-lysine, and L-pipecolic acid has been investigated in Neurospora crassa. Kinetic experiments show that radioactivity from D-lysine is efficiently incorporated into L-pipecolic acid and that this metabolite is converted to L-lysine. The alpha-amino group from D-[alpha-15N]lysine is lost in the course of its conversion to L-pipecolic acid and is trapped by pyruvate and alpha-keto glutarate to give L-[alpha-15N]alanine and L-[alpha-15N]glutamic acid. These amino acids are devoid of any label, however, when D-[epsilon-15N]lysine is applied to the fungus. As determined by mass and 15N NMR spectrometry the label from D-[epsilon-15N]lysine migrate via L-pipecolic acid into the alpha position of L-lysine, i.e. D-[epsilon-15N]lysine is converted to L-[alpha-15N]lysine. L-Pipecolic acid functions as an intermediate in this conversion.     

4-Aminobutyrate aminotransferase: identification of lysine residues connected with catalytic activity

Bis-PLP (P'P2-bis[5'-pyridoxal]diphosphate) was used as a probe of the catalytic site of 4-aminobutyrate aminotransferase. It reacts with lysine residues connected with aminotransferase activity and the binding of 1 mol of reduced bis-PLP/enzyme monomer abrogates catalytic activity. The reactive lysine residues are characterized by low pK values (pK = 7.3). The presence of substrate 2-oxoglutarate (4 mM) prevents inactivation of the aminotransferase treated with bis-PLP. After tryptic digestion of the enzyme modified with bis-PLP and reduced with tritiated NaBH4, a radioactive peptide absorbing at 320 nm was separated by reverse-phase high-performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysine residue of monomeric 4-aminobutyrate aminotransferase has reacted with bis-PLP. The sequence of the modified peptide differs from the sequence of the peptide bearing the cofactor pyridoxal-5-P covalently attached to a lysine residue. It is postulated that the modified lysine residue is involved in direct interactions with negatively charged carboxylic groups of 2-oxoglutarate.     

Selectivity in the modification of the alpha-amino groups of hemoglobin on reductive alkylation with aliphatic carbonyl compounds. Influence of derivatization on the polymerization of hemoglobin S

The reactivity of the alpha-amino groups of the alpha- and beta-chains of hemoglobn toward reductive alkylation using limiting concentrations of the aliphatic carbonyl compounds, acetaldehyde (ethylation), glyoxylic acid (carboxymethylation), glycolaldehyde (hydroxyethylation), glyceraldehyde (dihydroxypropylation), and dihydroxyacetone (dihydroxyisopropylation) has been investigated. Hemoglobin A reductively ethylated at the alpha-amino groups eluted on CM-52 ahead of unmodified hemoglobin A, and hemoglobin A reductively ethylated at the epsilon-amino groups. This observation is similar to that seen on hydroxyethylation and dihydroxypropylation of the alpha-amino group of hemoglobin A. The presence of the alpha-hydroxyl or the carboxyl group in the carbonyl component used in the reductive alkylation influences considerably the selectivity pattern during the derivatization. The alpha-amino groups of the alpha- and beta-chains are modified to nearly the same degree during reductive hydroxyethylation as well as during reductive dihydroxypropylation. Reductive ethylation (aldehyde lacking the alpha-hydroxyl group) exhibited a slight preferential reaction at Val-1(beta). The presence of a negatively charged carboxyl group in the carbonyl component, i.e. glyoxylic acid, made this preferential reaction at Val-1(beta) even more pronounced. When the reductive alkylation is carried out with dihydroxyacetone (a ketone instead of an aldehyde), the dihydroxyisopropylation occurred at a slower rate and exclusively at Val-1(beta). The ethylation, hydroxyethylation, carboxymethylation, and dihydroxypropylation of the alpha-amino groups of hemoglobin S increased its solubility from the value of 16 g/dl for the unmodified protein to about 25 g/dl for the modified protein. Thus, the alkyl chains on the alpha-amino groups on the polymerization have a strong inhibitory influence. In order to determine the influence of the alkyl chains at the alpha-amino groups of alpha- and beta-chains on polymerization, hybrid hemoglobin S tetramers with hydroxyethylation either at Val-1(alpha) or at Val-1(beta) have been prepared. The solubility of each hybrid is about 26 g/dl. Thus, the hydroxyethyl group either on the alpha- or the beta-chain appears to interfere with the polymerization of deoxygenated HbS to the same degree. The inhibitory influence of the hydroxyethyl chain at Val-1(alpha) on the polymerization, compared with the lack of such an influence when this alpha-amino group is modified by cyanate, suggests that a carbamoyl group on Val-1(alpha) can be accommodated in the intermolecular contact region involving this segment of the molecule without seriously perturbing the mo     

Synthesis and Properties of Modified Oligodeoxyribonucleotides Containing 2"-O-(2,3-Dihydroxypropyl)uridine and 2"-O-(2-Oxoethyl)uridine

A new uridine derivative, 2"-O-(2,3-dihydroxypropyl)uridine, and its 3"-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2"-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2"-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2" position of the carbohydrate moiety was confirmed by the interaction withp-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units does not practically differ from that of the natural analogues.     

The structures of the serine-linked sugar chains on human chorionic gonadotropin

The human chorionic gonadotropin beta-subunit tryptic COOH-terminal peptide (residues 123-145) which contains 3 serine-linked sugar chains was isolated. The sugar chains were cleaved by beta-elimination and then separated by gel filtration. The peaks were pooled and their compositions determined. The products of serial glycosidase digestion and periodate oxidation of the intact glycopeptide were also characterized. Of the serine-linked sugar chains, 13% were the hexasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,3 Gal beta 1,4 GlcNAc beta 1,6) GalNAc, 34% the tetrasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,6) GalNAc, 43% the trisaccharide NeuAc alpha 2,3 Gal beta 1,3 GalNAc and 10% the disaccharide NeuAc alpha 2,6 GalNAc.     

Selective 'in synthesis' labeling of peptides with biotin and rhodamine

A new method is described for the selective 'in synthesis' labeling of peptides by rhodamine or biotin at a single, predetermined epsilon-amino group of a lysine residue. The alpha-amino group and other lysyl residues of the peptide remain unmodified. Peptides are assembled by the Fmoc approach, which requires mild operative conditions for the final deprotection and cleavage, and ensures little damage of the reporter group. The labeling technique involves the previous preparation of a suitable Lysine derivative, easily obtained from commercially-available protected amino acids. This new derivative, where the reporter group (biotin, or rhodamine) acts now as permanent protection of lysyl side chain functions, is then inserted into the synthesis program as a conventional protected amino acid, and linked to the preceding residue by aid of carbodiimide. A simpler, alternative method is also described for the selective 'in synthesis' labeling of peptides with N-terminal lysyl residues. Several applications of labeled peptides are reported.     

The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1.

1. When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37 degrees, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50 degrees and 1 h at 60 degrees. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation. 2. Upon tryptic digestion at 50 degrees, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. 3. When the trinitrophenylated enzyme, in which the alpha-amino group of alanine-1 and the episolone-amino group of lysine 41 were selectively modified, was treated with trypsin at 37 degrees, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.     

Protected natural peptides as intermediates for preparing semisynthetic peptides and protein analogs. Selective acylation of epsilon-amino groups in cyanogen bromide peptides of cytochrome c using azidoformates.

Treatment of CNBr peptides 66--80, 81--104 and 66--104 from cytochrome c with t-butyloxycarbonyl azide leads to selective acylation of the epsilon-amino groups of lysine residues and the phenolic hydroxyl groups of tyrosine residues with less than 25% acylation of the alpha-amino groups. Similar selectivity was obtained for reactions of benzyloxycarbonyl azide, p-nitrobenzyloxycarbonyl azide and p-methoxybenzyloxycarbonyl azide with peptide 81--104. All of these protective groups can be removed under mild conditions, and thus, the partially protected natural peptides are desirable intermediates for the preparation of semisynthetic peptides. Model condensation reactions of N alpha t-butyloxycarbonyl methionine N-hydroxysuccinimide ester with Z-protected peptide 81--104 produced a peptide corresponding to residues 80 to 104 of cytochrome c in 94% yield.     

Glycation of amino groups in protein. Studies on the specificity of modification of RNase by glucose

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.     

Structure of 2-hydroxy-5-nitrobenzylated carboxypeptidase A.

Bovine pancreatic carboxypeptidase A (EC 3.4.12.2) was treated with dimethyl (2-hydroxy-5-nitrobenzyl)sulfonium chloride at pH 7.5, resulting in a preparation which consisted primarily of a monohydroxynitrobenzylated derivative of the enzyme. Samples of the hydroxynitrobenzylated enzyme were subjected to tryptic digestion and to cyanogen bromide cleavage, and resulting peptides were isolated chromatographically. One tryptic hydroxynitrobenzyl-containing peptide was isolated; its amino acid composition was that of the N-terminal tryptic segment of carboxypeptidase Agamma (residues 8--35). Likewise, CNBr cleavage of the hydroxynitrobenzylated enzyme revealed that the hydroxynitrobenzyl group resided in the N-terminal fragment, FN (residues 8--22). Neither of these hydroxynitrobenzylated peptides contains Trp, the amino acid residue which is characteristically the site of hydroxynitrobenzylation in proteins, and each was found to contain approximately one less Asx than the corresponding native peptide. Both dansylation and automated Edman degradation procedures revealed that the N-terminal Asn of carboxypeptidase Agamma had been modified by hydroxynitrobenzylation of the enzyme. Thus the sulfonium salt reacts with carboxypeptidase A in the same manner as that established earlier for 2-hydroxy-5-nitrobenzyl bromide (Radhakrishnan, T.M., Bradshaw, R.A., Deranleau, D.A. and Neurath, H. (1970) FEBS Lett. 7, 72--76). Such reactivity of the alpha-amino group presumably reflects its unique location with respect to Trp residues in the tertiary structure of the enzyme.     

The primary structure of the glycan moiety of pseudomurein from Methanobacterium thermoautotrophicum

After treatment of isolated cells walls of Methanobacterium thermoautotrophicum with sodium hydroxide or anhydrous hydrazine, water soluble glycan strands were obtained. These consisted of alternating (beta 1-3)-linked D-glucosamine and (alpha 1-3)-linked L-talosaminuronic acid residues and their length was about 25 disaccharides. Some of the L-talosaminuronic acid residues remained linked to either glutamic acid or the peptides N-gamma-glutamylalanine and N epsilon-(gamma-glutamylalanyl)lysine, indicating that the peptide moiety of pseudomurein is bound to the carboxyl group of talosaminuronic acid via the amino group of glutamic acid.     

Bioconjugation of ribonuclease A: a detailed chromatographic and mass spectrometric analysis of chemical modification by a cross-linking reagent

The modification of ribonuclease A with the heterobifunctional cross-linker, 4-succinimdyloxycarbonyl-methyl-alpha-[2-pyridyldithio]-toluene (SMPT) is described. RNase A has 11 potential sites of modification by the SMPT reagent. Tracking the two-dimensional separation and proteolytic digestion of SMPT-modified RNase A with ESI/FTICR-MS and HPLC/ESI/QIT-MS demonstrates the detailed information about number of SMPT modifications and sites of modification that can be obtained by application of these techniques. Analysis of native and modified RNase A tryptic digests by ESI/FTICR-MS resulted in the identification of the sites of modification. Semiquantitative results of the reactivity of certain lysine residues toward the coupling reagent SMPT are presented. Two sites (lysines 1 and 37) are highly reactive, while three sites (lysines 41, 61, and 104) appear to be unreactive toward SMPT under the conditions used. Experimental results demonstrate that quantitative comparison of relative intensities of peptide sequences of different charge states is not possible. No correlation was found between number of basic residues and sensitivity to detection. Digestion of the modified and unmodified RNase A by subtilisin followed by examination by HPLC/ESI/QIT-MS and MS(n) enabled further investigation of modification on lysines 1 and 7, including modification at the epsilon- and alpha-amino positions on lysine 1.     

Preparation and ESR study of spin-labeled derivatives of Naja naja oxiana neurotoxin II

After neurotoxin II Naja naja oxiana reaction with N-hydroxysuccinimidyl 2,2,6,6-tetramethyl-4-carboxymethylpiperidine-1-oxyl, six derivatives were isolated, each containing one spin label. Their analysis (reduction, carboxymethylation, tryptic hydrolysis, isolation and identification of the spin labeled peptide) allowed to localize the label position: the epsilon-amino groups of Lys15, Lys25, Lys26, Lys44, Lys48, and alpha-amino group of Leu1. The neurotoxin II reaction with N-hydroxysuccinimidyl 2,2,5,5-tetramethyl-3-carboxypyrrolin-1-oxyl followed by chromatography afforded 10 derivatives, each having two labeled lysine residues, wherein the position of the modified residues was determined. The reactivity and microenvironment of amino groups are discussed basing on the dependence between the reaction conditions and yields. For di-spin labeled derivatives of the pyrroline series, the inter-label distances were determined by EPR from the standard curve and used for refinement of the neurotoxin conformation in solution.     

Interaction of pyridoxal-5-phosphate with human serum albumin and pancreatic ribonuclease

Pyridoxal-5-phosphate (in a lesser degree, pyridoxal) interacts with both non-protonated and protonated exposed epsilon-amino groups of lysine residues and with alpha-amino groups in human serum albumin and pancreatic ribonuclease A. The reaction of Schiff base formation proceeds within a wide pH range--from 3.0 to 12.0. At a great pyridoxal-5-phosphate excess in ribonuclease A in neutral or slightly acidic aqueous media all the ten epsilon-amino groups of lysine residues and the alpha-amino groups of Lys-1 become modified. The formation of aldimine bonds of pyridoxal-5-phosphate with protonated amino groups in acidic media is determined by ionization of its phenol hydroxyl and phosphate residues. Acetaldehyde, propionic aldehyde and pyridine aldehyde interact only with non-protonated amino groups of the proteins. The equilibrium constants of pyridoxal-5-phosphate and other aldehydes binding to proteins and amino acids were determined. The rate constants of Schiff base formation for pyridoxal-5-phosphates with some amino acids and primary sites of proteins for direct and reverse reactions were calculated.     

Reductive alkylation of lysine residues in subtilisin DY

Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.     

Inactivation of brain myo-inositol monophosphate phosphatase by pyridoxal-5'-phosphate

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.