Dihydroxypropylation of amino groups of proteins: use of glyceraldehyde as a reversible agent for reductive alkylation

The mode of derivatization of amino groups of proteins by glyceraldehyde, an aldotriose, depends on the presence or absence of reducing agent. In the presence of sodium cyanoborohydride, the Schiff base adducts of the aldehyde with the amino groups are reduced, and dihydroxypropylation of amino groups takes place (reductive mode). The reductively glycated lysine residue, N epsilon-(2,3-dihydroxypropyl)lysine, is a substituted alpha-amino alcohol. This alpha-amino alcoholic function of the derivatized lysine should be susceptible to periodate oxidation, and this oxidation is anticipated to result in the regeneration of the lysine residue. This aspect has been now investigated. Indeed, on mild periodate oxidation (15 mM periodate, 15 min at room temperature) of dihydroxypropylated ribonuclease A, nearly 95% of its N epsilon-(2,3-dihydroxypropyl)lysine residues were regenerated to lysine residues. The removal of the dihydroxypropyl groups by periodate oxidation could be accomplished within a wide pH range with little variation in the recovery of lysines. The possible usefulness of this reversible chemical modification procedure in the primary structural studies of proteins was investigated with a tryptic peptide of dihydroxypropylated streptococcal M5 protein, namely, DHP-T4. This 12-residue tryptic peptide contains one internal N epsilon-(dihydroxypropyl)lysine. The dihydroxypropylated peptide released most of its dihydroxypropyl groups on mild periodate oxidation. Redigestion of the periodate-treated peptide with trypsin generated the two expected peptides, demonstrating the generation of a trypsin-susceptible site. Reductive dihydroxypropylation of amino groups of RNase A resulted in the loss of its enzyme activity, the extent of inactivation increasing with the concentration of the glyceraldehyde used.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the exocyclic amino groups of conserved purines in hammerhead ribozyme cleavage.

A series of 2 stranded hammerhead ribozymes has been synthesized in which single conserved adenosine residues have been replaced by nebularine or single guanosine residues by inosine. Comparison of the rates of trans-cleavage for the modified structures suggests the presence of interstrand non-Watson-Crick hydrogen bonding interactions including a GA:AG double mismatch. The exocyclic amino group of one of the guanosine residues is essential for cleavage.     

Fluorometric determination of carbohydrate with 2-aminothiophenol

The 2-aminothiophenol-based fluorometric assay of Nakano et al. (1973, J. Pharm. Soc. Jpn. 93, 350-353) for monosaccharides has been modified to improve the speed, applicability, and sensitivity of the method. The improved assay is applicable to complex carbohydrates as well as to monosaccharides. Less than 50 ng of carbohydrate in a final volume of 2 ml can be quantitatively measured within 30 min. The assay is reasonably compatible with the presence of a variety of reagents commonly used in aqueous buffer solutions. The assay is especially useful for monitoring column eluents during the purification of small quantities of carbohydrates or their conjugates.     

The reversible reaction of protein amino groups with exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride. The reaction with lysozyme

1. The reaction of exo-cis-3,6-endoxo-Delta(4)-tetrahydrophthalic anhydride with amino groups of model compounds and lysozyme is described. 2. Reaction with the in-amino group of N(alpha)-acetyl-l-lysine amide gives rise to two diastereoisomeric products; at acid pH the free amino group is liberated with anchimeric assistance by the neighbouring protonated carboxyl group with a half-time of 4-5h at pH3.0 and 25 degrees C. 3. The amino groups of lysozyme can be completely blocked, with total loss of enzymic activity. Dialysis at pH3.0 results in complete recovery of the native primary and tertiary structure of lysozyme and complete return of catalytic activity. 4. The specificity of reaction of this and other anhydrides with amino groups in proteins is discussed.     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Reactions of the amino groups in ribonuclease A: II. Identification of reactive amino groups in ribonuclease

Ribonuclease A has been trinitrophenylated to varying degrees by reaction with trinitrobenzenesulfonic acid. The reactive amino groups were identified by use of the peptides obtained from the oxidized TNP-RNase by tryptic and chymotryptic hydrolysis. From a quantitative study of the TNP-peptides it was possible to associate each amino group with values of pK_ma. It was shown that the lys-41 amino group had a pK_a of 9.03 in TEA buffer. The pK_a values of all of the other amino groups were dependent on the nature of the buffer (triethanolamine and phosphate) and on the pH.     

A convenient route for the preparation of peptide nucleic acid monomers carrying acid-labile groups for the protection of the amino function

Summary A convenient route for the preparation of peptide nucleic acid (PNA) monomers is described. Two different baselabile protecting groups (2-cyanoethyl and 4-nitrophenylethyl) are described for the protection of the carboxylic function of theN-(2-aminoethyl)glycine backbone during the assembly of the monomers. These groups are selectively removed yielding the desired PNA monomers in high yields, the 2-cyanoethyl group being faster and cleaner than the 4-nitrophenylethyl group. The use of PNA monomers for the preparation of DNA-PNA chimeric molecules is also discussed.     

A convenient route for the preparation of peptide nucleic acid monomers carrying acid-labile groups for the protection of the amino function

A convenient route for the preparation of peptide nucleic acid (PNA) monomers is described. Two different base-labile protecting groups (2-cyanoethyl and 4-nitrophenylethyl) are described for the protection of the carboxylic function of the N-(2-aminoethyl)glycine backbone during the assembly of the monomers. These groups are selectively removed yielding the desired PNA monomers in high yields, the 2-cyanoethyl group being faster and cleaner than the 4-nitrophenylethyl group. The use of PNA monomers for the preparation of DNA–PNA chimeric molecules is also discussed.     

On-sequencer pyridylethylation of cysteine residues after protection of amino groups by reaction with phenylisothiocyanate

Cysteine residues in polypeptides are not easily identified during automated N-terminal sequence analysis. Reaction of cysteine side chains with 4-vinylpyridine and identification as the pyridylethylated phenylthiohydantion derivative (PE-PTH-Cys) were proposed. However, after this reaction a desalting step is necessary. If limited sample amounts do not allow this desalting step, on-sequencer pyridylethylation is an alternative, although preview of the consecutive amino acid is usually observed in this case. We describe an on-sequencer procedure that avoids such preview formation by derivatizing the peptide with phenylisothiocyanate (PITC) prior to reaction with 4-vinylpyridine. The pyridylethylation is performed in the cartridge of the sequencer after immobilization of the protein or peptide on a polybrene-coated glass fiber filter and thiocarbamylation with PITC. Preview caused by N-alkylation is not observed and PE-PTH-Cys is detected in much higher yields than usual. The procedure reported here is significantly shortened, optimized to reduce side products, and avoids losses during sample handling. It can easily be adapted to any automated version of the sequencers.     

The use of maleic anhydride for the reversible blocking of amino groups on polypeptide chains

1. Maleic anhydride was shown to react rapidly and specifically with amino groups of proteins and peptides. Complete substitution of chymotrypsinogen was achieved under mild conditions and the extent of reaction could be readily determined from the spectrum of the maleyl-protein. 2. Maleyl-proteins are generally soluble and disaggregated at neutral pH. Trypsin splits the blocked proteins only at arginine residues and there is frequently selectivity in this cleavage, e.g. in yeast alcohol dehydrogenase and pig glyceraldehyde 3-phosphate dehydrogenase. 3. The group is removed by intramolecular catalysis at acid pH. The half-time was 11-12hr. at 37 degrees at pH3.5 in in-maleyl-lysine or in maleyl-chymotrypsinogen. 4. The unblocking reaction can be used as the basis for a ;diagonal'-electrophoretic separation of lysine peptides and N-terminal peptides, as shown by studies with beta-melanocyte-stimulating hormone.     

Dinitrophenylation as a probe for the determination of environments of amino groups in protein. Reactivities of individual amino groups in lysozyme

Dinitrophenylation of hen egg white lysozyme with 2,4-dinitrofluorobenzene (DNFB) was carried out at pH 7-11 and room temperature in order to examine whether dinitrophenylation could be applied to determine the environments of individual amino groups in lysozyme or not. Lightly dinitrophenylated lysozyme was reduced, S-carboxymethylated and then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). All tryptic peptides, which contained dinitrophenylated amino groups (one alpha-amino group, Lys 1(alpha), and six epsilon-amino groups, Lys 1(epsilon), Lys 13, Lys 33, Lys 96, Lys 97, and Lys 116), could be separated and monitored by absorbance measurement at 360 nm on RP-HPLC. The relative reactivities of individual amino groups, determined from the relative peak areas of dinitrophenylated tryptic peptides at 360 nm, were found to be sensitive to the reaction pH and to the presence of the trimer of N-acetyl-D-glucosamine or NaCl. It was concluded that dinitrophenylation of a protein with DNFB followed by peptide analysis by RP-HPLC with detection at 360 nm is a good method for probing the environments of individual amino groups in the protein.     

The structure and function of ribonuclease T1. XXIII. Inactivation of ribonuclease T1 by reversible blocking of amino groups with cis-aconitic anhydride and related dicarboxylic acid anhydrides.

Ribonuclease T1 [EC 3.1.4.8] was inactivated rapidly by treatment at pH 8.0 and 0 degrees C with cis-aconitic anhydride and related dicabroxylic acid anhydrides, including citraconic, maleic, and succinic anhydrides. Under reaction conditions used, roughly 90% inactivation occurred within 30 min. Analyses of the inactivated enzymes indicated that the reaction took place fairly specifically at the alpha-amino group of the N-terminal alanine and the epsilon-amino group of lysine-41. Upon incubation of these inactivated enzymes at pH 3.6 and 37 degreeC, the activity was regenerated to various extents, depending on the nature of the introduced acyl groups. Under these conditions, the enzyme modified with cis-aconitc anhydride or citraconic anhydride recovered much of the origninal activity after 48 h whereas the enzyme modified with maleic anhydride recovered its activity only partially. Practically no activity was regenerated in the case of the enzyme modified with succinic anhydride under these conditions. The inactivation appears to be due mainly to the effect of the carboxyl group introduced at the epsilon-amino group of lysine-41. The results suggest the usefulness of cis-aconitic anhydride as a reversible blocking reagent for amino groups in proteins.     

Synthesis and application of chemically reactive proteins by the reversible modification of protein amino groups with exo-cis-3,6-endo-epoxy-4,5-cis-epoxyhexahydrophthalic anhydride.

The reversible reaction of exo-cis-3,6-endo-epoxy-4,5-cis-epoxyhexahydrophthalic anhydride (EEHPA) with free protein amino groups is described. The free protein amino groups of lysozyme can be completely blocked through the reaction of the anhydride EEHPA. The chemically less reactive epoxy groups in EEHPA-modified lysozyme remain intact during modification of the protein and can be used for many subsequent chemical reactions. Hydrolysis of the modified inactive lysozyme at pH 2.5 results in deblocking and almost complete recovery of the enzymic activity of the protein. The epoxy groups in EEHPA-modified proteins have a great many potential uses: disaggregation of supramolecular structures, conversion of hydrophobic membrane proteins or tryptic peptides into water-soluble coloured proteins or peptides, inhibition of tryptic cleavage at lysine residues, synthesis of chemically reactive proteins or enzymes for affinity chromatography or immobilized-enzyme technology, two-dimensional separation techniques for complex protein mixtures, detection of specific protein-binding sites for organic substrates or tumour diagnostics, synthesis of defined artificial glycoproteins for biophysical and cytochemical studies and chemical synthesis of radioactively labelled proteins.     

Selective DNA cleavage by elsamicin A and switch function of its amino sugar group.

We report guanine-specific recognition and selective cleavage of DNA by the antitumor antibiotic elsamicin A equipped with an amino sugar and compare these results with cleavage by chartarin and chartreusin antibiotics. The preferential cutting sites of DNA strand scission with elsamicin A are on the bases adjacent to the 3'-side of guanine residues such as 5'-GN sites, in particular 5'-GG sites. The present results also indicate that (1) the aglycon portion binds intercalatively to the 3'-side of guanine in host DNA, (2) the guanine 2-amino group has an important effect on selective DNA binding of elsamicin A, and (3) the amino sugar residue of elsamicin A facilitates the drug binding into the minor groove of B-DNA. In addition, we found that an acetylation of the amino group on the elsamicin A sugar portion plays an interesting switch function for the activity of elsamicin A. The biological implication of this switch has also been discussed.