Reversible fluorescence labeling of amino groups of protein using dansylaminomethylmaleic anhydride

The reversible fluorescence labeling of insulin, catalase and lysozyme has been demonstrated. As a derivatizing reagent, dansylaminomethylmaleic acid (DAM) has been used after investigating the precolumn and precapillary derivatization conditions. This reagent (DAM) reacts with the amino groups of proteins via its anhydride in the presence of a suitable dehydrating reagent, which then could be liberated under mild acidic conditions and the native proteins are regenerated. After the derivatization of insulin, catalase and lysozyme with DAM, no peaks of these native proteins were observed while several peaks of the derivatized proteins due to the multiple labeling were observed. However, after the regeneration, increasing amounts of the native proteins were observed as the regeneration period increased. For the lysozyme, the bacteriolytic activity of the enzyme decreased after the derivatization, and only 0.9% of the activity remained. The activity increases by the regeneration, and 95.6% of the bacteriolytic activity of the native enzyme was observed after a 48-h regeneration at pH 2.5 and 40 degrees C.     

2-Vinylquinoline, a reagent to determine protein sulfhydryl groups spectrophotometrically

A reagent has been sought for the selective derivatization of protein sulfhydryl groups that will allow the spectrophotometric determination of the cysteine and cystine content of intact proteins. 2-Vinylquinoline appears to be that reagent. Protein sulfhydryl groups were reacted with 2-vinylquinoline to yield the protein-linked S-2-(2-quinolylethyl)-l-cysteine (Qe-cysteine). After urea and other excess reagents were removed, the modified proteins were examined spectrophotometrically. The extinction coefficient (10,000) and absorption maximum (318 mμ) of the protein-linked vinylquinoline derivatives were identical to those of the model Qe-cysteine. Optimum conditions for the reaction require an equimolar concentration of 2-vinylquinoline to all sulfhydryls and a 4 hr reaction period. The total cysteine and cysteine contents of the proteins, when determined under these conditions, were in excellent agreement with standard literature values.     

A quantitating reagent for methanethiolation of protein sulfhydryl groups

p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH₃S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH₃S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups.     

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.     

Single-step, quantitative derivatization of amino, carboxyl, and hydroxyl groups in iodothyronine amino acids with ethanolic pivalic anhydride containing 4-dimethylaminopyridine

Reaction of thyroxine with ethanol and pivalic anhydride in the presence of 4-dimethylaminopyridine quantitatively forms N,O-dipivalyl thyroxine ethyl ester. Other iodothyronines react similarly and the procedure is moisture insensitive. Apparently this reaction is successful, in contrast to similar procedures reported for the derivatization of alpha-amino acids, because it overcomes the problem in other procedures of irreversible side reactions arising from an oxazolone intermediate.     

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 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.     

Synthesis and application of a fluorescent imido ester for specific labelling of amino groups in proteins

2-(5'-Dimethylaminonaphthalene-1'-sulfonamido)methylimidic acid methyl ester has been synthesized for fluorescence labelling of amino groups in proteins. The incorporation of the dansyl group serving as an extrinsic fluorescent probe is determined spectrophotometrically. Glucose dehydrogenase (beta-D-glucose: NAD(P+) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium having a reactive lysine residue which belongs to the active site has been labelled. To give proof of the selectivity of the modification, the enzyme preparation having 1.3 dansyl groups per subunit has been digested with trypsin and the major labelled peptide has been isolated and sequenced.     

The action of tellurite, a reagent for thiol groups, on mitochondria oxidative processes

Tellurite, a reagent for thiol groups, added to rat kidney, or liver mitochondria at the concentration of 1 mM, selectively inhibits the oxidation of NAD dependent substrates (pyruvate, α-ketoglutarate, glutamate, etc.) without affecting the oxidation of succinate, α-glycerophosphate and ascorbate. Moreover NADH oxidation by rat heart or by aged liver mitochondria is not affected by tellurite.     

Optimised amino acid specific weighting factors for unbound protein docking

Background  

One of the most challenging aspects of protein-protein docking is the inclusion of flexibility into the docking procedure. We developed a postfilter where the grid-representation of proteins for docking is extended by an optimised weighting factor for each amino acid.     

Synthesis of lactones using substituted benzoic anhydride as a coupling reagent

This protocol describes a procedure for the synthesis of a 29-membered macrolactone. The facile mixed-anhydride method is very effective for the preparation of carboxylic esters and lactones using substituted benzoic anhydrides by the promotion of Lewis acid or basic catalysts under mild reaction conditions. Owing to the reaction rapidly proceeding to produce the monomeric compounds with high chemoselectivity, the protocol is quite powerful for the synthesis of not only the giant-sized lactones but also the highly strained cyclic compounds such as medium-sized lactones. The remarkable efficiency of the lactonizations promoted by the substituted benzoic anhydrides has been already shown in the synthesis of many natural complex molecules. It takes approximately 19 h to complete the protocol: 0.5 h to set up the reaction, 13.5 h for the reaction and 5 h for isolation and purification.     

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.