Intramolecular cross-linkage of lysozyme. Imidazole catalysis of the formation of the cross-link between lysine-13 (epsilon-amino) and leucine-129 (alpha-carboxyl) by carbodiimide reaction

The salt bridge between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme was converted to an amide bond by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) reaction in the presence of imidazole (0.3-1 M) at pH 5 and room temperature, followed by dialysis at pH 10. Absence of imidazole under a similar condition did not give this intramolecularly cross-linked lysozyme derivative (CL-lysozyme) but resulted in the formation of intermolecularly cross-linked lysozyme oligomers. From the mechanistic studies on the formation of CL-lysozyme, imidazole was suggested to play the following three roles. (1) Some carboxyl groups activated by EDC in lysozyme were converted to acylimidazole groups which protected them from the reaction with amino groups in other lysozyme molecules at pH 5. These could be hydrolyzed at pH 10 to regenerate free carboxyls. (2) High concentrations of imidazole (pH 5) increased the ionic strength of the solution which weakened the salt bridge in lysozyme and facilitated the activation of the alpha-carboxyl group by EDC. (3) The alpha-carboxyl group activated by EDC was converted to an acylimidazole group which could react with the epsilon-amino group of Lys-13 in the same molecule to form an amide bond. The last step may involve some conformational change of the backbone of lysozyme and be slower than the hydrolysis reaction of the alpha-carboxyl group activated by EDC itself. However, acylimidazole groups are stable against hydrolysis at pH 5. This may afford enough time to allow the epsilon-amino group of Lys-13 to attack the acylimidazole group of Leu-129.     

Mode of interaction between lysozyme and the other proteins of the hen's egg vitelline membrane

Salt solutions and charged detergents are efficient solubilizing agents for ovovitelline membrane lysozyme. Reassociation experiments with chemically modified lysozymes indicate that positively charged amino acid residues of lysozyme (the epsilon-amino group of lysine and the guanidino group of arginine) are involved in the interaction with other proteins of the vitelline membrane. Exogenous proteins are adsorbed to lysozyme-free vitelline membranes, only if they have a high pI, comparable to that of lysozyme. It is concluded that the lysozyme-ovovitelline membrane interaction is predominantly ionic. An ovomucin-lysozyme complex is postulated as the major component of the outer layer of the membrane.     

Investigation of protein structure by means of 19F-NMR. A study of hen egg-white lysozyme

A 19F-labeled derivative of hen egg-white lysozyme, in which the six epsilon-amino groups are trifluoroacetylated (LF6), was prepared by reaction of lysozyme with S-ethyltrifluorothioacetate. The reaction mixture was fractionated by cation-exchange chromatography at pH 7.3. A comparison of the circular dichroic spectra and the activity towards Micrococcus lysodeikticus of both LF6 and native lysozyme reveals that the labeling causes no major conformational changes of the polypeptide backbone. Assignment of the six resonances present in the 19F-NMR spectrum of LF6 was accomplished by using a variety of techniques: specific chemical modifications, the effect of the inhibitor (GlcNAc)3, 19F-shift/pH information and relaxation parameters.     

The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.

A procedure using 2,4,6-trinitrobenzenesulfonic acid (TNBS) for the determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials is presented. The major modification from previous procedures is an extended TNBS reaction time to allow a stoichiometric reaction with amino groups. In addition, autoclave hydrolysis is used to assure sample dissolution for spectrophotometric measurements. The assay accuracy was evaluated by determining epsilon-amino groups of insulin and bovine albumin. The determinations differed from literature values by < or = 3.3%. The epsilon-amino group content of Type B gelatin was found to be 33.0 mol/gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen. The coefficient of variation for determinations on all three materials was < or = 5.3%. The assay should be applicable to a broad range of proteinaceous materials.     

pH dependence of individual tryptophan N-1 hydrogen exchange rates in lysozyme and its chemically modified derivatives

Nuclear magnetic resonance analyses have been made of the individual hydrogen-deuterium exchange rates of tryptophan indole N-1 hydrogens in native lysozyme and its chemically modified derivatives including lysozyme with an ester cross-linkage between Glu-35 and Trp-108, lysozyme with an internal amide cross-linking between the epsilon-amino group of Lys-13 and the alpha-carboxyl group of Leu-129, and lysozyme with the beta-aspartyl sequence at Asp-101. The pH dependence curves of the exchange rates for Trp-63 and Trp-108 are different from those expected for tryptophan. The pH dependence curve for Trp-108 exchange exhibits the effects from molecular aggregation at pH above 5 and from a transition between the two conformational fluctuations at around pH 4. The exchange rates for tryptophan residues in native lysozyme and modified derivatives are not correlated with the thermodynamic or kinetic parameters in protein denaturation, suggesting that the fluctuations responsible for the exchange are not global ones. The exchange rates for tryptophan residues remote from the modification site are perturbed. Such tryptophan residues are found to be involved in a small but distinct conformational change due to the modification. Therefore, the perturbations of the N-1 hydrogen exchange rates are related to the minor change in local conformation or in conformational strain induced by the chemical modification.     

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.     

A quantitative determination by capillary gas-liquid chromatography of neutral and amino sugars (as O-methyloxime acetates), and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries

Pyridine borane has been reported as a superior reagent over a wide pH range, 5-9, for the reductive methylation of amino groups of proteins with formaldehyde [J. C. Cabacungan , A. I. Ahmed , and R. E. Feeney (1982) Anal. Biochem. 124, 272-278]. It has also been reported to reduce tryptophan to dihydrotryptophan and to inactivate lysozyme in trifluoroacetic acid [M. Kurata , Y. Kikugawa , T. Kuwae , I. Koyama , and T. Takagi (1980) Chem. Pharm . Bull 28, 2274-2275]. In the present study the specificity of pyridine borane for the two different modifications under different reaction conditions has been demonstrated, and extended to the application to the synthesis of protein containing reductively attached carbohydrates. In the acid reduction, pyridine borane selectively reduced all six tryptophans in lysozyme to dihydrotryptophan while all other amino acids remained intact. On similar treatment no cleavage of the carbohydrate moiety from chicken ovomucoid, and no losses of activity of ovomucoid or ribonuclease, two proteins devoid of tryptophan, were observed. Nearly complete methylation of the lysines of lysozyme, chicken ovomucoid, and ribonuclease was achieved with formaldehyde at pH 7.0 after 2 h at room temperature, with the retention of full activity of the protein without any destruction of tryptophan. The same chemistry was applied to covalently attach glucose and lactose to bovine serum albumin. Parameters, including pH, temperature, and methanol, that affect the reactions were investigated. Incremental additions of pyridine borane during the course of the reactions increased the rate of modification. The covalent attachment of sugar to the epsilon-amino group of lysine was demonstrated by the synthesis of N-alpha- acetylglucitollysine and comparison with acid hydrolysates of the bovine serum albumin-sugar derivatives.     

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.     

Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha-(t-butoxycarbonyl)-l-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.     

Analysis of glycated insulin by MALDI-TOF mass spectrometry

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.     

Oxidative deamination of epsilon-aminolysine residues and formation of Schiff base cross-linkages in cell envelopes of Escherichia coli.

Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates. The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links. This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli. Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB[3H]4 or labeled with [14C]lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine. When [6-3H]lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred. This suggests that an enzymatic reaction catalyzes the deamination of lysine in E. coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links.     

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.     

Unusual chemical properties of the amino groups of insulin: implications for structure-function relationship.

The chemical properties of the three amino groups of insulin were obtained at 10 and 37 degrees C using the competitive labelling technique with acetic anhydride as the labelling reagent. At 10 degrees C, pK values of 7.9, 7.2, and 7.8 were found for the glycyl A1, phenylalanyl B1, and lysyl B29 amino groups. When compared with standard amino compounds by means of a Br?nsted plot, the two amino-termini were found to be 'super-reactive' and the lysyl epsilon-amino group buried. In the presence of carbon dioxide at physiological pH values, all three amino groups became much less reactive indicating that they had reacted to form carbamino derivatives. Above pH 8 the reactivities of the glycyl amino terminus and epsilon-amino group increase sharply indicating that insulin is undergoing a conformational change which is most likely a change in its association state. At 37 degrees C the amino groups do not titrate normally but exhibit sharp increases in reactivity over the physiological pH range with the midpoints in the pH reactivity profiles between pH values of 7.0 and 7.3. This behaviour is interpreted as a rapid disaggregation of insulin to form monomers as a result of the ionization of the amino groups. It is concluded that at physiological pH and temperature all three amino groups are deprotonated.     

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.     

Scavenging of Active Oxygen Species by Glycated Proteins

Scavenging of active oxygen species by glycated proteins was investigated. Glycated proteins were prepared from bovine serum albumin (BSA), insulin, and lysozyme incubated with glucose. Glycated BSA at concentration of 0.5% scavenged 34% of hydroxyl radicals by ESR experiments using DMPO as a spin-trapping reagent. The ability to scavenge hydroxyl radicals by glycated BSA was higher than that by BSA. Hydrogen peroxides also were largely scavenged with an increase in the concentration of glycated proteins. However, the ability to scavenge superoxides by glycated BSA was lower than that by BSA because glycated proteins produced superoxides. Experiments using model compounds such as Amadori compound and caproyl pyrraline suggested that the scavenging ability of glycated proteins against hydroxyl radicals depends on Maillard reaction products in the advanced stage, while the ability against hydrogen peroxides is dependent upon Maillard reaction products in the early stage and brown pigments.     

Drug delivery systems employing 1,6-elimination: releasable poly(ethylene glycol) conjugates of proteins

Using lysozyme as a representative protein substrate that loses its activity when PEGylation takes place on the epsilon-amino group of lysine residues, various amounts of a novel releasable PEG linker (rPEG) were conjugated to the protein. rPEG-lysozyme conjugates were relatively stable in pH 7.4 buffer for over 24 h. However, regeneration of native protein from the rPEG conjugates occurred in a predictable manner during incubation in high pH buffer or rat plasma, as demonstrated by enzymatic activity and structural characterization. The rates of regeneration were also correlated with PEG number: native lysozyme was released more rapidly from the monosubstituted conjugate than from the disubstituted conjugate, suggesting possible steric hindrance to the approach of cleaving enzymes. Recovery of normal activity and structure for the regenerated native lysozyme was shown by a variety of assays.     

Synthesis, characterization and biological activity of chemically modified insulin derivative with alpha lipoic acid

A novel chemically-modified insulin, epsilon-N(B29)-lipoyl insulin, was selectively prepared by the covalent linkage of alpha-lipoic acid (LA) to the epsilon-amino group of Lys(B29) of insulin without any protecting agent and analyzed by PAGE, HPLC, MALDI-TOF-MS. Monolipoyl- insulin maintained the glucose-lowering effect as well as native insulin and showed a longer duration of action than native insulin and an inhibitory effect towards trypsin degradation.     

Protein purification with vapor-phase carbon dioxide

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.     

An intramolecular cross-linkage of lysozyme. Formation of cross-links between lysine-1 and histidine-15 with bis(bromoacetamide) derivatives by a two-stage reaction procedure and properties of the resulting derivatives

Hen egg white lysozyme was treated at pH 5.5 with four bifunctional reagents, bis(bromoacetamide) derivatives [BrCH2CONH(CH2)nNHCOCH2Br, 1-n, n = 0, 2, 4, and 6], to alkylate His-15 monofunctionally. The excess bifunctional reagent was then removed, and the pH was raised to 9.0 to allow the other end of the reagent molecule to react. The shortest reagent (1-0) gave no intramolecularly cross-linked lysozyme derivative but only histidine-15-modified lysozyme monomer and intermolecularly cross-linked lysozyme dimer. However, the reagents with longer arms (1-2, 1-4, and 1-6) gave lysozyme derivatives cross-linked intramolecularly between the nitrogen at epsilon 2 of His-15 and the epsilon-amino group of Lys-1 without formation of any other intramolecularly cross-linked lysozyme derivative. These results are consistent with our previous proposal that lysozyme has a small hydrophobic pocket that binds small molecules in the direction from His-15 to Lys-1 [Yamada, H., Uozumi, F., Ishikawa, A., & Imoto, T. (1984) J. Biochem. (Tokyo) 95, 503-510]. The thermal stabilities of three cross-linked lysozymes thus obtained were investigated in 0.1 M acetate buffer containing 3 M guanidine hydrochloride at pH 5.5. All derivatives were stabilized but to different degrees. The derivative cross-linked with 1-4 was most stabilized (2.3 kcal/mol), but the derivatives cross-linked with the reagents both shorter (1-2) and longer (1-6) than 1-4 were less stabilized (both 1.6 kcal/mol).(ABSTRACT TRUNCATED AT 250 WORDS)