Nuclear-magnetic-resonance-spectroscopic studies of the amino groups of insulin.

The amino groups of insulin have been studied by 1H and 13C nuclear magnetic resonance spectroscopy in insulin, zinc-free insulin and methylated insulin. By difference spectroscopy it is possible to follow the shift with pH of the epsilon-CH2 and delta-CH2 proton resonances of lysine-B29 in insulin. In methylated insulin the dimethyl proton resonances of glycine-A1, phenylalanine-B1 and lysine-B29 can be followed as a function of pH. In native insulin pKapp values of 6.7 and 8.0 are obtained for phenylalanine-B1 and glycine-A1 (the assignment is tentative) and 11.2 for lysine-B29. Separate resonances have been observed from the lysine-B29 Nepsilon-(CH3)2 group for the monomeric and dimeric forms of methylated insulin, which indicates a small change in the environment of lysine-B29 on dimerisation. The nuclear magnetic resonance spectral characteristics of these groups are, in general, consistent with the overall structure of the crystal form of the 2-zinc insulin hexamer.     

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.     

B-chain shortening of matrix-bound insulin by pepsin, I:Preparation and properties of bovine des-pentapeptide(B26-30) insulin (suthor's transl)]

Insulin was adsorbed to a strongly acidic ion exchanger and incubated with pepsin. The digestion of the matrix-bound insulin was found to be restricted to the cleavage of the peptide bond between phenylalanine-B25 and tyrosine-B26. Factionation of the reaction products was achieved by gel filtrationon Sephadex G-50 at pH 8 where des-pentapeptide(B26-30)-insulin does not aggregate. Another way to purify this compound was ion-exchange chromatography, which was easy due to the loss of one positive charge on the modified insulin. Crystallization could be achieved in a phenol-containing buffer. Des-pentapeptide(B26-30)-insulin was found to be molecularly uniform by electrophoresis at pH 2.2 and 8.6, thin-layer chromatography, performic acid oxidation, end group analysis and amino acid analysis. The CD-spectrum indicated conformational changes compared to insulin. The biological activity was considerably reduced: fat cell assay 20%, blood sugar depression 30%.     

Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on the central nervous system.

Specific chemical modifications of apamin have been used to study the residues involved in its toxic action. Transformation of Lys4 into homoarginine did not affect toxicity. Modification of the alpha-amino group of Cys1 and of the epsilon-amino group of Lys4 by acetic anhydride or fluorescamine decreased toxicity only by a factor of 2.5-2.8. Modification of the gamma-carboxylate of Glu7 with glycine ethyl ester in the presence of a soluble carbodiimide decreased toxicity by a factor of 2. Diethyl pyrocarbonate treated of the imidazole side chain of His18 decreased toxicity by a factor of 2.6. Thus none of these residues is essential for toxicity. However, combined modification of amino groups and of the imidazole side chain of His-18 completely abolished biological activity. Complete loss of toxicity also resulted from reduction and alkylation of both disulfide bridges, from chemical modification with cyclohexanedione of Arg-13 and Arg-14, and from removal of Arg-14 of acetylated apamin by digestion with trypsin. Incorporation of radioactive acetyl groups on both amino groups of apamin gave an active labeled toxin which has been used to localize the site of action of apamin in the spinal cord, principally in the lumbar part of the neuraxis.     

红花菜豆(矮生红花变种)凝集素分子的色氨酸残基修饰与其活性的关系

用各种化学试剂修饰红花菜豆（Ｐｈａｓｅｏｌｕｓｃｏｃｃｉｎｅｕｓｖａｒｒｕｂｒｏｎａｎｕｓ,Ｂｅｒｒｙ）凝集素（简称ＰＣＬ）分子,测定与其活性相关的氨基酸残基．经ＮＢＳ修饰表明ＰＣＬ具有８个Ｔｒｐ残基,其中４个暴露于分子表面,此４个Ｔｒｐ残基被修饰后,ＰＣＬ的凝血活性完全丧失．比较ＰＣＬ修饰前后的ＣＤ光谱表明修饰不改变其二级结构。修饰Ｔｙｒ,Ａｒｇ,Ｈｉｓ残基和游离氨基及羧基不影响ＰＣＬ的血凝活性．巯基也不是血凝活性所必需,但是ＰＣＬ分子中的二硫键被还原,或被ＣＮＢｒ分解为两个片断则使蛋白质丧失血凝活性,提示分子的完整结构对ＰＣＬ的血凝活力是重要的     

Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type 1 DNA-binding protein with single-stranded DNA.

Modification of the herpes simplex virus type 1 major DNA-binding protein (ICP8) with reagents and conditions specific for arginine, lysine, and tyrosine residues indicates that surface lysine and tyrosine residues are required for the interaction of this protein with single-stranded DNA. Modification of either of these two amino acids resulted in a loss and/or modification of binding activity as judged by nitrocellulose filter assays and gel shift. Modification specific for arginine residues did not affect binding within the limits of the assays used. Finally, quenching of the intrinsic tryptophan fluorescence of ICP8 in the presence of single-stranded DNA either suggests involvement of this amino acid in the binding reaction or reflects a conformational change in the protein upon binding.     

The reaction of protein amino groups with methyl 5-iodopyridine-2-carboximidate. A possible general method of preparing isomorphous heavy-atom derivatives of proteins

1. The synthesis of methyl 5-iodopyridine-2-carboximidate and its reaction with amino groups of model compounds and performic acid-oxidized insulin are described. The reagent was designed to introduce heavy atoms into specific sites in proteins. 2. Specific reaction with the amino groups of oxidized insulin can be achieved under reasonably mild conditions giving rise to the corresponding N-monosubstituted amidines. 3. The extent of reaction of this reagent with protein amino groups can be readily determined by difference spectroscopy. Modification of lysine residues inhibits tryptic cleavage at such residues, and this can be of assistance in establishing the site of modification in the primary structure. 4. Evidence is presented to show that methyl 5-iodopyridine-2-carboximidate can react specifically, at pH5.0, with the aromatic amino group of 3-amino-l-tyrosine; the final product of this reaction is a 2-arylbenzoxazole. 5. The use of this reagent as a general method for preparing heavy-atom isomorphous derivatives of proteins is discussed.     

Reductive methylation of insulin. Production of a biologically active tritiated insulin

Reductive methylation of the three amino groups of porcine insulin was accomplished by incubation with formaldehyde and sodium cyanoborohydride. The two amino termini and the epsilon amino group of B29 lysine were each dimethylated within 1 h of incubation. The fully methylated insulin bound more tightly to a reverse phase column than did native insulin, had a slightly more acid isoelectric point, and maintained approximately 50% biological activity when examined with an insulin sensitive cultured cell line. Reductive methylation with sodium cyanoboro [3H] hydride resulted in a [3H] methylated insulin with a specific activity of 6 Ci/mmol.     

Studies on the chemical modification of potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity

1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN′N″-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with α-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem. 242, 2447–2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.     

蚕豆β－半乳糖苷酶活性必需氨基酸残基分析

 蚕豆β－半乳糖苷酶活性必需氨基酸残基分析龚笑海,孙册（中国科学院上海生物化学研究所,上海２０００３１）化学修饰作为一种研究酶活性基团的方法,对研究糖苷酶的催化机理有其独到的优点,并已有这方面的报道．从蚕豆纯化的β－半乳糖苷酶,分子量为７０ｋＤ,由两个...     

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.     

Effect of Chemical Modifications on the Gushing-inducing Activity of a Hydrophobic Protein Produced by a Nigrospora sp

A beer-gushing inducing protein (NGF) produced by Nigrospora sp. No. 207 was found to be characterized by a relatively higher content of hydrophobic amino acid residues, a considerably high content (about 10 %) of half-cystine residue, and the lack of tyrosine, tryptophan, methionine and histidine. Cleavage of disulfide bonds in NGF, either oxidative or reductive, resulted in complete loss of the gushing-inducing activity, indicating that the disulfide bonds are essential for exhibiting the activity.

Modification of amino groups in NGF with maleic anhydride or O-methylisourea did not affect the gushing activity, whereas modification with tri nitrobenzene sulfonate reduced the activity to some extent. Modification of arginine-guanidino groups in NGF with phenyl- glyoxal markedly reduced the gushing activity. When the free carboxyl groups of NGF were modified by incorporating glycine or tyrosine, the gushing activity was also drastically destroyed, implying that the acidic groups of the NGF protein are important for the gushing activity.

It appeared that maintenance of a definite molecular conformation is essential for the gushing-inducing activity of this hydrophobic protein.     

Chemical modification studies on Ricinus communis (Castor Bean) agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.     

Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.     

Diethyl pyrocarbonate inactivates swine pepsinogen by reaction at amino groups

One of the histidine residues in swine pepsinogen can be modified in a rapid reaction with diethyl pyrocarbonate (DEP). At the same time, the potential proteolytic activity of the zymogen is reduced to 40% of its initial value. Reaction at the histine residue is reversed by neutral hydroxylamine, but the loss in activity is not. Fractionation of DEP-pepsinogen activation mixtures gave fully active pepsin in reduced yield, not modified enzyme with reduced specific activity. This was taken to indicate that the reaction had produced a mixture of products. Irreversible incorporation of [¹⁴C]DEP indicates that carbethoxy groups are incorporated into the molecule at amino acids other than histidine. The positions of these carbethoxy groups were determined by tryptic digestion and hplc. Modification was found to have occurred, in nonstoichiometric amounts, at lysine residues 3 and 9 and at leucine 1. Control experiments showed that activation was not affected by reaction at leucine 1, indicating that inactivation is caused by reaction at one or more of the lysine residues.     

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin.     

Chemical modification studies of Artocarpus lakoocha lectin artocarpin.

The effect of chemical modification on an anti T-like lectin, artocarpin isolated from Artocarpus lakoocha seeds was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of carboxyl groups, arginine and lysine residues, did not affect the lectin activity. However, modification of tryptophan, tyrosine and histidine residues led to a complete loss of its activity, indicating the involvement of these amino acids in the saccharide-binding ability. A protection was observed in the presence of inhibitory sugar. A marked decrease in the fluorescence emission was found when the tryptophan residues of lectin were modified. The circular dichroism spectra showed the presence of an identical pattern of conformation in the native and modified lectin, indicating that the loss in activity was due to modification only. The effect of pronase on artocarpin showed loss of activity whereas papain and trypsin had no effect. The specific activity of artocarpin remained unaltered on treatment with glycosidases but remarkable increase in the activity (of the same) was observed with xylanase treatment. Immunodiffusion studies with chemically modified lectin showed no gross structural changes, indicating that the group specific modifying agents did not alter the antigenic sites of the modified lectin.     

Role of arginine residues in ovine lutropin: Reversible modification by 1,2-cyclohexanedione

The modification of arginine residues of ovine pituitary lutropin by 1,2-cyclohexanedione has been studied. This alteration did not disrupt the quaternary structure of the hormone. Modification of the first set of about five reactive arginines resulted in 50% loss of hormonal activity. Further alteration in which seven to eight residues of arginine were modified led to 85% loss in activity. Hydroxylamine treatment of the derivative restored a significant amount (70%) of biological activity. Modification of isolated subunits did not appear to affect recombination. Recombinants in which either the α or β subunit was modified showed approximately 30% of the activity of the native hormone. The recombinant in which both of the subunits were derivatized had about 10% hormonal activity.     

Purification and active site modification studies on glyoxalase I from monkey intestinal mucosa

Glyoxalase I ((R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from monkey intestinal mucosa was purified to homogeneity. The purified enzyme had a molecular weight of 48,000, composed of two apparently identical subunits. Active-site modification was carried out on the purified enzyme in presence and absence of S-hexylglutathione, a reversible competitive inhibitor of glyoxalase I. Modification by tetranitromethane and N-acetylimidazole caused inactivation of the enzyme. Inactivation by N-acetylimidazole was reversible with hydroxylamine treatment, suggesting the importance of tyrosine residues for the activity of the enzyme. The enzyme was inactivated by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, 2,4,6-trinitrobenzenesulphonic acid, pyridoxal phosphate and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, indicating the importance of tryptophan, lysine and glutamic acid/aspartic acid residues for the activity of the enzyme. The enzyme was inactivated by diethyl pyrocarbonate and the activity was not restored by hydroxylamine treatment, suggesting that histidine residues may not be important for activity. Modification by N-ethylmaleimide and p-hydroxymercuribenzoate did not affect its activity, indicating that sulphydryl groups may not be important for activity. These studies indicated that the amino acids present in the active site of glyoxalase I from intestinal mucosa which may be important for activity are tyrosine, tryptophan, lysine and glutamic acid/aspartic acid residues.     

Identification of formaldehyde-induced modifications in proteins: reactions with model peptides

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.