Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.     

Specific chemical modifications of link protein and their effect on binding to hyaluronate and cartilage proteoglycan

Specific chemical modifications of amino acid residues were performed on purified, native link protein from bovine articular cartilage. The effects of these on link protein's interactions with hyaluronate and bovine articular cartilage proteoglycan were assayed by gel chromatography. Interaction with hyaluronate was significantly perturbed by modification of lysine, arginine, tyrosine and aspartic/glutamic acid residues, but not histidine and tryptophan residues. No free, accessible sulphydryl group was found on native link protein. The requirement for unmodified lysine and arginine residues resembles that of the hyaluronate-binding site of pig laryngeal cartilage proteoglycan (Hardingham, T.E., Ewins, R.J.F. and Muir, H. (1976) Biochem. J. 157, 127-143). In contrast, proteoglycan binding was only significantly perturbed by the loss of arginine residues. This resistance may reflect hydrophobicity of the binding site or masking of the site from chemical modification by link protein self-association. Amidation of carboxyl groups, which destroyed hyaluronate binding but left proteoglycan binding intact, provides a means of generating a monofunctional link protein molecule of potential use in proteoglycan aggregation studies.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

Chemical modification studies on the glucose/mannose specific lectins from field and lablab beans.

Seeds of Dolichos lablab var. lignosus (field beans) and variety typicus (lablab beans) contain glucose/mannose specific lectins that have been affinity purified and well characterised (Siva Kumar N., and Rajagopal Rao, D., J.Biosci., 1986, 10, 95-109, (1) Rajasekhar et al., (Biochem.Archives. 1997, 13, 233-240) (2). Purified lectins are glycoproteins with a native molecular mass of 60 kDa and are made of two types of subunits (Gowda et al., 1994, J.Biol.Chem. 269, 18789-18793) (3). Chemical modifications of various groups in purified lectins (using group specific reagents) such as lysine (citraconic anhydride), carboxyl groups (water soluble carbodiimide) tyrosine (N-acetyl imidazole) and tryptophan (2-hydroxy 5-nitro benzylbromide) revealed that 14 out of 21 residues of lysines 7 out of 92 residues of carboxyl groups, 16 out of 24 tyrosine residues and 2 out of 10 tryptophan residues were modified. Lysine and carboxyl group modification led to 95% loss in haemaglutinating activity compared to control while tyrosine and tryptophan modifications led to complete loss of lectin activity. Arginine and histidine modifications led to only 50% loss in activity. The extent of modification and loss in activity was same when the lysine and carboxyl groups were modified in the presence and absence of the inhibitory sugar methyl alpha-D-glucopyranoside at 0.1 M concentration. However protection of modification and lectin activity was observed when the tyrosine and tryptophan residues were modified in the presence of the inhibitory sugar. Earlier CD studies carried out (1) and extensive chemical modification studies reported here substantiate the involvement of tyrosine and tryptophan residues in the sugar binding site of these lectins.     

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.     

Iodination of ovotransferrin and its iron complex. Extent of involvement of tyrosine phenolic groups in the iron binding

Short-time iodination of metal-free ovotransferrin indicated that the tyrosine groups involved in the iron-binding activity are indistinguishable from other structural tyrosines. Modification of a minimum of 14 tyrosine residues per molecule of protein was required to achieve a complete loss of metal-binding activity. In contrast, a maximum modification of 10 tyrosine residues in iron-ovotransferrin complex could be produced with no loss of iron-binding activity. The difference in the extent of modification of tyrosines, therefore, indicated the involvement of four tyrosines in the binding of two atoms of iron. A minimal modification of histidine residues was also found, which was limited to one residue per molecule of both ovotransferrin and its iron complex. The possible participation of two tryptophan residues in the iron-binding activity is also suggested in the present study.     

Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.     

粘质赛氏菌胞外蛋白酶的化学修饰

用九种化学修饰剂研究了粘质赛氏菌ＳｅｒｒａｔｉａＭａｒｃｅｓｃｅｎｓ４１００３（２）胞外蛋白酶分子中氨基酸侧链基团与酶催化活性的关系,结果表明组氨酸、丝氨酸、赖氨酸、精氨酸、谷氨酸及天冬氨酸等残基与酶活性无关;半胱氨酸残基与酶活性也无直接关系;而酪氨酸和色氨酸残基侧链的修饰引起酶活力大幅度下降,说明酪氨酸和色氨酸残基为酶活力必需．     

Hemagglutinating activity and conformation of a lactose-binding lectin from mushroom Agrocybe cylindracea

A lactose-binding lectin (Agrocybe cylindracea Lectin, ACL) purified from fruiting bodies of the mushroom A. cylindracea was investigated to determine the hemagglutinating activity and conformation changes after chemical modification, removal of metal ion and treatment at different temperatures and pH. ACL agglutinated both rabbit and human erythrocytes and its hemagglutinating activity could be inhibited by lactose. This lectin was stable in the pH range of 6-9 and temperature up to 60 degrees C. Fluorescence quenching and modification of tryptophan residues indicated that there were about two tryptophan residues in ACL molecule and one of them might be located on the surface, while the other was buried in the hydrophobic shallow groove near the surface. Chemical modification of serine/threonine and histidine showed that the partial necessity of these residues for the hemagglutinating activity of ACL. However, modifications of arginine, tyrosine and cysteine residues had no effect on its agglutinating activity.     

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.     

Effect of amino acid residue and oligosaccharide chain chemical modifications on spectral and hemagglutinating activity of Millettia dielsiana Harms. ex Diels. lectin

The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in λmax. The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively. However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I^- was 0.15M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.     

Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2

In an effort to identify the anticoagulant region of venom phospholipases A2, we have systematically compared the amino acid sequences of strong, weak and non-anticoagulant phospholipases. The comparison disclosed several significant substitutions in the region between residues 54 and 77 (homology numbers). This proposed anticoagulant region is positively charged in strong, but negatively charged in weak and non-anticoagulant phospholipases. The microenvironment of a tryptophan residue falls within the proposed region, accounting for the differential characteristics of intrinsic fluorescence changes observed at 335 nm after the binding of phospholipid vesicles to strong and weak anticoagulants. Four lysine residues are located in specific positions in the "anticoagulant" region of strong anticoagulants, and should form a cationic surface, based on analogy with the available crystallographic structures. The chemical modification of lysine, arginine, tyrosine, and tryptophan residues and carboxylate groups, performed by other investigators, not only provides added support for the predicted site, but also confirms the essentiality of the positive charges in the site. This region may participate in the formation of a specific preferential hydrolytic complex leading to the strong anticoagulant effect. The anticoagulant region is distinct and separate from the predicted neurotoxic and myotoxic sites, and is located on the opposite surface of the phospholipase molecule.     

Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli.

The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques. Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts. Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane. Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium. More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore. In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all. Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule.     

On the reaction of papain and succinylpapin with diazo-1-H-tetrazole.

1. The reaction of papain and succinylpapain with diazo-1-H-tetrazole was investigated under different conditions. The extent of modification of the amino acids histidine, tyrosine, tryptophan and lysine was determined spectrophotometrically and/or by amino acid analysis. 2. Only one of the two histidine residues present in the enzyme reacts with diazo-1-H-tetrazole forming a monoazo derivative. The pH dependence of the coupling reaction reveals a normal pK of this reactive histidine. There are several arguments suggesting that this may be histidine 159 near the essential SH-group of papain. 3. All five tryptophan residues of the protein react with the diazonium ion below pH 7 forming a monoazo derivative with an absorption maximum at 370 nm, above pH 7 only four residues couple with diazo-1-H-tetrazole. The reaction of one tryptophan and one histidine are correlated as can be concluded from the pH dependence of the coupling rate of both amino acids and the parallel impairment of the catalytic acitivity. 4. 10-11 tyrosine residues out of 19 react with diazo-1-H-tetrazole to give bisazo compounds. 5 residues involved in hydrogen bridges form monoazo compounds. Only 12 tyrosines can be acylated by acetylimidazole. A relationship between the extent of modification of tyrosine and the activity of the enzyme could not be found.     

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.     

Evidence for essential histidine and dicarboxylic amino-acid residues in the active site of UDP-glucose : solasodine glucosyltransferase from eggplant leaves

Effects of several chemical probes selectively modifying various amino-acid residues on the activity of UDP-glucose : solasodine glucosyltransferase from eggplant leaves was studied. It was shown that diethylpyrocarbonate (DEPC), a specific modifier of histidine residues, was strongly inhibitory. However, in the presence of excessive amounts of the enzyme substrates, i.e. either UDP-glucose or solasodine, the inhibitory effect of DEPC was much weaker indicating that histidine (or histidines) are present in the active site of the enzyme. Our results suggest also that unmodified residues of glutamic (or aspartic) acid, lysine, cysteine, tyrosine and tryptophan are necessary for full activity of the enzyme. Reagents modifying serine and arginine residues have no effect on the enzyme activity.     

Calcium-ion-binding activity in human small-intestinal mucosal cytosol. Purification of two proteins and interrelationship of calcium-binding fractions.

Butane-2,3-dione inactivates the aspartyl proteinases from Penicillium roqueforti and Penicillium caseicolum, as well as pig pepsin, penicillopepsin and Rhizopus pepsin, at pH 6.0 in the presence of light but not in the dark. The inactivation is due to a photosensitized modification of tryptophan and tyrosine residues. In the dark none of the amino acid residues, not even arginine residues, is modified even after several days. In the light one arginine residue in pig pepsin is lost at a rate that is comparable with the rate of inactivation; however, the loss of the single arginine residue in the aspartyl proteinase of P. roqueforti and the second arginine residue of pig pepsin is slower than the loss of activity; penicillopepsin is devoid of arginine. Loss of most of the activity is accompanied by the following amino acid losses: P. roqueforti aspartyl proteinase, about two tryptophan and six tyrosine residues; penicillopepsin, about two tryptophan and three tyrosine residues; pig pepsin, about four tryptophan and most of the tyrosine residues. Modification of histidine residues was too slow to contribute to inactivation. None of the other residues, including half-cystine and methionine residues (when present), was modified even after prolonged incubation. The inactivation of P. roqueforti aspartyl proteinase and pig pepsin appears due to non-specific modification of several residues. With penicillopepsin, however, the reaction is more limited and initially affects only those tryptophan and tyrosine residues that lie in the active-site groove. In the presence of pepstatin the rate of inactivation is considerably diminished. After prolonged reaction a general structural breakdown occurs.     

Effects of denaturation and amino acid modification on fluorescence spectrum and hemagglutinating activity of Hericium erinaceum Lectin

In a broad sense, lectins are proteins or glycoproteins ofnon-immune origin that bind specifically to carbohydrates[1]. But most lectins are usually multivalent, which meansthey have more than one carbohydrate-binding site in onemolecule, a property that enables them to agglutinate eryth-rocytes and other cells [2,3]. Some lectins exhibit blood-group specificity [4] and can be used in blood grouping;some agglutinate transformed cells better than the normalones [5]. Therefore, clinical research…     

柞蚕抗菌肽D中精氨酸、赖氨酸和色氨酸等氨基酸残基的化学修饰

用不同的化学试剂修饰了柞蚕抗菌肽D分子中的色氨酸、精氨酸和赖氨酸等氨基酸残基。NBS修饰抗菌肽D,以及氨肽酶M对抗菌肽D作用的结果表明色氨酸残基对抗菌肽D抑制E.coli D31的作用影响不大。CHD和MLH对精氨酸和赖氨酸残基的修饰,导致抗菌肽D失去抑制E.coli的作用,但可逆地消除CHD和MLH的修饰作用后,抗菌肽D恢复了对E.coli D31的抑菌作用。这些结果初步认为,抗菌肽D抑菌作用与分子中的荷电性有关,改变了分子的电荷,也就同时失去了其抑菌功能。 此外,对精氨酸残基修饰的结果还表明,抗菌肽D的免疫原性与精氨酸残基有关。但是,抗菌肽D的免疫决定簇与其生物活性中心并不完全平行。     

Involvement of lysine and arginine residues in the binding of yeast ribosomal protein YL3 to 5S RNA

The contribution of lysine and arginine residues to the formation of yeast ribonucleoprotein complex 5S RNA. protein YL3 has been investigated by determining the effects on complex formation of modification with chemical reagents specific for either lysine or arginine. Treatment of protein YL3 with acetic anhydride, malefic anhydride or phenylglyoxal is accompanied by loss of its capacity to bind to 5S RNA. This effect is accomplished by modification with phenylglyoxal of only 3 arginine residues per YL3 molecule. In contrast, a large number of protein YL3 amino groups [16] must be modified by acetic anhydride to prevent complex formation.