Role of tyrosine and tryptophan in chemically modified serum albumin on its tissue distribution. (1/15)

To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased. After intravenous injection into mice, all synthetic BSA derivatives were eliminated very slowly from the systemic circulation. However, (111)In-TNM(6.6)- and (111)In-HNB(2.0)-BSA, derivatives with a high degree of modification, showed a slightly faster disappearance from the systemic circulation and slightly higher accumulation in the liver than (111)In-unmodified BSA. Pharmacokinetic analyses also demonstrated that the modification of Tyr or Trp residues on BSA had only marginal effects on tissue distribution. These results indicate that the Tyr and Trp residues have little effect on the tissue distribution characteristics of serum albumins, and that the specific modification of these residues may be a promising approach to designing sustained drug delivery systems using serum albumins.  (+info)

The role of tryptophanyl residues in heavy meromyosin as studied by chemical modification with 2-hydroxy-5-nitrobenzyl bromide. (2/15)

1. Two moles of 2-hydroxy-5-nitrobenzyl group bound selectively to one mole of heavy meromyosin when it was treated with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for tryptophanyl residues. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum with and without ADP of the bound 2-hydroxy-5-nitrobenzyl groups were measured with heavy meromyosin modified with various amounts of reagent. The properties of the modified heavy meromyosin did not change until the molar binding ratio of the reagent, rH, was about 1, but the properties changed remarkably when rH increased from 1 to 2. 2. Subfragment-1 was prepared from the modified heavy meromyosin by trypsin [EC 3.4.21.4] digestion. The molar binding ratio of the reagent in subfragment-1, rS, was found to be less than 0.1 when rH of the starting heavy meromyosin was less than 0.8. However, rS was about 0.5 in subfragment-1 prepared from heavy meromyosin of rH about 2. The results indicate that only one mole of 2-hydroxy-5-nitrobenzyl group, which was bound with lower reactivity than the other, was bound to a head part of heavy meromyosin. 3. Subfragment-1 fraction prepared from the modified heavy meromyosin could be separated into two fractions by DE-32 cellulose column chromatography; the subfragment-1 portion which eluted later showed a higher rS than that eluted in front. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum induced by ATP were measured with the modified subfragment-1 separated by DE-32 cellulose column chromatography. The ADP-binding ability and the size of the initial burst were not dependent on rS, and coincided with those of subfragment-1 prepared from unmodified heavy meromyosin. 4. The results of ADP binding studies suggest that heavy meromyosin is constituted from nonidentical subunits, and that there is an interaction between them which controls the ADP binding. Two tryptophanyl residues having specific reactivity toward 2-hydroxy-5-nitrobenzyl bromide are assumed to be involved in the interaction.  (+info)

Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6. (3/15)

Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.  (+info)

The preparation and some properties of mammalian cytochrome c modified with 2-hydroxy-5-nitrobenzyl bromide. (4/15)

2-Hydroxy-5-nitrobenzyl bromide reacts with horse heart cytochrome c at acid pH to yield a chemically modified protein. Chromatography of the protein on CM-cellulose allows separation of a single chemically modified species. This species is shown by gel chromatography to be monomeric, and isoelectric focusing shows the pI to be lowered from 10.5 to 9.8 on introduction of the reagent molecule. The changes observed in the u.v. region of the spectrum are consistent with the introduction of a single residue of the reagent, and the normal fluorescence of tryptophan is lost. The chemically modified protein exhibits marked changes in its functional properties as compared with native cytochrome c. Unlike the native monomer, the modified cytochrome c has a pH-dependent spectrum which is typical of a high-spin species in the alpha/beta region at low pH, changing to a low-spin species with an apparent pK of 7.5. The modified protein is autoxidizable and the ferrous form binds CO at neutral pH with an affinity constant of 2.6 X 10(5)M-1. The ferrous form of the modified cytochrome c binds CN- at pH 10.0 with an affinity constant of 3.5 X 10(2)M-1. The modified cytochrome c was incapable of restoring the electron-transfer activity to mitochondria depleted of cytochrome c.  (+info)

Kinetic studies on mammalian cytochrome c modified with 2-hydroxy-5-hydroxy-5-nitrobenzyl bromide. (5/15)

The reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c by the chromous ion was studied by stopped-flow techniques. At pH6.5 the reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c is complex, showing the presence of three distinct phases. Two chromium concentration-dependent phases are observed (1.1 X 10(5) M-1-S-1, phase 1; 1.25 X 10(4)M-1-S-1, phase 2) and one slow first-order process (0.25S-1, phase 3). A comparison of the static and kinetic difference spectra, along with the data from the reduction of the reoxidized reduced protein, suggests that the slow chromium concentration-independent phase is due to a slow conformational event after fast reduction of the NO2 group. The rates of the chromium concentration-dependent phases show a marked variation with pH above 7.5. The activation energies for the three processes were also measured at 33.2, 38.6 and 69.7 kJ-mol-1 for phases 1, 2 and 3 respectively. The reaction of reduced 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c with CO was foollowed by means of both stopped-flow and flash photolysis. The combination with CO at pH 6.8 as measured in stopped-flow experiments showed two phases, one CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and one CO-independent phase (phase 1, 0.015S-1). Investigation of the pH-dependence of the phases showed both the rates and amounts of each phase to be pH-invariant. CO recombination, after photolytic removal, was found to be biphasic; a CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and a CO-independent phase (phase 1, 1.0s-1) were observed. A tentative model which can accommodate these observations is proposed.  (+info)

Identification of amino acid residues essential for the enzymatic activities of pertussis toxin. (6/15)

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.  (+info)

Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site. (7/15)

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.  (+info)

Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities. (8/15)

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.  (+info)

• 1
• 2