Effect of reductive alkylation on thermal stability of ribonuclease A and chymotrypsinogen A

In order to probe changes in the structural stability induced by the introduction of hydrophobic groups into proteins, the amino groups of ribonuclease A and chymotrypsinogen A were reductively alkylated by reaction with various aliphatic aldehydes, formaldehyde, acetaldehyde, n-butylaldehyde and n-hexylaldehyde, and their thermal stabilities were investigated by differential scanning calorimetry (DSC) at different acidic pH values. Ribonuclease A was thermally unstabilized by reductive alkylation, while chymotrypsinogen A was slightly stabilized, depending on both the size of the introduced alkyl groups and the extent of modification. These observations suggest that the effects induced by alkylation involve not only steric hindrance due to the entering bulky groups but also certain other factors such as the participation of the chemically introduced alkyl groups in hydrophobic interactions.     

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50°C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.     

THE PH DEPENDENCE OF BINDING OF α, α‘-DIBROMO-P-XYLENESULFONIC ACID TO LYSOZYME

The chemical modification of lysozyme (I) has been accomplished with α,α'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28°C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues of DBX-³⁵S were bound per molecule of I at both pH 9 and pH 8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intramolecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.     

Effect of glutathione on the covalent binding of the 13C-labeled skin sensitizer 5-chloro-2-methylisothiazol-3-one to human serum albumin: identification of adducts by nuclear magnetic resonance, matrix-assisted laser desorption/ionization mass spectrometry, and nanoelectrospray tandem mass spectrometry

The covalent binding of 4-[(13)C]- and 5-[(13)C]-5-chloro-2-methylisothiazol-3-one (MCI) toward human serum albumin (HSA) was followed by (13)C and (1)H[(13)C] NMR spectroscopy. MCI was found to react with histidine through an addition-elimination at position 5, leading to stable substitution adducts, and with lysine to form open adducts of the thioamide or amide type. No other modification could be detected on either cysteine or tyrosine. In the presence of glutathione (GSH), we observed an increased covalent binding to lysine residues. This could be explained by the rapid reaction of GSH with MCI to form a chlorothioacyl intermediate very reactive toward primary amino groups of lysine residues. To further confirm these observations and map covalent binding sites, HSA samples modified by MCI with or without GSH were analyzed by matrix-assisted laser desorption/ionization mass spectrometry of tryptic digests and electrospray tandem mass spectrometry of modified peptides purified by reverse phase HPLC. About 80% of the HSA sequence was mapped, and several modified peptides were identified. When HSA was incubated with MCI without GSH, three peptides modified at histidine residues were characterized while when HSA was incubated in the presence of GSH, five peptides modified at histidine and lysine residues were identified. These experiments confirmed that modifications on lysine residues were of the amide and thioamide types. Observed modifications were in accordance with mass increases corresponding to structures identified by NMR, and an extra adduct corresponding to a double modification of His 338 was observed. Comparison of HSA-MCI and HSA-MCI-GSH samples confirmed that the presence of GSH increased the modification of lysine residues.     

Influence of some flavonoids on reticulation of collagen fibrils in vitro

Reconstituted collagen fibrils obtained from neutral-salt soluble type I collagen of lathyritic rat skin are incubated at 37° with flavanoids, dihydroquercetin, rutin, naringin and mainly (+)-catechin, alone or in presence of cupric ions. The influence of these compounds on the solubility, thermal stability and cross-links of the collagen fibrils is studied. With (+)-catechin, collagen fibrils become insoluble, but their thermal stability is only slightly modified. In presence of (+)-catechin-Cu(II) complex, collagen fibrils become totally insoluble and their thermal stability increases with formation of many cross links precursors (allysine and hydroxyallysine) and reducible cross-links (hydroxylysinonorleucine and lysinonorleucine). These last compounds are also found with the other flavonoids. The formation of these compounds indicates the oxidative desamination of lysine and hydroxylysine residues of collagen molecules by flavonoid and Cu(II) mixture. This reaction has also been observed with type II collagen and with albumin and is not specific to connective tissue proteins.     

Synthesis and stabilization of amino and carboxy terminal constrained collagenous peptides

Short collagenous peptides cross-linked at their amino and carboxy termini with Lys-Lys-dimer template(s) were synthesized, and the effect of the cross-linking on their stabilities was investigated by thermal denaturation experiments. Two chemoselective ligations were used for the construction of the amino and the carboxy cross-linked peptides. The thermal transition temperature (T_m) and the standard free energies (ΔG°) of the cross-linked collagenous peptides increased, and the thermal stabilization effect corresponded to an elongation by two units of the Gly-Pro-Hyp triad. The van't Hoff enthalpy (ΔH°) and the entropy (ΔS°) values of the cross-linked peptides increased with chain elongation, although the increments were smaller than those of the linear peptides. When the same chain lengths were compared, the ΔH° was increased and the ΔS° was nearly the same or increased by the cross-linking. These results suggest that the cross-linking of the collagenous peptides with the Lys-Lys-dimer template(s) for stabilization contributes to the enthalpic effect, rather than the entropic effect.     

The influence of molecular size and pH on the macrocationic inhibition of pepsin by polylysine

Polylysines of 7–371 lysine residues inhibited pepsin over the pH range 3·6-5·0 in a system using azocoll as substrate. Tetralysine was inactive. An almost 1: 1 molar ratio with pepsin gave maximum inhibition for a polylysine containing 59 lysine residues but increase in polylysine molecular size beyond this size was not accompanied by increase in activity on a weight basis although the polylysine: pepsin molar ratio for maximum inhibition decreased and inhibition mechanism varied. Polylysines of 59 and 158 lysine residues which were intermediate in the series were non-competitive inhibitors, whilst polylysines of greater and smaller molecular size were competitive inhibitors, although only the smallest inhibitory polylysine, containing 7 lysine residues, was a pure competitive inhibitor. Polylysine inhibition of pepsin was found to be strongest at pH 5·0 and the pH dependence appeared to be associated with the relative concentrations of the enzyme and inhibitors in ionized form. For each polylysine it was possible to detect a polylysine: pepsin concentration ratio for which inhibition was pH-independent over the range 3·6-5·0.     

Isolation and characterization of β-galactoside specific lectin from Korean mistletoe (Viscum album var.coloratum) with lactose-BSA-Sepharose 4B and changes of lectin conformation

Lectins and its A- and B-chains from Korean mistletoe (Viscum album var.coloratum) were isolated by affinity chromatography on the Sepharose 4B modified by lactose-BSA conjugate synthesized by reductive amination of ligand (lactose) to ε-amino groups of lysine residues of spacer (BSA) after reduction by NaCNBH₃. The lactose-BSA conjugate was coupled to Sepharose 4B activated by cyanogen bromide. The molecular weight determined by SDS-PAGE were a 31 kD of A-chain and a 35kD of B-chain. Amino acid analysis andN-terminal sequencing were performed. The effects of pH, temperature and guanidine chloride on the conformation of the lectin were investigated by measuring its intrinsic fluorescence and compared with its hemagglutinating activities. Blue shift was detected on the acidic pH and there was a close relationship between activities and conformation of the lectin. Under denaturing conditions, the tryptophan emission profile of lectin showed typical denaturational red shift which also correspond to the conformations and activity of lectin.     

Conjugation of Emtansine Onto Trastuzumab Promotes Aggregation of the Antibody–Drug Conjugate by Reducing Repulsive Electrostatic Interactions and Increasing Hydrophobic Interactions

The impact of drug conjugation on intra- and intermolecular interactions of trastuzumab (TmAb) was determined by comparing the conformational and colloidal stabilities of TmAb and trastuzumab emtansine (T-DM1). In low ionic strength formulations, drug conjugation to native lysine residues of TmAb significantly reduced the repulsive electrostatic interactions between T-DM1 molecules. When these electrostatic interactions were screened in solutions with high ionic strength, intermolecular interactions between T-DM1 molecules were found to be more attractive than those between TmAb molecules. Drug conjugation lowered the colloidal stability of T-DM1 compared to TmAb, making T-DM1 more susceptible to agitation-induced aggregation. The presence of polysorbate-20 in the formulations inhibited aggregation of TmAb and T-DM1 induced by the hydrophobic air-water interface. Furthermore, the effect of increased hydrophobic interactions between T-DM1 molecules was studied by monitoring aggregation in TmAb and T-DM1 solutions that were incubated at 4°C, 25°C, and 50°C. Conjugating DM1 to TmAb increased the hydrophobicity of the molecule, and faster aggregation of T-DM1 at 50°C could be attributed to a temperature-dependent increase in hydrophobic interactions between T-DM1 molecules.     

Inhibition of acetylcholinesterase by dibenamine and dibenzyline

Dibenamine and dibenzyline are irreversible inhibitors of acetylcholinesterase (AChE). Kinetic studies show that at pH 9.5 a fast reaction occurs between a group on the enzyme with pK_a 9.1 and the ethyleniminium ion derived from the inhibitor. Either the ε-amino-group of a lysine residue is alkylated or else a lysine residue catalyses the alkylation of a non-ionisable group (e.g. hydroxyl). At pH 6.5 there is a slow reaction between a carboxyl anion on the enzyme and the ethyleniminium ion. Studies of the alkylation reactions in the presence of the reversible competitive inhibitor of the enzyme, tetramethylammonium ion, show that alkylation occurs at some distance from the anionic site and probably on the borders of the active site.     

Kinetics of reversible reactions of ampicillin with various aldehydes and ketones with formation of 4-imidazolidinones

The reaction of ampicillin with various aldehydes and ketones has been studied in aqueous solution at 37°C. A reversible 4-imidazolidinone formation occurred through condensation between the carbonyl compounds and the α-aminoamide side-chain function of ampicillin. Equilibrium constants and rate constants for the formation and hydrolysis of the imidazolidinones were obtained at pH 7.45. The rate of imidazolidinone formation was found to be depressed by increasing steric effects within the carbonyl compounds whereas the rate of hydrolysis of the imidazo-lidinones showed only a small dependence on the carbonyl component except for the compound derived from formaldehyde. This derivative showed a half-life of hydrolysis of 29 h at pH 7.45 whereas the imidazolidinones derived from other aldehydes and ketones had half-lives of 4–31 min. The results obtained are discussed in relation to the possible in vivo formation of imidazolidinones by reaction of ampicillin with biogenic aldehydes or ketones and in relation to the possible utility of 4-imidazolidinones as prodrug forms for peptides containing an α-aminoamide moiety.     

Biophysical Properties and Heating-Induced Aggregation of Lysine-Conjugated Antibody-Drug Conjugates

The commercially available antibody-drug conjugate (ADC) product, Kadcyla^® is synthesized using a 2-step reaction, wherein the linker is conjugated to native lysines on the mAb in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine-conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla^® using a 1-step reaction. Mass spectrometry of both products revealed a subpopulation of Kadcyla^® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the 1-step reaction ADC product. Differential scanning calorimetry thermograms showed that the drug and linker conjugation significantly reduced the thermal stability and energies of activation for the denaturation of the C_H2 domain of the ADCs. The heating induced aggregation events started as early as ～57°C and ～45°C for Kadcyla^® and Syn-ADC, respectively, compared with 71°C for Herceptin^®. The colloidal stability measurements clearly showed that the hydrophobic drug payload on ADCs significantly reduced the repulsive interprotein interactions when compared to the unconjugated antibody under formulation buffer conditions (pH 6.0). Attaching hydrophobic drug and linker moieties onto the antibody lowered the thermal and colloidal stabilities and increased the aggregation propensity of the ADCs.     

Protein binding of sotalol enantiomers in young and elderly human and rat serum using ultrafiltration

The protein binding of sotalol (STL) enantiomers was evaluated using an ultrafiltration technique with serum from young (32±2 years, n=5) and elderly (73±6 years, n=5) male and female humans, and young (8 weeks, n=4) and elderly (60 weeks, n=3) male Sprague—Dawley rats. Serum samples were collected and immediately frozen at ?20°C. Within 1 week, the serum samples were thawed at room temperature, and adjusted to pH 7.4 using 0.05 M phosphate buffer, pH 5.0. Aliquots were spiked with 250 ng mL^?1 and 500 ng mL^?1 of each STL enantiomer, placed in ultrafiltration sets (Microsep, 30K molecular weight cut-off), capped, equilibrated to 37°C, and centrifuged at 1850g for 1.5h at 37°C. Aliquots of ultrafiltrate and unspun serum were analysed for STL enantiomer concentration using a stereospecific HPLC assay. In all groups, bound fraction was less than 7% for both STL enantiomers. There were no significant differences in bound fraction between groups, or between enantiomers. Adsorption of STL enantiomers to the ultrafiltration device and membrane, evaporative loss of serum samples during centrifugation, and protein concentration in each ultrafiltrate sample were all negligible. It is concluded that the binding of STL in human and rat serum at therapeutic concentrations and physiological temperature and pH is negligible and non-stereoselective.     

Acetylation of bovine growth hormone by N-acetylimidazole

Bovine growth hormone was chemically modified with N-acetyhmidazole and the acetylated tyrosine residues were identified. Tyrosine 42 was the most reactive followed by tyrosines 158, 35 and 109 and to a lesser degree by tyrosines 141 and 174. Several lysine residues were also acetylated. The modified hormone retained the native α-helix content but showed a weak growth promoting activity and was unable to compete in vivo with ¹²⁵I-labeled bovine growth hormone for the liver binding sites. The loss in growth promoting activity induced by acetylation was prevented by the amidination of lysine residues. These findings combined with results already published suggest that tyrosine residues are not important in the manifestation of the hormone growth promoting activity.     

Crystal and molecular structure of l-valyl-l-lysine hydrochloride

l -Valyl-l -lysine hydrochloride, C₁₁N₃O₃H₂₃ HCl, crystallizes in the monoclinic space group P2, with a = 5.438(5), b = 14.188(5), c = 9.521(5) Å, β= 95.38(2)° and Z = 2. The crystal structure, solved by direct methods, refined to R = 0.036, using full matrix least-squares method. The peptide exists in a zwitterionic form, with the N atom of the lysine side-chain protonated. The two γ-carbons of the valine side-chain have positional disorder, giving rise to two conformations, χ¹¹₁= -67.3 and 65.9°, one of which (65.9°) is sterically less favourable and has been found to be less popular amongst residues branching at β-C. The lysine side-chain has the geometry of g^? tgt, not seen in crystal structures of the dipeptides reported so far. Interestingly, χ³₂ (63.6°) of lysine side-chain has a gauche⁺ conformation unlike in most of the other structures, where it is trans. The neighbouring peptide molecules are hydrogen bonded in a head-to-tail fashion, a rather uncommon interaction in lysine peptide structures. The structure shows considerable similarity with that of l -Lys-l -Val HO in conformational angles and H-bond interactions [4].     

Evidence for the steric proximity of Tyr 174 and Lys 111 in bovine growth hormone

Bovine growth hormone was modified by reaction with 1,5-difluoro-2,4-dinitro-benzene under conditions favouring production of intramolecularly crosslinked derivatives from monomeric molecules. The monomeric fraction, isolated by chromatography on Sephadex G-100, was oxidized or reduced and carbamido-methylated and trypsin digested. The resulting peptides were fractionated on SP-Sephadex and further purified by peptide mapping or HPLC. Two modified peptides containing sequences 108–112 or 108–113 and 171–176 of bGH were obtained, including a dinitrophenylene bridge between lysine 111 and tyrosine 174, thus suggesting the stereochemical proximity of these residues.     

Trypsin-mediated semisynthesis of salmon calcitonin

Salmon calcitonin, CT(1-32)·NH₂, was synthesised by the trypsin-mediated coupling of the peptide fragments CT(1-24) and CT(25-32)·NH₂, prepared by conventional Fmoc solid-phase chemistry. Optimal conditions regarding reaction time course, pH, proportion of catalyst, substrate concentration and composition of the reaction medium were determined from initial studies on the coupling of CT(1-11) to CT(12-24) and of CT(12-24) to CT(25-32)·NH₂. For the final successful semisynthesis, we found that it was unnecessary to protect lysine residues not involved in the coupling, and that secondary hydrolysis at these sites could be prevented by increasing the pH of the reaction medium. The reaction achieved equilibrium after 30-45 min, with overall conversion of around 30% of the initial amount of CT(1-24) substrate into product. Yields were depressed due to cyclisation of the CT(1-24) substrate via air-oxidation of the Cys¹ and Cys⁷ residues.     

Ampicillin biosynthesis by immobilized enzyme

Ampicillin was synthesized from 6-aminopenicillanic acid (6-APA) and D-α-phenylglycine methyl ester by using ampicillin synthesizing enzyme fromPseudomonas melanogenum (IAM 1655). The whole cell enzyme was immobilized by entrapping it in the polyacrylamide gel lattices. The polymer used in the enzyme entrapment was made from 150 mg per ml of acrylamide monomer and 8 mg per ml of N,?-methylenebisacrylamide. About 200 mg/whole cell enzyme was mixed in the polymer for entrapment. The maximal activity retention after immobilization was 56%. The optimal pH values for the whole cell enzyme and the immobilized whole cell enzyme were 6.0 and 5.9, respectively. The optimal temperature for the enzyme activity were the same for both type of preparations. The enzyme stabilities against pH and heat increased for immobilized whole cell enzyme. Immobilized cell was more stable especially in the acidic condition while both type were found to be very susceptible to thermal inactivation at a temperature above 40°C. The kinetic constants obtained from Lineweaver-Burk plot based on two substrate reaction mechanism showed somewhat higher value for immobilized whole cell enzyme as compared to the whole cell enzyme: the Km value for 6-APA were 7.0 mM and 12.5 mM while Km values for phenylglycine methyl ester were 4.5 mM and 8.2 mM, respectively. Using the immobilized whole cell enzyme packed in a column reactor, the productivity of ampicillin was studied by varying the flow rate of substrate solution. At the space velocity, SV, 0.14 hr^?1 the conversion was 45%. Operational stability found in terms of half life was 30 hr at SV=0.2 hr.     

Impact of industrial stress factors on lysozyme enzyme: Role of denaturation processes and initial protein activity

Protein instability and efficiency loss are the major drawbacks industry faces in medical, pharmaceutical and food sectors. Understanding how processes induce denaturation and consequently modify enzymatic activity can lead to more sustainable pharmaceutical or food manufacturing by avoiding the loss of active molecules. In the present investigation, the influence of some stress factors (heating, chemical denaturation, ultrasound application, and ultrafiltration) on the final lysozyme antibacterial activity was evaluated to determine the limiting conditions of industrial processes. Two commercial lysozymes with different initial enzymatic activities were compared. The temperature of denaturation was higher than 70 °C, and a reduction of 25% of activity was observed after treatment at 90 °C. Chemical denaturants (urea and guanidine hydrochloride) induced different LSZ conformational changes, and the modification was dependent on the concentration for guanidine hydrochloride (activity decrease of 30% using a concentration of 10 M). The difference between the two lysozymes was observed for the ultrasound and ultrafiltration treatments. During ultrasonic treatment, one lysozyme was stable for 15 min, while a loss of activity was observed after 3 min for the other. The same difference in stability was observed during ultrafiltration process. The activity decreases linearly with transmembrane pressure (around 50% for 12 bar) during filtration of the less stable lysozyme, and no change in the antibacterial activity was observed for the second protein. This study shows that stress factors induce different denaturation states and that for some of them (ultrasound and ultrafiltration), the presence of small residues in the protein formulation is important.     

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups

The carboxylate groups in an acidic and in a basic phospholipase A2 (PLA2) enzyme, purified, respectively, from Naja naja atra and Naja nigricollis snake venoms, were modified with carbodiimide and semicarbazide. The derivatives modified at pH 3.5 and pH 5.5 had less than 1% (N. nigricollis) or 2% (N. n. atra) residual enzymatic activity, whereas 12-16% enzymatic activity remained following modification at pH 5.5 in the presence of Ca2+. In marked contrast, these derivatives retained variable, but significantly greater, levels of lethal potency, hemolytic and anticoagulant activities, and abilities to block indirectly and directly induced contractions of the diaphragm. By this modification of aspartic and glutamic acid residues we have, for the first time, obtained derivatives of PLA2 which selectively retain greater pharmacological activity relative to enzymatic activity. Previously, we had found that modification of lysine and arginine residues produced derivatives which retain enzymatic activity but show a loss of pharmacological properties. These findings suggest that some pharmacological effects of snake venom PLA2 enzymes are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.