Screening of Reducing Agents for the PEGylation of Recombinant Human IL-10

PEGylation is a technology commonly used to enhance the bioavailability of therapeutic proteins in patients. Reductive alkylation of a protein amino terminal alpha amine in the presence of a polyethylene glycol (PEG) chain derivatized with propionaldehyde and a reducing agent, typically sodium cyanoborohydride, is one of the technologies available to achieve quantitative and site specific PEGylation. While cyanoborohydride has proven to be a robust and efficient reagent for this type of reaction, it generates aqueous cyanide as a reaction by-product (and its corollary, the very volatile hydrogen cyanide). We report here the screening of reducing agents such as dimethylamine borane, trimethylamine borane, triethylamine borane, tert-butylamine borane, morpholine borane, pyridine borane, 2-picoline borane, and 5-ethyl-2-methyl-pyridine borane as alternatives to cyanoborohydride for the PEGylation of recombinant human IL-10. The results of our study show that pyridine borane and 2-picoline borane promote rhIL-10 PEGylation at levels comparable to those observed with cyanoborohydride.     

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.     

Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis

Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endonuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80%. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the alpha NH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.     

Reductive methylation as a tool for the identification of the amino groups in alpha-bungarotoxin interacting with nicotinic acetylcholine receptor

alpha-Bungarotoxin (alpha Bgt) is a postsynaptic neurotoxin which blocks cholinergic transmission at the neuromuscular junction by binding tightly to the acetylcholine receptor (AcChR). The number of methylation sites in alpha Bgt has been shown to decrease significantly upon binding of the toxin to the AcChR [Soler, G., Farach, M. C., Farach, H. A., Mattingly, J. R., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872-878]. We have compared the chemical reactivities of amino groups in free and AcChR-bound alpha Bgt in an attempt to identify the regions in the alpha Bgt molecule that become masked upon binding to the AcChR. Free alpha Bgt and AcChR-bound alpha Bgt were reductively methylated with formaldehyde and sodium cyanoborohydride, and the rate of modification of each one of the available amino groups was followed by cleaving the methylated toxin with V8 protease and resolving the resulting peptides by reversed-phase, high-performance liquid chromatography. Under conditions of limited reagent availability, five of seven amino groups in free alpha Bgt reacted readily, whereas two other amino groups, probably those corresponding to Lys-51 and Lys-70, displayed lower reactivity. Upon binding to the AcChR, the rates of reductive methylation of residues Ile-1, Lys-26, and Lys-38 were considerably reduced (although to differing extents). The degree of protection was most pronounced for Lys-26. The rates of methylation of the amino groups in all other positions remained unchanged. These results allow further definition of the minimal binding surface of a representative neurotoxin.     

Conjugation of N-acylated amino sugars to protein by reductive alkylation using sodium cyanoborohydride: application to an azo derivative of alpha-amanitin.

Reductive alkylation mediated by cyanoborohydride is an attractive approach to the conjugation of small molecules, such as drugs, to proteins. This reaction is specific for protein amino groups and can be conducted under mild conditions with little risk of protein polymerization. However, the lability of the aldehyde function that is needed in such reactions presents a difficulty. We have investigated the use of derivatives of D-galactosamine and D-glucosamine in reductive alkylation, since these sugars contain aldehyde groups that are inherently protected and that may be readily linked to other molecules through their amino groups. The amino groups of these sugars were acylated with N-4-nitro-benzoylglycylglycine. Studies of the reductive coupling of the resultant adducts to bovine serum albumin revealed that conjugation to albumin is strongly dependent on cyanoborohydride, is much faster in the presence of borate, and shows a marked increase in rate between pH 7.0 and 9.0. In the presence of borate, the glucosamine derivative coupled much more rapidly than did the galactosamine derivative. The aryl nitro group of the glucosamine adduct was selectively reduced to an amine, diazotized, and reacted with alpha-amanitin to form an azo compound. This azo derivative was reductively coupled to form conjugates that inhibit calf thymus RNA polymerase II.     

Amine boranes as alternative reducing agents for reductive alkylation of proteins

The use of several commercially available amine-borane complexes was investigated for the reductive methylation of amino groups of several proteins. An earlier study in our laboratory, using turkey ovomucoid as the model protein, showed that dimethylamine borane is a slightly weaker reducing agent, but a good, less toxic substitute for sodium cyanoborohydride (K. F. Geoghegan, J. C. Cabacungan, H. B. F. Dixon, and R. E. Feeney, 1981, Int. J. Peptide Protein Res.17, 345–352). N-α-Acetyl-l-lysine, poly-l-lysine, turkey ovomucoid, bovine serum albumin, chicken ovalbumin, β-lactoglobulin, casein, and soybean protein were reductively methylated with dimethylamine borane and trimethylamine borane. The latter produced a consistently lower degree of modification even in the presence of sodium dodecyl sulfate. In a comparison that included the boranes triethylamine, t-butylamine, morpholine, and pyridine, pyridine borane was found to be slightly stronger than sodium cyanoborohydride. In a pH 7 solution containing 2 mmN-α-acetyl-l-lysine and 20 mm formaldehyde, complete dimethylation was achieved with about 10 mm pyridine borane after 2 h incubation at 22°C, while more than 15 mm was necessary with sodium cyanoborohydride. Like dimethylamine borane, both pyridine borane and triethylamine borane showed a reducing capacity at pH 7 which was as high as that at pH 9. Reductive alkylation under neutral to mild acid conditions allows modification of alkaline labile proteins and also limits the side reactions between proteins and formaldehyde.     

Derivatization of gamma-glutamyl semialdehyde residues in oxidized proteins by fluoresceinamine

Oxidative modification of proteins is implicated in a number of physiologic and pathologic processes. Metal-catalyzed oxidative modification usually causes inactivation of enzymes and the appearance of carbonyl groups in amino acid side chains of the protein. We describe use of fluoresceinamine to label certain of those carbonyl groups. Fluoresceinamine reacted with those carbonyl groups to form a Schiff base which was reduced by cyanoborohydride to yield a stable chromophore on the oxidized residue. The high molar absorbtivity of the fluorescein moiety conferred high sensitivity upon the method. Labeled peptides were readily identified after tryptic digestion of oxidized glutamine synthetase. Further, acid hydrolysis of labeled glutamine synthetase allowed isolation of the derivatized, oxidized residue. The oxidized amino acid was identified as gamma-glutamyl semialdehyde. During metal-catalyzed oxidation, the inactivation of glutamine synthetase paralleled the appearance of gamma-glutamyl semialdehyde.     

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.     

Rapid sodium cyanide depletion in cell culture media: outgassing of hydrogen cyanide at physiological pH

During the course of in vitro studies on cyanide exposure with SH-SY5Y human neuroblastoma cells, we found that sodium cyanide (NaCN) up to a concentration of 10 mM had no significant toxic effect under our culture conditions. Further investigation of this apparent cyanide resistance revealed that the sodium cyanide was being rapidly depleted from the cell culture medium. Cyanide was interacting with constituents of the cell culture medium and was somehow being detoxified or removed from solution. The reaction of cyanide with cell culture media in 96-well culture plates reduced cyanide concentrations rapidly (80-90% in 2 h at 37 degrees C). Running the same reaction in capped tubes significantly reduced cyanide loss from solution. Incubation of cyanide with individual constituents of the cell culture medium in solution showed that glucose, phenol red, and amino acids all acted to detoxify or remove cyanide from solution. When amino acids or buffers were incubated with sodium cyanide in aqueous solution at pH 7.4, hydrogen cyanide (HCN) was found to degas from the solutions. We compared HCN outgassing over a range of pH values. As expected, HCN remained very soluble at high pH, but as the pH was reduced to 7.0, the rate of HCN formation and outgassing increased dramatically. Acid-base reactions involving cyanide and proton donors, such as amino acids and other cell culture media constituents, at physiological pH result in rapid HCN outgassing from solution at 37 degrees C. These results indicate that previous in vitro cyanide toxicity studies done in standard culture media with prolonged incubation times using gas-exchanging culture containers might have to be reevaluated in light of the fact that the effective cyanide concentrations in the culture media were significantly lower than reported.     

Lectin purification on affinity columns containing reductively aminated disaccharides.

A method is described for isolating lectins in pure form and quantitative yield in a single step by affinity chromatography on aminoethyl polyacrylamide gels containing reductively aminated disaccharide residues. The affinity columns were prepared in two steps: (a) direct reductive amination of the disaccharide and aminoethyl gel with sodium cyanoborohydride in aqueous solution at pH 9; (b) N-acetylation of excess amino groups. Affinity columns prepared by reductive amination of lactose, melibiose, maltose, and di-N-acetylchitobiose were used to purify the following lectins: lactose, peanut, castor bean; melibiose, Bandeiraea simplicifolia; maltose, jack bean, common lentil; di-N-acetylchitobiose, wheat germ. These columns are extremely stable, have good flow rates, and high binding capacities.     

The site of attachment of retinal in bacteriorhodopsin. The epsilon-amino group in Lys-41 is not required for proton translocation

Chymotryptic fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71) of bacteriorhodopsin have been shown previously to reassociate so as to regenerate the native bacteriorhodopsin chromophore in lipid/detergent mixtures and to form functional proton-translocating vesicles. The fragment C-2 has now been selectively methylated with formaldehyde and sodium cyanoborohydride to give the epsilon-dimethylamino derivatives of Lys-30, 40, and 41 in 96-99% average yield. The methylated and unmethylated C-2 fragments were identical in their ability to reassociate with fragment C-1 and retinal to regenerate the bacteriorhodopsin chromophore and to form functional proton-translocating vesicles. In contrast, dimethylation of the lysine residues of the C-1 fragment gave a derivative which did not form an active complex with unmethylated C-2. We conclude that the epsilon-amino group in Lys-41 is not required for Schiff's base formation with retinal at any step in the light-driven proton-translocation cycle.     

Protein labeling by reductive methylation with sodium cyanoborohydride: Effect of cyanide and metal ions on the reaction

Previous studies (N. Jentoft and D. G. Dearborn, 1979, J. Biol. Chem.254, 4359) have demonstrated that reductive methylation with labeled formaldehyde and NaCNBH₃ provides a simple method for specifically labeling the amino groups of proteins using extremely mild reaction conditions. However, cyanide, which is one of the products of the reaction, reduces labeling effciency by reacting with formaldehyde to from the formaldehyde cyanohydrin addition product. Certain transition metal ions are able to prevent this secondary reaction by forming stable coordination complexes with cyanide. Inclusion of millimolar quantities of Ni(II) in reaction mixtures leads to a 20–30% increase in protein labeling so that maximal derivatization of amino groups can be realized with only a 3- to 4-fold ratio of formaldehyde to amine rather than the 5- to 10-fold excess necessary in the absence of metal ions.     

亲水化法稳定超氧化物歧化酶

在氰基硼氢化钠存在下用乙醛酸修饰含铁超氧化物歧化酶（Ｆｅ－ＳＯＤ）的氨基可使酶的热稳定性显著提高,酶在８０℃时的半寿期增加一倍。     

The effect of heme-linked ionizable groups on cyanide binding to methemoglobin

The pH dependence of the kinetics of the binding of cyanide ion to methemoglobins A and S and to guinea pig and pigeon methemoglobins appears to be not directly correlated with the net charges on the proteins. The kinetics can, however, be adequately explained in terms of three sets of heme-linked ionizable groups with pK1 ranging between 4.9 and 5.3, pK2 between 6.2 and 7.9, and pK3 between 8.0 and 8.5 at 20 degrees C. pK1 is assigned to carboxylic acid groups, pK2 to histidines and terminal amino groups, and pK3 to the acid-alkaline methemoglobin transition. Kinetic second order rate constants have also been determined for the binding of cyanide ion by the four sets of methemoglobin species present in solution. The pKi values and the rate constants of methemoglobin S are strikingly different from those of methemoglobin A. This result is explained in terms of different electrostatic contributions to the free energy of heme linkage arising from differences in the environments of ionizable groups at the surfaces of the two molecules.     

Proton and nitrogen-15 NMR studies of ferricytochrome c cyanide complexes: remarkable conservation of the heme environment among organisms of diverse origin

The native ferric and cyanide-bound ferric forms of nine vertebrate and two yeast cytochromes c have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy. Spectral comparisons have been made among the cytochromes with emphasis on the signal positions for heme and amino acid ligand protons. Consistent with earlier more limited studies of native ferric cytochromes c, the paramagnetically shifted proton NMR signals show little variation among species with up to 50% substitution of amino acids. Proton NMR spectra for the cyanide complexes also show little variation among species. The nitrogen-15 signal for the coordinated cyanide ion is known to be highly variable among other hemoproteins, but the signal covers a range of only 855 to 865 ppm (nitrate ion reference) for vertebrate cytochromes c and 884 to 886 ppm for yeast cytochromes c. The cyanide ligand probe thus reports an amazing conservation of the heme and proximal ligand environment among the cytochromes. Comparative proton and nitrogen-15 chemical shift values are consistent with a slightly stronger proximal histidine imidazole hydrogen bond to an amino acid carbonyl function than is the case for hemoglobin and myoglobin.     

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.     

Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.

Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum characteristic of pyridoxal-containing enzymes, and it has now been found to contain bound pyruvate, the carbonyl function of which is essential for catalytic activity. The decarboxylase is inactivated by treatment with a number of reagents that attack carbonyl groups, including sodium borohydride. Reduction with tritiated borohydride leads to the introduction of stably bound radioactivity, which, after acid hydrolysis, has been identified as tritiated lactate by several chromatographic procedures and by treatment with lactate dehydrogenase. The enzyme resists inactivation by cyanoborohydride in the absence of substrate, but is readily inactivated by this reagent in the presence of phosphatidylserine. Under the conditions of treatment of neutral pH, cyanoborohydride does not react with carbonyl residues at an appreciable rate, but reduces imino groups much more rapidly. This finding, together with demonstrated dependence of the enzyme upon the carbonyl residue of pyruvate for activity, strongly suggests that a Schiff base is formed by addition of the amino group of phosphatidylserine to the pyruvate residue of the enzyme as an essential step in the action of the decarboxylase.     

The demonstration of two acid groups in juxtaglomerular granules with basic triphenyl methane dyes,aldehyde-fuchsin and schiff's reagent

Summary Oxidation and bromination of mouse kidney JG cell-granules result in the production of cysteic acid from cystine; cysteic acid is capable of taking up rapidly and selectively certain basic triphenyl methane dyes including aldehyde fuchsin at lower pH levels.After treatment with periodic acid, bromine and hydrochloric acid, the JG granules or the nuclear chromatin also take up the basic triphenyl methane dyes (including aldehyde fuchsin) which contain amino groups, probable as a result of the production of aldehyde groups. Basic triphenyl methane lacking amino groups does not react with aldehydes.Some substance present in JG granules could be stained by aldehyde fuchsin after prior oxidation; HCl methyl violet 2B was taken up both with or without prior oxidation. Only strong methylation completely abolished these affinities which were restored after demethylation. These reactions are attributed to cystine.The staining of JG granules with dilute aldehyde fuchsin and dilute methyl violet 2B is not affected by oxidation, bromination, aldehyde blocking and hydrolysis; these reactions are abolished by mild methylation, but restored by subsequent saponification. These staining properties are due to the presence of carboxylic acid in JG granules.The positive PAS reaction of JG granules is due to the presence of 1.2-glycol in the same granules.     

Excitation and emission wavelength ratiometric cyanide-sensitive probes for physiological sensing

We characterize three new fluorescent probes that show both spectral shifts and intensity changes in the presence of aqueous cyanide, allowing for both excitation and fluorescence emission wavelength ratiometric and colorimetric sensing. The relatively high binding constants of the probes for cyanide enables a distinct colorimetric change to be visually observed with as little as 10 microM cyanide. The response of the new probes is based on the ability of the boronic acid group to interact with the CN(-) anion, changing from the neutral form of the boronic acid group R-B(OH)(2) to the anionic R-B(-)(OH)3 form, which is an electron-donating group. The presence of an electron-deficient quaternary heterocyclic nitrogen center and a strong electron-donating amino group in the 6 position on the quinolinium backbone provides for the spectral changes observed upon CN(-) complexation. We have determined the binding constants for the ortho-, meta-, and para-boronic acid probes to be 0.12, 0.17, and 0.14 microM(-3). In addition we have synthesized a control compound that does not contain the boronic acid moiety, allowing for structural comparisons and a rationale for the sensing mechanism to be made. Finally we show that the affinity for monosaccharides, such as glucose or fructose, is relatively low as compared to that for cyanide, enabling the potential detection of cyanide in physiologies up to lethal levels.     

化学修饰对胰激肽释放酶稳定性及其性质的影响

以自制高活性ＰＰＫ为材料,在氰基硼氢化钠存在下经水溶性乙醛酸修饰其表面氨基,使其抗不可逆热失活稳定性有显著提高。结果表明,修饰ＰＰＫ在等电点等基本性质方面都有变化。修饰ＰＰＫ的ＢＡＥＥ活性为天然酶的８２％,氨基修饰度为５８％,抗蛋白酶水解,贮藏和冻干稳定性都有加强。