Mechanism of activation of Sepharose and Sephadex by cyanogen bromide

The mechanism of CNBr activation of polysaccharide resins like Sepharose and Sephadex has been elucidated using recently published analytical procedures for the determination of cyanate esters and imido carbonates. It was found that on agarose-based resins coupling of ligand occurs predominantly via cyanate esters, and not via imidocarbonates as in the case of Sephadex. This explains the different behaviours of Sepharose and Sephadex during CNBr activation.     

The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N(2)-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500+/-1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500+/-1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400+/-1400. 7. The N-terminal amino acid composition is 0.64+/-0.04mol of valine and 0.36+/-0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis.     

Ion chromatographic quantification of cyanate in urea solutions: estimation of the efficiency of cyanate scavengers for use in recombinant protein manufacturing

The chaotrope urea is commonly used during recombinant protein manufacturing as a denaturant/solublizing agent. The adventitious accumulation of cyanate in urea solutions during product manufacturing can cause unwanted carbamylation of proteins, leading to alterations in drug product structure, stability and function. We have developed an ion chromatographic method to quantify cyanate production in urea solutions, suitable for analysis of samples from manufacturing process buffers. We discuss assay development, system suitability criteria and limitations on assay applicability. The assay has a linear range from 2 to 250 microM, with LOQ/LOD values of 6 and 2 microM, respectively. Assay accuracy through spike/recovery testing were established and both precision and intermediate precision were estimated. We assessed the utility of the assay by testing a variety of biological buffers and potential cyanate scavengers, which could be used during protein purification processes, for their ability to control the level of cyanate in 8 M urea solutions buffered over the range of pH 5-10. Our results demonstrate pH dependence for prevention of cyanate accumulation by these buffers/scavengers and indicate useful buffers, pH ranges, and additives for controlling cyanate accumulation during recombinant protein manufacturing. The pertinence of these approaches in preventing protein carbamylation during manufacturing are discussed.     

Analysis of plasma cyanate as 2-nitro-5-thiocarbamylbenzoic acid by high-performance liquid chromatography

A method for the determination of cyanate concentration in blood plasma over the range 1 to 1000 microM is presented. Cyanate present in the dried residue of acetone-deproteinized plasma is converted to a chromophoric thiocarbamyl derivative by addition of pH 3.0-buffered thionitrobenzoic acid. The derivative is then analyzed by reversed-phase high-performance liquid chromatography with detection at 313 nm, near the absorption maximum. Carbamyl thionitrobenzoic acid peak height is quantified by comparison to a standard curve made by analysis of plasma samples to which known quantities of cyanate have been added. This technique is sensitive and linear with respect to cyanate concentration, and is faster than other reported methods; sample analysis and column regeneration are accomplished within 20 min.     

Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764.

Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth. Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia. Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), as described previously in Escherichia coli and Flavobacterium sp.     

Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.     

Fate and transformation of thiocyanate and cyanate under methanogenic conditions

The fate of thiocyanate (SCN⁻) and cyanate (OCN⁻) under methanogenic conditions was investigated at 35 °C. Thiocyanate and cyanate were added to mixed methanogenic cultures along with an organic mixture. Thiocyanate was stable under these conditions, and had no adverse effect on methanogenesis at a concentration as high as 2.5 mM. In contrast, cyanate at a concentration as low as 0.3 mM initially inhibited methanogenesis but, after the complete removal of cyanate, methanogenesis gradually recovered. The inhibitory effect of cyanate on methanogenesis became more profound with repeated additions of cyanate. The transformation of cyanate followed the hydrolytic route to ammonia and bicarbonate under anaerobic conditions and its hydrolysis rate was enhanced by microbial activity. Cyanide was not detected as a cyanate transformation product under the methanogenic conditions of this study. Received: 13 June 1997 / Received revision: 29 August 1997 / Accepted: 15 September 1997     

Identification and characterization of a cyanate permease in Escherichia coli K-12.

Escherichia coli contains an inducible enzyme, cyanase, that catalyzes the decomposition of cyanate into ammonia and bicarbonate. The gene encoding cyanase, cynS, was cloned and found to be on a DNA fragment that contained the lac operon. Characterization of a plasmid encoding cyanase indicated that a 26-kilodalton (kDa) protein of unknown function was also induced by cyanate (Y-C. Sung, D. Parsell, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 169:2639-2642, 1987). The gene encoding the 26-kDa protein was located between cynS and its promoter, indicating the existence of a cyn operon. The 26-kDa protein was identified as a cyanate permease that transports exogenous cyanate by active transport. E. coli was shown to contain a cyanate transport system that is energy dependent and saturable by cyanate.     

Inhibition by cyanate of the processing of lysosomal enzymes

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.     

The effect of sodium cyanate-modified hemoglobin oxygen affinity on the heat resistance of rats]

Regression analysis of relationship between rat hemoglobin-oxygen affinity (HOA) and rectal temperature has shown a close positive correlation of these parameters. Heat resistance (HR) was examined in rats with HOA elevated by sodium cyanate in order to recognize the contribution of HOA to a process of body heat adaptation. Our data suggest that HR of treated rats was larger than in control animals. These results are discussed in relation with antioxidant type of the cyanate elevation in HOA.     

A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.     

Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B. Guilloton, A.F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate.     

Release of mitochondrial respiratory control by cyanate salts

Mitochondrial respiration is stimulated by 5–40 mM potassium cyanate in the presence or absence of oligomycin. When the cyanate concentration is increased over 40 mM, the mitochondria respire at progressively lower rates. In these respects, although at relatively high concentrations, cyanate behaves as an uncoupler of oxidative phosphorylation.     

Cyanate modification of essential lysyl residues of the diphosphopyridine nucleotide-specific isocitrate dehydrogenase of pig heart.

The DPN-specific isocitrate dehydrogenase of pig heart is totally and irreversibly inactivated by 0.05 M potassium cyanate at pH 7.4 A plot of the rate constant versus cyanate concentration is not linear, but rather exhibits saturation kinetics, implying that cyanate may bind to the enzyme to give an enzyme-cyanate complex (K equal 0.125 M) prior to the covalent reaction. In the presence of manganous ion the addition of isocitrate protects the enzyme against cyanate inactivation, indicating that chemical modification occurs in the active site region of the enzyme. The dependence of the decrease of the rate constant for inactivation on the isocitrate concentration yields a dissociation constant for the enzyme-manganese-isocitrate complex which agrees with the Michaelis constant. The allosteric activator ADP, which lowers the Michaelis constant for isocitrate, does not itself significantly affect the cyanate reaction; however, it strikingly enhances the protection by isocitrate. The addition of the chelator EDTA essentially prevents protection by isocitrate and manganous ion, demonstrating the importance of the metal ion in this process. The substrate alpha-ketoglutarate and the coenzymes DPN and DPNH do not significantly affect the rate of modification of the enzymes by cyanate. Incubation of isocitrate dehydrogenase with 14C-labeled potassium cyanate leads to the incorporation of approximately 1 mol of radioactive cyanate per peptide chain concomitant with inactivation. Analysis of acid hydrolysates of the radioactive enzyme reveals that lysyl residues are the sole amino acids modified. These results suggest that cyanate, or isocyanic acid, may bind to the active site of this enzyme as an analogue of carbon dioxide and carbamylate a lysyl residue at the active site.     

Cyanate specifically inhibits arginine biosynthesis in Escherichia coli K12: a case of by-product inhibition?

Growth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mM-cyanate. This inhibition could be specifically reversed by arginine. Citrulline (but not ornithine, N-alpha-acetylornithine or N-acetylglutamate) could also restore a normal growth rate. Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine. The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase. A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion. Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.     

Isolation and characterization of Escherichia coli mutants lacking inducible cyanase

To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.     

Modifying action of carbamoylation reaction on mutagenic and toxic effects of alkylating compounds and heavy metal salts

Using mammalian somatic cells (CHO-AT3-2) we have demonstrated a synergistic effect of ethyl methane sulfonate and a carbamoylating agent, potassium cyanate. Potassium cyanate aggravated the toxic action of EMS and the induction of predominantly micro- and macroaberrational mutation, whereas the rate of point mutations of the base substitution type was not affected. No synergistic effect was observed when potassium cyanate was used in combination with heavy metal salts, regardless of the test employed.     

Cyanate as an inactivator of complement proteins.

Sodium cyanate added to normal human serum or serum from patients with sickle-cell disease resulted in the functional inactivation of C3, C5, C6, C7, and the C3b inactivator, but not C8 and C9. Final concentrations as low as 0.5 mM in serum caused inactivation of 12 to 64% of the C3 after 8 hr at 37 degrees C. The activity of the inactivated C3, C5, and C3b inactivator was not restored by dialysis. Most of the functional activity of C3 in cyanate-treated sera was destroyed by very small quantities of 14C-labeled cyanate that was bound to the protein. C3 inactivation by cyanate occurred in heated sera (50 degrees C, 30 min) and sera treated with EDTA, probably indicating that one mechanism for inactivation was by a direct carbamylation reaction. Both C3 and C5 showed two anodal-migrating forms in two dimensional antigen-antibody crossed electrophoresis in some sera treated with low concentrations of cyanate. Measurements of circular dichroism of highly purified carbamylated C3 showed no detectable changes in structure even though most of the functional activity was destroyed. Purified, inactive C3 that was carbamylated with 14C-labeled cyanate was capable of binding to EAC142, but the resulting EAC1423 was weakly positive for immune adherence and negative for agglutination with anti-C3 antiserum. Unlabeled, cell-bound C3b on EAC142 was not susceptible to cyanate action as shown by no loss in immune adherence and positive agglutination with anti-C3 antiserum. The C3b inactivator was more susceptible to cyanate than C3 in a short time period, whereas both were inactivated after 8 hr. Since cyanate is currently being evaluated as a treatment for sickle-cell disease, the inactivation of C3 by the drug is an important consideration for such patients who are already deficient in C3 dependent heat-labile opsonins that aid in host defense.