Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

Purification of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose.

Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.     

Affinity chromatography of alpha-chymotrypsin, subtilism, and metalloendopeptidases on carbobenzoxy-L-phenylalanyl-triehtylenetetraminyl-sepharose.

Carbobenzoxy-L-phenylalanyl-triethylenetetraminyl-Sepharose (Z-L-Phe-T-Sepharose) was found to be an effective affinity adsorbent for bovine pancreatic alpha-chymotrypsin [EC 3.4.21.1] as well as neutral [EC 3.4.24.4] and alkaline [EC 3.4.21.14] proteases of Bacillus species. These enzymes were adsorbed in the neutral pH range. alpha-Chymotrypsin was recovered by elution with 0.1 A acetic acid while neutral subtilopeptidase was eluted with 0.5 M NaCl at pH 0. Thermolysin and subtilisin were found in eluates with 1.5 and 2.0 M guanidine-HCl at pH 7.2, respectively. The resulting enzymes appeared homogeneous on disc-electrophoresis and showed higher specific activities than those of crystalline or highly purified preparations available commercially. Modifications of the active site serines of alpha-chymotrypsin and subtilisin by treatment with diisopropylfluorophosphate (DFP) or phenylmethanesulfonyl fluoride (PMSF) resulted in loss in their binding abilities to the adsorbent. Complexes of porcine alpha2-macroglobulin with each of these four enzymes and that of Streptomyces-subtilisin inhibitor (S-SI) with subtilisin were also found in nonadsorbed fractions.     

Elution of Acid Phosphatase from the Cell Surface of Saccharomyces mellis by Potassium Chloride.

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Elution of acid phosphatase from the cell surface of Saccharomyces mellis by potassium chloride. J. Bacteriol. 90:82-94. 1965.-Acid phosphatase of Saccharomyces mellis may be eluted from intact resting cells by 0.5 m KCl or other salts. However, the enzyme is not eluted at higher salt concentrations of about 2 m unless a thiol, such as beta-mercaptoethanol, is included in the reaction mixture. These treatments do not significantly affect viability of the cells. Neutral compounds like sorbitol or sucrose cannot substitute for ionic compounds in eluting the enzyme from resting cells. Furthermore, the neutral compounds are also inadequate for stabilizing the protoplast structure. It is suggested that the enzyme is held on the cell surface by a combination of electrostatic forces and disulfide bonds. Thiol alone dissociates protein and carbohydrate from the cell surface, but the eluate has no acid phosphatase activity. Salts also remove protein and carbohydrate from the cell surface, but the amount of protein removed is considerably less than that dissociated by thiol. A concentration of 0.5 m KCl elutes more protein than does a 2 m concentration, and enzymatic activity is present only in the 0.5 m KCl eluate. The carbohydrate eluted by either reagent has been identified as a mannan. Conditions for eluting acid phosphatase from acetonedried cells of S. mellis are essentially the same as those for resting cells. Significantly, though, thiol is required at all salt concentrations to dissociate the enzyme. Pretreatment of the cells with thiol, followed by KCl, elutes acid phosphatase, whereas the reverse procedure does not. Acid phosphatase is excreted by growing cells of S. mellis into growth media if the medium contains 0.25 m KCl. The total yield of enzymatic activity may be 8 to 10 times greater than is usually present on derepressed cells grown in a salt-free medium. The enzyme can be precipitated from the culture fluid with acetone. The acetone-precipitated fraction contains mannan and protein in a ratio of 12:1 by weight. Partial purification of the enzyme by calcium phosphate gel and elution resulted in an enzyme fraction in which the specific activity on the basis of protein increased 12-fold, and the carbohydrate-protein ratio was reduced to 1:1.     

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.     

Structural and functional comparisons of pH-sensitive liposomes composed of phosphatidylethanolamine and three different diacylsuccinylglycerols

The titratable, double-chain amphiphiles 1,2-dipalmitoyl-sn-3-succinylglycerol (1,2-DPSG), 1,2-dioleoyl-sn-3-succinylglycerol (1,2-DOSG) and 1,3-dipalmitoylsuccinylglycerol (1,3-DPSG) have been used in combination with phosphatidylethanolamine (PE) to form pH-sensitive liposomes. The effect of the compounds on dielaidoyl PE bilayer stabilization was examined by differential scanning calorimetry. Only 1,2-DPSG showed bilayer stabilization activity; whereas the other two are destabilizers at pH 7.4. All three amphiphiles became strong destabilizers at pH 5.0. The ability of the amphiphiles to stabilize DOPE liposomes was examined by light scattering and calcein entrapment. In general, 1,2-DPSG is the most potent stabilizer of PE bilayers while 1,3-DPSG is the weakest liposome stabilizer. All three compounds can be combined with DOPE to generate liposomes which are stable at neutral and basic pH. At weakly acidic pH, the liposomes are leaky and exhibit extensive lipid mixing, with protons and calcium showing synergistic effects on lipid mixing. DOPE/1,2-DPSG liposomes are stable in human plasma and remain acid-sensitive even after prolonged plasma incubation. Immunoliposomes prepared from either DOPE/1,2-DPSG or DOPE/1,2-DOSG can deliver diphtheria toxin A fragment to the cytoplasm of cultured cells in a process which involves endocytosis of the liposomes. Immunoliposomes prepared with 1,2-DPSG are more effective drug carriers than those prepared with 1,2-DOSG. These results indicate that the bilayer- and, hence the liposome-stabilization activity of the diacylsuccinylglycerol depends on the structure of the compounds. The potential drug delivery activity of the pH-sensitive liposomes composed of these lipids is discussed.     

High-level production and characterization of a novel β-1,3-1,4-glucanase from Aspergillus awamori and its potential application in the brewing industry

A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was extracellularly expressed in Pichia pastoris. AaBglu12A showed amino acid identity of 96 % with a glycoside hydrolase family 12 cellulase from A. kawachii and 48 % with a β-1,3-1,4-glucanase from Magnaporthe oryzae. The highest β-1,3-1,4-glucanase activity of 159,500 ± 500 U/mL with protein concentration of 31.7 ± 0.3 g/L was achieved in a 5-L fermentor. AaBglu12A was purified until homogeneous with recovery yield of 92 %. Its maximal activity was found at 55 °C and pH 5.0. The enzyme was stable up to 60 °C and within the pH range of 2.0-9.0. It also demonstrated strict substrate specificity towards oat- and barley-glucans as well as lichenan. The K_m values for oat-, barley-glucans, and lichenan were 2.82, 3.51, and 2.53 mg/mL, respectively. The V_max values for oat-, barley-glucans, and lichenan were 12,068, 10,790, and 7236 μmol/min·mg, respectively. AaBglu12A hydrolyzed oat- and barley-β-glucans to produce tetra- and tri-saccharides. However, lichenan was hydrolyzed to yield trisaccharides as the main end product. The addition of AaBglu12A to the mashing process substantially decreased filtration time by 34.5 % and viscosity by 9.6 %. Therefore, the high-level production of AaBglu12A might be a promising strategy for the brewing industry owing to its favorable properties.     

Effect of neuraminidase on the chromatographic behaviour of eleven acid hydrolases from human liver and plasma.

1. The elution profiles of eleven acid hydrolases from human liver and plasma were directly compared using a system whereby a single salt gradient was simultaneously applied to two DEAE-cellulose chromatographic columns. 2. Plasma alpha-L-fucosidase, alpha-mannosidase, alpha-galactosidase and alpha-glucosidase isoenzymes were eluted at higher salt concentrations than the corresponding liver isoenzymes whereasbeta-N-acetylglucosaminidase, beta-galactosidase, beta-glucosidase, exo-1,4-beta-xylosidase and alpha-L-arabinofuranosidase isoenzymes were eluted at lower salt concentrations. The elution profiles of beta-glucuronidase and acid phosphatase weremore complex. 3. After incubation with neuraminidase most plasma hydrolases were eluted at lower salt concentrations, however the elution patterns of beta-glucosidase, beta-xylosidase and acid phosphatase were not altered. 4. Preincubation with neuraminidase had no effect on the elution profiles of six liver hydrolases whereas the major isoenzymes of alpha-mannosidase, beta-galactosidase and alpha-L-arabinofuranosidase were eluted at markedly lower salt concentrations. Liver alpha-fucosidase and alpha-galactosidase were eluted at slightly lower salt concentrations afterincubation with neuraminidase. 5. The results are discussed in relation to thepathogenesis of Mucolipidosis II (I-cell disease), and the synthesis and packaging of lysosomal enzymes.     

Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.     

Affinity chromatography of bovine trypsin. A rapid separation of bovine α- and β-trypsin

Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p′-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6–8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure β-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both α- and β-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-α-(N′-methyl-N′-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.     

Synthesis of 2- (and 6-) -dithian-2-yluracil nucleosides and their conversion into nucleoside derivatives

Addition of 2,2′-anhydro-[1-(3-O-acetyl-5-O-trityl-β-D-arabinofuranosyl)uracil] (1) to excess 2-litho-1,3-dithiane (2)in oxolane at ?78° gave 2-(1,3-dithian-2-yl)-1-(5-O-trityl-β-D-arabinofuranosyl)-4(1H)pyrimidinone (3), O²,2′-anhydro-5,6-di-hydro-6-(S)-(1,3-dithian-2-yl)-5′-O-trityluridine (4), and 2-(1,4-dihydroxybutyl)-1,3-dithiane (5) in yields of 15, 30, and 10% respectively. The structure of 3 was proved by its hydrolysis in acid to give 2-(1,3-dithian-2-yl)-4-pyrimidinone (6) and arabinose, and by desulfurization with Raney nickel to yield the known 2-methyl-1-(5-O-trityl-β-D-arabinofuranosyl)-4(1H)-pyrimidinone (7). Detritylation of 3 without glycosidic cleavage could only be effected by prior acetylation to 1-(2,3-di-O-acetyl-5-O-trityl-β-D-arabinofuranosyl)-2-(1,3-dithian-2-yl)-4(1H)-pyrimidinone (8) which, after treatment with acetic acid at room temperature for 65 h followed by the action of sodium methoxide gave 2-(1,3-dithian-2-yl)-1-β-D-arabinofuranosyl-4(1H)-pyrimidinone (10) in 45% yield. Detritylation of 4 in boiling acetic acid gave 5,6-dihydro-6-(S)-(1,3-dithian-2-yl)-1-β-D-arabinofuranosyluracil (12) and 3-[(S)-1-(1,3-dithian-2-yl)]propionamido-(1,2-dideoxy-β-D-arabinofurano)-[1,2-d]-2-oxazolidinone (13) in 10 and 90% yields, respectively. When 12 was kept in water or methanol for 7 days, quantitative conversion into 13 occurred. Acid hydrolysis of 12 afforded arabinose and 5,6-di-hydro-6-(1,3-dithian-2-yl)uracil (14), which was desulfurized with Raney nickel to the known 5,6-dihydro-6-methyluracil (15). Treatment of 13 with trifluoroacetic anhydride-pyridine yielded 77% of the cyano derivative 17. Similar dehydration of 3-(R)-1-methylpropionamido-(1,2-dideoxy-β-D-arabinofurano)-[1,2-d]-2-oxalidinone (18), obtained by desulfurization of 13, gave 60% of the nitrile 19. Hydrogenation of 19 over platinum oxide in acetic anhydride gave the acetamide derivative 20 in 95% yield. Nitrobenzoylation of 13 gave 3-[(S)-1-(1,3-dithian-2-yl)]cyanomethyl-3,5-di-O-p-nitrobenzoyl-(1,2-dideoxy-β-D-arabinofurano)-[1,2-d]-2-oxazolidinone (22), which was converted in 37% yield by treatment with methyl iodide in dimethyl sulfoxide into the aldehyde 24, characterized as the semicarbazone 25. The purification of 5 and its characterization as 2-(1,4-di-O-p-nitrobenzoylbutyl)-1,3-dithiane (27) is described.     

Characterisation of a novel Bacillus sp. SJ-10 β-1,3–1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish

A novel β-1,3–1,4-glucanase gene was identified in Bacillus sp. SJ-10 (KCCM 90078) isolated from jeotgal, a traditional Korean fermented fish. We analysed the β-1,3–1,4-glucanase gene sequence and examined the recombinant enzyme. The open reading frame of the gene encoded 244 amino acids. The sequence was not identical to any β-glucanases deposited in GenBank. The gene was cloned into pET22b(+) and expressed in Escherichia coli BL21. Purification of recombinant β-1,3–1,4-glucanase was conducted by affinity chromatography using a Ni-NTA column. Enzyme specificity of β-1,3–1,4-glucanase was confirmed based on substrate specificity. The optimal temperature and pH of the purified enzyme towards barley β-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30–70 °C and pH values of 4–9, which differed from all other bacterial β-1,3–1,4-glucanases. The degradation products of barley β-glucan by β-1,3–1,4-glucanase were analysed using thin-layer chromatography, and ultimately glucose was produced by treatment with cellobiase.     

Phosphate and pepsin adsorptions by a new boehmite compound and aluminum hydroxide

A new microcrystalline compound of aluminum oxide hydroxide (tentatively named PT-A) was synthesized in the hope of providing a better phosphate adsorbent for future clinical use than the currently marketed aluminum hydroxide gels (ALG). An X-ray diffraction study demonstrated a boehmite structure in PT-A but an amorphous structure in ALG. PT-A was more stable in pH change than ALG; in elution tests in artificial gastric and intestinal solutions, aluminum ion eluted from PT-A was maximally 10% of the amount from ALG at pH 1.2; and was undetectable at pH 6.8, at which point ALG still showed some aluminum elution. Phosphate-adsorbing efficacy of PT-A and ALG in vitro was about the same at pH 1.2; however, it was four times greater in PT-A than in ALG at pH 6.8, indicating that PT-A will be effective in the intestine. PT-A also adsorbed pepsin but the amount was at most the same or much less than that adsorbed by ALG, which depended on pH in solution.     

Non-inducibility of the glutathione transferases of the earthworm Eisenia andrei

1. Earthworms were exposed to solutions of potential biotransformation enzyme inducers, i.e. trans-stilbene oxide (0.1 mM), 3-methylcholanthrene (50 μM) or 1,4-bis[2-(3,5-dichloropyridoxyl)]benzene (30 μM) for 1 hr each day in three successive days.2. Glutathione transferase (GST) was determined in cytosolic extracts of the worms the day after the last treatment, with chloro-2,4-dinitrobenzene, l,2-dichloro-4-nitrobenzene and ethacrynic acid as electrophilic substrates. Activity toward 1,2-epoxy-3-[p-nitro-fenoxy]propane was not detected.3. With all three substrates, the specific activity in the cytosol was similar in treated animals and controls.4. Ion exchange chromatography was performed on a NeoBar AQ^TMcolumn (The “Low Pressure High Performance” technique), and GST eluted in four distinct peaks.5. The elution profiles of GST activity were almost identical for exposed earthworms and controls.6. The results suggest that earthworms may not have inducible GST, perhaps because they are detritus eaters, and do not need to detoxicate poisonous secondary plant metabolites.     

Affinity chromatography of arabinogalactan-proteins

Arabino-(1→3), (1→6)-β-d-galactan-proteins (AGPs) and related compounds from Lolium multiflorum (ryegrass) endosperm cell suspension culture, wheat endosperm, larchwood and Gladiolus stigma extract were shown to bind selectively at neutral pH to a column of Sepharose to which the anti-galactan myeloma protein J539 had been covalently linked, Elution was achieved with buffer at pH 3 or with a pulse of p-nitrophenyl-β-d-galactopyranoside at neutral pH. These observations formed the basis for an affinity chromatographic purification of AGPs from natural sources. Some heterogeneity in a ryegrass AGP preparation was indicated by its incomplete elution by d-galactose.     

Isolation of β-Xylanase from whole broth of Bacillus amyloliquefaciens by adsorption on a matrix in fluidized bed with low degree of expansion

-1,4-Xylanase, produced extracellularly by Bacillus amyloliquefaciens MIR 32, was isolated directly from the culture broth by adsorption on a cation exchanger, Amberlite IRC-50, in fluidized bed with a low degree of expansion. The enzyme was eluted from the adsorbent by increase in pH, with a recovery of 82.3% and purification of 5.3 fold. About 99.99% of the colony forming units, 82% of the contaminating neutral protease activity, and 100% of the reducing sugars present in the crude feedstock were removed at the end of the purification cycle.     

Effect of Hydroxyl Groups and Rigid Structure in 1,4-Cyclohexanediol on Percutaneous Absorption of Metronidazole

In a previous study, a synergistic retardation effect of 1,4-cyclohexanediol and 1,2-hexanediol on percutaneous absorption and penetration of metronidazole (MTZ) was discovered. A complex formation between 1,4-cyclohexanediol and 1,2-hexanediol was proposed to be responsible for the observed effect. The objective of this study was to investigate the necessity of hydroxyl group and the ring structure in 1,4-cyclohexanediol on percutaneous absorption and penetration of MTZ. Eleven formulations were studied in an in vitro porcine skin model using glass vertical Frans Diffusion Cell. 1,4-Cyclohexanediol was changed into 1,4-cyclohexanedicarboxylic acid, trans (and cis)-1,2-cyclohexanediol and 1,6-hexanediol, respectively, to study if H-bonding or ring structure would influence the retardation effect. MTZ was applied at infinite dose (100 mg), which corresponded to 750 μg of MTZ. Based on modifier ratios (MR) calculated by the flux values, the retardation effect on percutaneous absorption and penetration of MTZ was found in the formulations containing 1,4-cyclohexanedicarboxylic acid or cis-1,2-cyclohexanediol (MR values were 0.47 for which only contains 1,4-cyclohexanedicarboxylic acid, 0.74 for the formulation containing both 1,4-cyclohexanedicarboxylic acid and 1,2-hexanediol, and 0.90 for the formulation containing cis-1,2-cyclohexanediol and 1,2-hexanediol, respectively). The results showed that the hydroxyl group and structure of 1,4-cyclohexanediol played a significant role in retardation effects and provided valuable insight on the mechanisms of retardation effect through structure–activity relationships.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5′-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.