Synthesis and properties of carrier-fixed enzymes. VII. Linking of glucoamylase to dialdehyde cellulose, carboxymethylcellulose hydrazide and carboxymethylcellulose azide]

Glucoamylase (alpha-1,4-glucan glucohydrolase, EC 3.2.1.3) has been covalently linked to dialdehyde cellulose resulting in an immobilized enzyme containing 0.98% protein and an activity of 4.5 mg of the native enzyme per g of matrix, i.e. 46% relative activity. The complex lost its activity in continuous and batch hydrolysis of starch at 55 degrees C down to a limit of 18% of its original value. In contrast, the activity of the complex did not change when working at a temperature of 25 degrees C. Glucoamylase-carboxymethylcellulose complexes synthesized via carboxymethylcellulose hydrazide and azide, in contrast to MAEDA und SUZUKI [1], showed only an activity of 1 mg of the native enzyme per g of matrix. We did not succeed in coupling periodate-oxidized glucoamylase to carboxymethylcellulose hydrazide because the enzyme used lost nearly all of its activity already during periodate oxidation.     

Studies on immobilized enzymes. IX. Preparation and properties of aminoacylase covalently attached to halogenoacetylcelluloses

Immobilization of mold aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated by covalently binding the enzyme to halogenoacetylcelluloses. As a result, the iodoacetylcellulose was found to be the best carrier among the halogenoacetylcelluloses. The yield of activity of the insoluble aminoacylase relative to that of the native aminoacylase used was 40–50%, and the specific activities of both enzyme preparations were the same within the limits of error of the estimation.     

Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.     

Covalent attachment of epoxide hydrolase to dextran

Epoxide hydrolase (EC 3.3.2.3) purified from rat liver microsomes has been immobilized by covalent linking to dextran activated by imidazolyl carbamate groups, under mild conditions. K^app_m values of free and dextran bound epoxide hydrolase toward benzo(a)pyrene-4,5-oxide were 0.5 and 0.35 μM respectively, while V^app_max was lowered from 300 to 120 nmol min^?1mg^?1protein. The activity lost upon coupling could not be restored by digestion of the support by dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) treatment. This fact, along with the similarity of the activation energy values for both native and bound epoxide hydrolase, indicated that steric hindrance effects due to the polymer support played only a minor role in this loss of activity. Evidences of changes in the conformation of epoxide hydrolase were obtained by a comparative study of u.v. circular dichroism and tryptophan fluorescence emission spectra of the native and dextran bound enzymes. On the other hand, the enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation.     

Influence of rheopolyglucin and dextran dialdehyde on physicochemical properties and thermostability of human hemoglobin

A study was made of the influence of rheopolyglucin and dextran dialdehyde derived therefrom on the structural characteristics and thermostability of human hemoglobin. The effects of solution pH, incubation time and temperature, and the degree of dextran oxidation on the conjugation between human hemoglobin and dextran dialdehyde were assessed. Formation of the hemoglobin-dextran dialdehyde complex resulted in shielding of the protein chromophore groups by the polysaccharide and transition of a part of hemoprotein molecules from a low-spin (HbO₂) to a high-spin (Hb and MetHb) state. It was found that the temperature of denaturation transition for the native protein and hemoglobin in the presence of rheopolyglucin was 60°C versus 80°C for the hemoprotein-dextran dialdehyde conjugate. Presumably, the latter is determined by the enhancement of hydrophobic interactions within the protein globule caused by dextran dialdehyde and the ability of the surface-bound carbohydrate components to prevent the association of hemoglobin molecules.     

Covalent binding of glutathione to hemoglobin. I. Inhibition of hemoglobin S polymerization

Thiol reagents react with cysteine beta 93 of hemoglobin and as a result increase the oxygen affinity of hemoglobin. In the present studies we have used a thiol-disulfide exchange between mixed disulfides of hemoglobin and reduced glutathione to attach intracellular glutathione to hemoglobin and to study its antisickling properties. The rates of production of glutathionyl hemoglobin (G-Hb) depend on the structure of the thiol reagent linked to cysteine beta 93. Up to 25% G-Hb can be produced in normal and sickle red cells because of the high intracellular concentration of reduced glutathione. This high level of G-Hb in normal cells increases the oxygen affinity by about 35% and reduces heme-heme interactions. In sickle cells the increased oxygen affinity is associated with an inhibition of sickling of about 70% at 21 mm Hg. Inhibition of polymerization of deoxy HbS is also due to a direct inhibition of intermolecular contacts in the fibers as demonstrated by the increased solubility and the increased delay time of G-HbS compared to deoxy HbS.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Covalent binding of enzymes to synthetic membranes containing acrylamide units, using formaldehyde

Synthetic membranes containing 10% acrylamide units were subjected to activation with formaldehyde at pH 7.5 and 45 degrees C. Trypsin, invertase, and urease were bound to this activated membrane and the kinetic properties of immobilized enzymes were studied. The permeability of the membrane for distilled water manifests certain differences depending on the enzyme bound. The membranes with immobilized enzymes stored at 4 degrees C in a moist state showed no change in their activity for 6 months. The membrane with immobilized invertase has preserved its activity even after 20 operations with 2% sucrose solution at 25 degrees C. The proposed method of binding enzymes to synthetic membranes containing acrylamide groups, through the introduction of N-hydroxymethyl groups, possesses several advantages with respect to the activation of the membrane in a one-step reaction with cheap and accessible reagent, high operative stability of the immobilized enzymes, no danger of bacterial rotting, and long shelf life of the membrane.     

Covalent fixation of hemoglobin to dextran phosphates decreases its oxygen affinity.

The interactions between various dextran phosphates and Hb (hemoglobin) were studied by measuring the oxygen-binding parameters of the mixtures. The effector properties of polymers were found to depend on the concentration of monoalkylmonophosphate groups on the polymers and also on their molecular weights. The covalent fixation of dextran phosphates bearing aldehydic groups to oxyHb and deoxyHb was carried out. The oxygen-binding properties of the conjugates thus obtained depended upon the initial form of the protein. Thus, only the conjugates synthesized from deoxyHb exhibited a low oxygen affinity, which means that, in this case, the linkages between the dextran phosphate and the protein allow a permanent interaction of the phosphate groups with amines of the 2,3-diphosphoglycerate binding site. The Hill coefficient values of these conjugates were smaller than that of free Hb, corresponding to a loss of the cooperativity of the protein upon fixation of polymers. However, as these new conjugates are capable of unloading more O2 than blood when subjected to oxygen pressures corresponding to physiological conditions, they can be regarded as potential erythrocyte substitutes.     

Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I.

Analysis of vaccinia topoisomerase mutants that are impaired in DNA relaxation has allowed the identification of amino acid residues required for the transesterification step of catalysis. Missense mutations of wild-type residues Gly-132----Asp and Arg-223----Gln rendered the protein inert in formation of the covalent enzyme-DNA complex and hence completely inactive in DNA relaxation. Mutations of Thr-147----Ile and Gly-132----Ser caused severe defects in covalent adduct formation that correlated with the extent of inhibition of relaxation. None of these point mutations had an effect on noncovalent DNA binding sufficient to account for the defect in relaxation. Deletion of amino- or carboxyl-terminal portions of the polypeptide abrogated noncovalent DNA binding. Two distinct topoisomerase-DNA complexes were resolved by native gel electrophoresis. One complex, which was unique to those proteins competent in covalent adduct formation, contained topoisomerase bound to the 5'-portion of the incised DNA strand. The 3'-segment of the cleaved strand had dissociated spontaneously. This complex was isolated and shown to catalyze transfer of the covalently bound DNA to a heterologous acceptor oligonucleotide, thereby proving that the covalent adduct between protein and duplex DNA is a true intermediate in strand breakage and reunion. The role of the active site region of eukaryotic topoisomerase in determining sensitivity or resistance to camptothecin was examined by converting the active site region of the resistant vaccinia enzyme (SKRAY274) to that of the drug-sensitive yeast enzyme (SKINY). The SKINY mutation did not alter the resistance of the vaccinia enzyme to the cleavage-enhancing effects of camptothecin.     

Some equilibrium and non-equilibrium properties of the allosteric interactions in enzymes. I. Ligand binding and saturation equations

Recent evidence and speculation regarding the dynamic structure of biological membranes is combined with information on the pharmacology and biochemistry of the allergic histamine release reaction to formulate a model which can explain many of the observed events in this reaction, and especially tie presumed early enzymological events to pharmacologically controlled subsequent events. It is proposed that the reaction of the cell-bound antibodies with suitable antigens causes a membrane deformation or a displacement of hydrophilic residues within the membrane in such a manner that there is a local clustering or polarization of charges. Biochemically, the earliest event may be the activation of a membrane-bound chymotrypsin-like proesterase. A speculative step in the proposed sequence is the activation of a membrane-bound pro-phospholipase A by the activated esterase. The local, limited action of the phospholipase A on the membrane lipids could influence the action of membrane-bound ATPase and nucleotide cyclases in several ways. The resulting local decrease in the concentration of cyclic AMP, is a condition which is known to modulate antigen-induced histamine release. It is proposed that the cyclic nucleotides may affect histamine release at more than one point in the sequence. First, they may regulate the contractility of the microtubles which have been shown to be involved in histamine release. Second, they may influence the state of aggregation and subcellular distribution of the microfilaments which play a role in the maintenance of the normal organization of the cell. As a result of the drop in the cyclic AMP concentration, or the accumulation of lysophosphatides, the cell membrane may be reorganized. This could lead to membrane invagination and an apparent “interiorization” of some of the aqueous milieu. The histamine-containing granules of the mast cells are thus brought into proximity of these deep invaginations by microtubule action, an energy-requiring process. The perigranular membranes fuse with the plasma membrane and the granules exchange their stored histamine for the extracellular sodium which enters the invaginations with the water. The histamine is then equilibrated with the external medium. A number of alternative mechanisms and testable corollaries of the theory are discussed.     

Effect of rheopolyglucin and dialdehyde dextran on thermostability of human haemoglobin molecules

Structural characteristics and thermostability of human haemoglobin molecules, modified by reopolyglukine and dialdehyddextrane (DAD) have been studied. Haemoglobin mainly preserves its oxyform when including reopolyglukine in mixture (the mol. ratio of protein to dextrane was 1:4), and disordering of polypeptide chains and increasing the volume of haemoproteids molecules are noticed. Assessment of the influence of mixture pH, exposure and temperature of incubation and the rate of dextrane oxidation on the intensity of the process of human haemoglobin conjugation with DAD have been chosen. Formation of the haemoglobin-DAD complex leads to shielding protein chromophoral groups by polysugar matrix and to transforming the part of haemoproteid molecules from lowspin form (HbO2) to highspin forms (Hb, MetHb). It has been detected that temperature of denaturational points for native protein and haemoglobin in the presence of reopoliglukine is 60 degrees C, but it is 80 degrees C for the haemoglobin-DAD conjugate. The latter is probably determined by increasing hydrophobic interactions inside the protein globule under the effect of DAD and by ability of the surface-bound carbohydrate components prevent association of haemoglobin molecules.