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Simonkolleite is a zinc-layered hydroxide salt with the formula Zn5(OH)8Cl2·H2O. It has a platelet morphology and can be used for many applications, owing to both its layered structure and its nature as a hydroxide salt. It can be prepared via a simple precipitation from ZnCl2 and NaOH in water thermostated at 50 °C. Depending on the synthesis conditions, we could obtain different sizes and a hybrid containing parts of ZnO. We studied the influence of the OH:Zn molar ratio, the addition order, and the maturation time after the reaction was completed. With the support of pH profiles, kinetic studies, and thermodynamic equilibrium data, we were able to propose a global synthesis mechanism explaining the influence of those three parameters and identify the range of conditions in which simonkolleite can be formed. Depending on the desired application, we were able to synthesize bigger or smaller layered crystals of simonkolleite, in the presence of absence of ZnO.

Some specific properties of pullulanases usually have a great deal to do with their specific N-terminal CBMs. This paper focusses on characters of the novel carbohydrate-binding module CBM68 from a thermostable pullulanase PulA. As to reveal its different properties with the common module CBM41/CBM41-X45 (from an acid pullulanase PulB), twelve recombinant pullulanases were constructed and named as Pul-1~Pul-12 respectively. Through comparative analysis of their thermostabilities, pH profiles, and kinetic parameters, some rules have been concluded. Compared with CBM41, CBM68 on the N-terminal of molecules could make pullulanases more thermostable, have higher substrate affinity, and have higher catalytic efficiency. While CBM41 had the advantage on improving the acidic stability of pullulanase comparing with CBM68. X45 could help pullulanases fold properly and has impact to the catalytic domains of pullulanase. The catalytic domain of PulA or PulB played important roles in the optimum pH and catalytic efficiency of pullulanases. Moreover, the optimal combination of CBM41-X45 and the catalytic domain of PulA made Pul-7 have the highest catalytic efficiency (1284.68 mL/mg·s) which was 1.74 times higher than that (737.78 mL/mg·s) of PulA. The characteristics of CBM68, CBM41 and CBM41-X45 revealed in this study would serve as a basis for rational design of pullulanases.

Understanding the pH effect of cellulolytic enzymes is of great technological importance. In this study, we have examined the influence of pH on activity and stability for central cellulases (Cel7A, Cel7B, Cel6A from Trichoderma reesei, and Cel7A from Rasamsonia emersonii). We systematically changed pH from 2 to 7, temperature from 20°C to 70°C, and used both soluble (4-nitrophenyl β- d-lactopyranoside [pNPL]) and insoluble (Avicel) substrates at different concentrations. Collective interpretation of these data provided new insights. An unusual tolerance to acidic conditions was observed for both investigated Cel7As, but only on real insoluble cellulose. In contrast, pH profiles on pNPL were bell-shaped with a strong loss of activity both above and below the optimal pH for all four enzymes. On a practical level, these observations call for the caution of the common practice of using soluble substrates for the general characterization of pH effects on cellulase activity. Kinetic modeling of the experimental data suggested that the nucleophile of Cel7A experiences a strong downward shift in pKa upon complexation with an insoluble substrate. This shift was less pronounced for Cel7B, Cel6A, and for Cel7A acting on the soluble substrate, and we hypothesize that these differences are related to the accessibility of water to the binding region of the Michaelis complex.

In the present study the solubility-pH profiles of sibutramine free base and its hydrochloride salt were determined in the pH range between 2.0 and 9.5 by means of the recommended shake-flask method, and the solids collected were dried and studied by X-ray diffraction in order to elucidate their free base or salt structure. Above pHmax (or Gibbs pKa) the solid collected was always identified as free base, whatever the sibutramine species (free base or hydrochloride salt) initially solved. However, in the pH range below pHmax different solids were isolated depending on the buffers employed.

Chloroperoxidase from the fungus Caldariomyces fumago was covalently immobilized on SBA-15 mesoporous material and assayed for the enzymatic oxidation of four azo dyes. All dyes were oxidized by the free and the immobilized enzyme to different extent. Acid Blue 120 and Direct Blue 85 dyes were decolorized almost completely. The catalytic efficiency, k cat/K M, of the immobilized enzyme was 27, 2.9, 137 and 28 times higher than the free enzyme for Basic Blue 41, Disperse Blue 85, Acid Blue 120 and Direct Black 22 oxidation, respectively. The immobilized enzyme displayed improved thermostability and a similar pH profile compared to the free enzyme. The immobilized chloroperoxidase showed excellent storage stability and maintained 100 % catalytic activity after 90 days at both 4 and 25 °C.

The objective of this study was to compare a novel sustained release tablet formulation that has the potential to be used for drugs of different physicochemical properties using a binary mixture of polymethacrylate polymers in their salt forms with the polymethacrylate interpolyelectrolyte complex (IPEC) tablets in terms of drug release and compactness. Also, we aimed to compare this formulation with an IPEC tablet in terms of drug release. Tablets prepared using Eudragit E-Citrate and Eudragit L-Sodium were more convenient, easier to prepare, and showed better sustained release and compactness characteristics compared to IPEC tablets of similar concentrations and preparation methods.

In this study, the zeta potential values of vermiculite and expanded vermiculite were measured to determine the effect of pH, clay concentration, and various mono- and multivalent electrolytes including NaCl, KCl, NH4Cl, NaNO3, NaClO4, Na2SO4, Na2CO3, Na3PO4·12H2O, MgCl2·6H2O, CaCl2·2H2O, BaCl2, SrCl2·6H2O, CuCl2·2H2O, CoCl2·6H2O, NiCl2, AlCl3, and CrCl3·6H2O on the electrokinetic properties of vermiculite samples. It was found that generally the measured zeta potential values of expanded vermiculite for the studied systems were slightly more negative than that of vermiculite. The pH profiles of vermiculite and expanded vermiculite at acidic, natural, and basic pH values were obtained to determine the effect of time on the pH values of clay suspensions. The zeta potential measurements showed that the surface charge of clay particles was negative in water. The isoelectric point of vermiculite and expanded vermiculite were determined as pH 2.30 and 2.57, respectively. Divalent cations (Mg2+, Ca2+, ...

Chloroperoxidase is a versatile fungal heme-thiolate protein that catalyzes a variety of one-electron and two-electron oxidations. We report here that the alkylation of an essential histidine residue showed no effect on the one-electron peroxidations but inhibited two-electron oxidations. The pH profiles of different peroxidative substrates showed optimal activities at varying pH values for the same enzyme. 2-Allylphenol and substituted ortho-phenolics showed efficient peroxidations. Also, substrates excluded from the active site (or with no favorable positioning at the heme center or heme edge) were converted in the peroxidation reaction. While hydrogen peroxide serves as the superior activator in the two-electron oxidations, small alkylhydroperoxides give much better rates for peroxidation reactions. All the above observations indicate that one-electron oxidations are mechanistically quite different from the two-electron oxidations catalyzed by chloroperoxidase. We propose that the peroxidatic substrates interact predominantly outside the heme active site, presumably at the surface of the enzyme.

The pH dependence of the reaction of various renins was investigated using sheep angiotensinogen as a substrate. Human renin showed two separate peaks, but rat and mouse Ren1 renins showed one peak with a shoulder. A comparison of the predicted subsite residues of human renin with those of rat and mouse Ren1 renins revealed that Arg82, Ser84, Thr85, Ala229, and Thr312 are unique in the human sequence. We examined the possible importance of these residues in the unique pH profile of the human renin reaction by replacing these residues with the corresponding residues of rat renin. The replacement of Ser84 of human renin with Gly changed the pH dependence of the reaction to one peak, similarly to rat and mouse Ren1 renins. Other mutant human renins kept two separate peaks, similarly to wild-type human renin. These results indicate that Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction.

The ribosome-catalyzed peptidyl transferase reaction displays a complex pH profile resulting from two functional groups whose deprotonation is important for the reaction, one within the A-site substrate and a second unidentified group thought to reside in the rRNA peptidyl transferase center. Here we report the synthesis and activity of the beta,beta-difluorophenylalanyl derivative of puromycin, an A-site substrate. The fluorine atoms reduce the pK(a) of the nucleophilic alpha-amino group (5.0) such that it is deprotonated at all pHs amenable to ribosomal analysis (pH 5.2-9.5). In the 50S modified fragment assay, this substrate reacts substantially faster than puromycin at neutral or acidic pH. The reaction follows a simplified pH profile that is dependent only upon deprotonation of a titratable group within the ribosomal active site. This feature will simplify characterization of the peptidyl transferase reaction mechanism. On the basis of the reaction efficiency of the doubly fluorinated substrate compared to the unfluorinated derivative, the Bronsted coefficient for the nucleophile is estimated to be substantially smaller than that reported for uncatalyzed aminolysis reactions, which has important mechanistic implications for the peptidyl transferase reaction.

The pH dependence of the kinetic parameters of recombinant acetohydroxy acid synthase catalyzed reaction was determined in order to obtain information about the chemical mechanism, particularly acid-base chemistry. The maximum velocity and V/K for pyruvate were bell-shaped with estimated pK values of 6.5-6.7 and 8.6-8.9, respectively. The maximum velocity and V/K for 2-ketobutyrate were also bell-shaped with estimated pK values of 6.6-7.0 and 8.4-8.6. The pH dependence of 1/Ki for 3-bromopyruvate, a competitive inhibitor of pyruvate, was also bell-shaped, giving pK values almost identical with those obtained for pyruvate. Since the same pK values were observed in the , V/K pH profiles and profiles, both enzyme groups must be in their optimum protonation state for efficient binding of reactants. These results reflect that two enzyme groups are necessary for binding of substrate and/or catalysis.

The pH control was important for curdlan production with Agrobacterium sp. ATCC31750. Specific cell growth rate was the highest at pH 7 and the specific curdlan production rate was at pH 5.5. The pH profiles maximizing curdlan production was changed from pH 7 optimal for cell growth to pH 5.5 optimal for curdlan production after ammonium consumption. The feedback inferential control methods, with easily measurable variables such as NaOH addition for pH control and dissolved oxygen (DO), were also applied. The pH was successfully controlled to follow optimal profiles and the maximal production of curdlan (60 g l−1 in 120 h) was achieved with feedback optimal control.

L-​Glutamine and glutamic acid were incorporated in a tetrahydro-​2H-​1,​3,​5-​thiadiazine-​2-​thione (THTT) skeleton contg. different alkyl, cycloalkyl and aralkyl groups at N3 of the ring system. These THTT derivs. revealed high lipophilicity compared with L-​glutamine and glutamic acid. The degrdn. kinetics were studied -​in vitro- using aq. buffer solns. of pH values: 1.2, 6.0, 7.4, 7.8 and 9.0 at 37°. From the pH profile, the degrdn. was accounted for, in terms of specific base-​catalyzed reactions. All of the compds. however, showed higher acid-​stability.

In order to gain some insights into durum wheat lipoxygenase (LOX) biochemistry and in particular in order to find out whether the antioxidants α-tocopherol and L-ascorbate can prevent the LOX-dependent semolina bleaching, which leads to a low quality of pasta product, a study of LOX activity in a crude protein extract from semolina was performed by means of spectrophotometric techniques. Measurements made of linoleate hydroperoxidation reaction (λ=234 nm) and of the coupled β-carotene bleaching reaction (λ=460 nm), demonstrated the existence of more than one enzyme exhibiting LOX activity as revealed by the different pH profiles. The linoleate hydroperoxidation reaction exhibited a sigmoidal dependence on the substrate concentration (K0·5=60±0·67 μ M ,Vmax =1·12±0·055 units/g of semolina), while β-carotene bleaching showed a hyperbolic relationship and followed the Michaelis-Menten equation (Km=3·3±0·53 μ M , Vm=0·030±0·0062 units/g). Both linoleate hydroperoxidation and β-carotene bleaching were strongly inhibited by added α-tocopherol. In both cases, the enzyme affinity for the substrate was reduced, while the Vmaxwas not affected. In particular, β-carotene bleaching was inhibited in a competitive manner (Ki=7·7±0·21 μ M ). L-ascorbate also inhibited both reactions: the Vmaxof linoleate hydroperoxidation was lowered, while theK0·5 remained unchanged; in the case of β-carotene bleaching the inhibition was uncompetitive (Ki=445±71·5 μ M ). Therefore, the use of both α-tocopherol and L-ascorbate to reduce semolina bleaching during pasta making is suggested. A purification procedure of semolina LOX is also reported, showing the existence of two isoforms.

The activity of a horse liver alcohol dehydrogenase catalysed reduction of cyclohexanone was investigated by using a central composite circumscribed design in which two parameters (pH and cyclohexanone concentration) were varied. By log transformation of the substrate concentration an adequate model could be obtained from which reliable kinetic constants and pH profiles were determined.

Using a random mutagenesis technique, the ferrous-ion-activated alcohol dehydrogenase of Zymomonas mobilis has been altered to produce more thermally stable variants. After three rounds of mutation, a variant over 10 degrees C more stable at pH 8, with essentially unaltered kinetic characteristics, was produced. However, the pH profile of thermostability of this variant was much altered compared with the wild-type, with a relatively small increase (4 degrees C) at pH 6. Sequencing of the variants indicated five amino acids changes which contributed to thermostability: F9S, M13I, K31R, F90L, and G250D. Four of these were contained in the final stable variant, and the changes were partially additive, with individual mutations causing between 2 and 3.5 degrees C stability increases (at pH 7.5). It is estimated that the most stable variant would have a half-life under physiological conditions at 70 degrees C of 15 min.

Most siderophores are catabolism resistant because of their non-peptidic or cyclic peptide chemical structures. Siderophore degradation is thus a rare event, and one which has received little attention. Based on literature precedence and the generation of monohydroxamic acid(s) by cell-free extracts of a deferrioxamine B degrading bacterium, a catabolic scheme of the siderophore is proposed. Data are also presented concerning parameters such as the pH profile, inducible nature of the siderophore-degrading metabolism and the ability of the deferrioxamine B degrading bacterium to metabolize some of the hypothesized products of deferrioxamine B dissimilation.

Unnatural selection: A fungal laccase was tailored by directed evolution to be active at neutral/alkaline pH. After five generations, the final mutant showed a broader pH profile while retaining 50 to 80 % of its activity at neutral pH., This study was based upon work funded by the EU Projects [NMP4‐SL‐2009–229255–3D‐Nanobiodevice, FP7‐KBBE‐2010–4–26537‐Peroxicats and COST Action CM0701] and a project of the Spanish Government [BIO2010–19697]. P.T.S. was supported by a grant from the “Comunidad de Madrid” and D.M.M. was supported by a JAE‐CSIC grant.

2-Hydroxypropyl-β-cyclodextrin (2HP-β-CyD) and 2,6-di-O-methyl-β-cyclodextrin (DIMEB) were tested as solubilizing agents for dihydroergotamine mesylate, with the aim of improving the pharmacotechnical properties of this drug. The pH dependence of the complexation was investigated on the basis of solubility/pH profiles. For uncomplexed dihydroergotamine mesylate, the solubility increased sharply in the acidic range. The same pH dependence was observed for 2HP-β-CyD and DIMEB. The shape of the pH profiles was not altered, but the pH profiles occurred at considerably higher concentrations than for dihydroergotamine mesylate alone. The solubilizing effect clearly increased in the acidic range, and the effects were stronger for DIMEB than for 2HP-β-CyD. The maximum stability of dihydroergotamine mesylate was shown to be pH 4.0. The degradation rates of dihydroergotamine mesylate in β-CyD solutions are not very different from those in pure dihydroergotamine mesylate solution. DIMEB has the most favourable effect on the stability, while 2HP-β-CyD causes higher degradation. During autoclaving, however, dihydroergotamine mesylate was to some extent protected against degradation by 2HP-β-CyD complexation. Nevertheless degradation was so strong that autoclaving is not possible.

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-β-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.

This review is an exhaustive description of the biochemistry and enzymology of all 17 known NAD(P)(+)-amino acid dehydrogenases. These enzymes catalyze the oxidative deamination of an amino acid to its keto acid and ammonia, with the concomitant reduction of either NAD+ or NADP+. These enzymes have many important applications in industrial and medical settings and have been the object of prodigious enzymological research. This article describes all that is known about the poorly characterized members of the family and contains detailed information on the better characterized enzymes, including valine, phenylalanine, leucine, alanine, and glutamate dehydrogenases. The latter three enzymes have been the subject of extensive enzymological experimentation, and, consequently, their chemical mechanisms are discussed. The three-dimensional structure of the Clostridium symbiosum glutamate dehydrogenase has been determined recently and remains the only structure known of any amino acid dehydrogenase. The three-dimensional structure and its implications to the chemical mechanisms and rate-limiting steps of the amino acid dehydrogenase family are discussed.

Summary A series of disubstituted tetrahydro-2 H -1,3,5-thiadiazine-2-thione (THTT) derivatives 4a–g were prepared and found to be promising prodrugs for amino acids and similar compounds. The pH profile for the degradation of the THTT derivatives in aqueous buffer solutions was determined using HPLC and was accounted for in terms of specific base-catalyzed reactions. The compounds, however, showed high acid stability. Enzymatic hydrolysis (human serum) of the derivatives offered an advantageous range of t 1/2 values, which may be useful in controlling the onset and the duration of action of drugs.

3,5-Disubstituted tetrahydro-2H-1,3,5-thiadiazine-2-thione (THTT) derivatives; 4a-g were prepared and found to be a promising prodrug approach for peptide drugs. The pH profile for their degradation in aqueous buffer solutions was determined using HPLC technique and accounted for, in terms of specific base-catalyzed reactions. All of the compounds however, showed high acid-stability. Enzymatic-(human serum) hydrolysis of the different derivatives offered an advantageous range of t1/2' s, the property that permits controlling onset and duration of actions of drugs.

The immobilized mercuric reductase covalently coupled to porous glass was prepared and its properties were investigated. Mercuric reductase immobilized on arylamino and carboxyl derivatives exhibited high relative activities about 20-30% compared with soluble enzymes. The optimum pH of mercuric reductase immobilized on these derivatives was 7.5, slightly higher than that of soluble state (7.0-7.2). The stability of mercuric reductase increased with immobilization onto arylamino derivative and the immobilized enzyme maintained at 30°C for 29 days showed 80-90% of initial activity. As the application of immobilized enzyme, mercuric ion sensor was prepared and it was found that mercuric ion higher than 0.5μM was detectable with this sensor.

One of the most important considerations in designing an integrated process for the production of organic acids is the strong pH dependence of the process parameters. The release of the acidic products during the fermenta- tion alters the pH of the broth and subsequently affects the kinetics of cell growth and product formation. In addition, some parameters (such as the distribution coefficient of the products during extraction, their solu- bility constants during precipitation, etc.) in the subse- quent separation processes following fermentation are also strong functions of pH.

The pH dependence and kinetics parameters of renin-angiotensinogen reactions were determined using wild-type and S84G mutant human renins and wild-type and H13Y mutant sheep angiotensinogens. It is explained in this report that (i) renin catalyzes acidic and basic reactions of which the optimum pHs are 5.5 and 7.5-8.2 respectively, both of which produce angiotensin I; (ii) Ser84 specific to human renin accelerates the acidic reaction by 75-110% through elevation of V(max), and shifts the optimum pH of the basic reaction from 7.5 to 8.0-8.2; and (iii) His13 specific to sheep angiotensinogen accelerates the acidic and basic reactions by 25-42% through reduction of K(m). It is concluded from these results that the coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks at pHs 5.5 and 8.2 to the reaction of human renin and sheep angiotensinogen.

A new cysteine endopeptidase (morrenain b I) has been purified and characterized from the latex of stems and petiols of Morrenia brachystephana Griseb. (Asclepiadaceae). Morrenain b I was the minor proteolytic component in the latex but showed higher specific activity than morrenain b II, which was the main active fraction. Both enzymes showed similar pH profiles and molecular masses, but kinetic parameters and N-terminal sequences were quite distinct, demonstrating that they are different enzymes instead of different forms of the same enzyme., Centro de Investigación de Proteínas Vegetales

Defatted flour was prepared from germinated and ungerminated melon seeds. Protein solubility, foaming and emulsification capacities were measured over the pH range of 2.0 to 11.0 and NaCl concentrations of O. I M to I .O. M. Below and above pH 4, protein solubility increased reaching maximum values of 60% and 70% for ungerminated and germinated flour respectively at pH 10.5. The emulsification capacity versus pH profile and the foaming capacity versus pH profile showed close resemblance to the protein solubility versus pH curve suggesting that those properties depended on solubilized protein. Protein solubility of ungerminated and germinated flour was reduced at higher salt concentrations of 0.6M and0.8M respectively, which also altered the emulsifying and foaming properties. The germinated flour showed higher protein solubility, foaming and emulsification capacities than the ungerminated flour at all pH and NaCl levels, may be due to protein modification occurring during germination.

Extracts of the red seaweed Porphyra umbilicalis are a valuable constituent of herbal medicines. The nucleotide complement of such an extract was purified and compounds identified by chromatographic behaviour, UV absorbance/pH profile, base-to-phosphate ratio, acid hydrolysis, and by fast-atom bombardment mass spectrometry with mass-analysed ion kinetic energy spectrum scanning. In addition to eighteen common nucleotides, and two cyclic nucleotides, fifteen novel nucleotides were identified, comprising eight deoxynucleotides and two cyclic deoxynucleotides, ten aminoacylnucleotides and two nucleoside trisphosphates together with 2'-phosphoadenosine-3',5'-cyclic pyrophosphate, 5'-phosphoadenosine-2',3'-cyclic monophosphate and N6,N6-dimethyladenosine-5'-monophosphate. The possible origin and potential actions of these novel compounds are discussed.

1. α-Chymotrypsin (CT), enclosed in positively and negatively charged polyelectroiyte gels based on methacrylate, with a preservation of high catalytic activity, was produced. 2. The pH profile of the enzymatic activity of CT enclosed in gel is shifted in absolute magnitude by approximately 1 pH unit, while the sign of the shift agrees with the theory. 3. The incorporation of CT into a negatively charged gel leads to a sharp (600-fold) increase in its stability with respect to monomolecular thermal inactivation. The stabilization is due to the influence of the polymer matrix on the protein globule of the enzyme.

The rate of inactivation of acid phosphatase (EC 3.1.3.2) from bovine brain by dithiobis-(2-nitrobenzoic acid) (Nbs2) is identical to the rate of titration of one of the two SH groups of this enzyme. The rate of inactivation of the enzyme by Nbs2 is pH dependent and, at 300 mM NaCl, can be described by the reaction of a single SH group of p K 8.4. At low ionic strength the p K determined from the k inactivation VS. pH profile is 7.7 and the results deviate markedly from the predicted values at pH values ⩽6. The decrease of V upon addition of salts is paralled by the decrease of inactivation rate by Nbs2. The relevance of SH groups in catalysis by bovine brain acid phosphatase is discussed in terms of these data.

Heat inactivation and pH optima of the enzymes peroxidase and catalase were studied in carrot, swede and Brussels sprouts. There were differences in the heat stabilities of the peroxidases from different vegetables, but all peroxidases were more heat stable than the catalases. From the pH profiles and the heat stability curves it was concluded that both the peroxidases and catalases in the three vegetable species are somewhat different. Lipoxygenase and phenolase activities were not detected by the methods used.

A spectrometer for recording 14N NMR spectra is described. The 14N NMR linewidth versus pH dependences of several amino acids and related molecules are presented. It appears that the linewidth pH profile of each amino acid is unique. Linewidth minima occur between pH 6 and 8. A discussion on how 14N NMR linewidths of amino acids might give information on weak association is given.

The phenotypes of 273 offspring of 56 matings of various genotypes of lysozyme-deficient and normal New Zealand white rabbits were analyzed. It was determined that lysozyme deficiency of rabbits is inherited as an autosomal recessive with complete penetrance. The symbol ld is suggested for the gene for lysozyme deficiency. Further studies on the nature of the condition revealed that the lysozyme deficiency was not due to a lysozyme that was cold labile, or to a lysozyme that was so tightly bound it was unavailable for enzymatic assay, or to a lysozyme with a variant pH optimum. It was demonstrated, however, that the pH profile of the lysozyme remaining in the ld/ld rabbits was different from that found in normal rabbits.

3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g−1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent Km values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced.

Bean plants (Phaseolus vulgaris L.) were cultured in nutrient solutions containing three concentrations of iron. The leaves of plants grown with a limiting supply of iron contained reduced levels of protein, chlorophyll and the iron‐containing enzyme, lipoxygenase (EC 1.13.11.12). Measurements of lipoxygenase made on leaf crude extracts did not correlate with iron supply; however, upon purification of the enzyme by affinity chromatography, a direct correlation was observed. The lack of correlation was partly due to the presence of chlorophyll, an inhibitor of lipoxygenase, in the crude extract. Lipoxygenase levels changed with age of the plants, but rose to a maximum at days 14–16. A comparison of leaf lipoxygenases grown with different iron supply showed that they were identical with the same pH profile, chlorophyll inhibition, reaction products, and electrophoretic mobility. It is suggested that lipoxygenase may be used as a probe to evaluate iron availability and to investigate iron metabolism...

Purified glucose isomerase from Actinoplanes missouriensis was immobilized on porous DEAE-cellulose beads by simple adsorption. The immobilized glucose isomerase retained over 70% of its original activity. The hinderance of immobilized enzyme activity due to pore diffusion and film diffusion was insignificant with the bead size at 35 mesh or smaller. The relative substrate flow rate can be kept at 0.04 cm/s or higher. The optimum pH of the imobilized enzyme did not change, however, the optimum pH range became broader. The broader pH profile indicated that immobilized enzymes are less sensitive to pH change in the substrate. The half life of the immobilized enzyme was at around 1,000 h at 60°C. Cobalt ions are not required for enzyme stability. The cost of using immobilized enzyme on DEAE cellulose beads should be less be than that of the whole cell immobilization system due primarily to the fact that DEAE-cellulose beads are reusable for immobilization as well as for enzyme purification. Auf DEAE-Celluloseperten immobilisierte Glucoseisomerase Gereinigte Glucoseisomerase aus Actinoplanes missouriensis, wurde durch einfache Adsorption auf porosen DEAE-Celluloseperlen immobilisiert. Die immobilisierte Glucoseisomerase behielt uber 70% ihrer ursprunglichen Aktivitat. Die Behinderung der immobili-sierten Enzymaktivitat durch Poren- und Filmdiffusion war bei einem Perlendurchmesser von 35 Maschen oder geringer unbedeutend. Die relative Fliesgeschwindigkeit kann bei 0,04 cm/s oder hoher gehalten werden. Das optimale pH des immobilisierten Enzyms anderte sich nicht, aber der optimale pH-Bereich wurde breiter. Das breitere pH-Profil deutete darauf hin, das immobilisierte Enzyme gegenuber pH-Anderungen weniger empfindlich sind. Die Halbwertzeit des immobilisierten Enzyms betrug bei 60°C etwa 1 000 h. Kobaltionen sind fur die Stabilitat des Enzyms nicht erforderlich. Die Kosten fur die Verwendung von auf DEAE-Celluloseperlen immobilisiertem Enzym sollten niedriger sein als bei dem vollstandigen Zellimmobilisierungssystem, wenn man berucksichtigt, das DEAE-Celluloseperlen sowohl fur die Immobilisierung als auch fur die Reinigung des Enzyms wiederverwendet werden konnen.

o-Diphenolase extracted from Triticum aestivum (cv. Nettuno) and purified in various steps gave a 250-fold purification over the crude extract. This purified enzyme showed maximum relative activity towards 4-methylcatechol, generally high or moderate activity towards di- and polyphenols, and very low activity towards monophenols. Total activity, determined at each step of purification, showed that treatment with ammonium sulphate and calcium phosphate gel gave rise to an activation of o-diphenolase, thus revealing a latency of enzymatic activity. In the pH profile activity towards 4-methylcatechol two optima at pH 5.3 and 6.9 were observed.

A pH-rate method, based on following the time dependence of hydroxide release by monitoring the pH drift as a funetion of time, is developed and applied to the construction of a detailed log activity vs pH profile for yeast pyruvate decarboxylase. In the pH range of 5 to 7 at 30°C this method reproduces the results of conventional initial rate studies but, in addition, produces a hitherto unreported pH independent activity region between pH 5.4–5.8. At least four pKs can be discerned in the pH-activity profile of this enzyme, instead of the two previously reported.

1. 1. This study was undertaken to characterize the α-glucosidase present in blood and seminal plasma of two strains of pure-bred rams which are known as seasonal breeders. 2. 2. pH profiles and activity levels were investigated in blood and seminal plasma using a sensitive spectrophotometric assay with para-nitrophenyl-α- d -glucoside as substrate. 3. 3. According to their pH optimum, blood plasma and seminal plasma α-glucosidases were typically neutral and acid enzymes and significant differences were recorded in their physico-chemical properties, establishing the tissue specificity of the enzyme. 4. 4. Notwithstanding the tissue under study, the nature of the α-glucosidase activity was similar in both strains of pure-bred rams.

The pH–solubility profile of a weak acid or base is shown to be a function of its pKsp, and pKa, and uncharged species solubility. Equations are presented that can be used to calculate the solubility as a function of pH. These equations can also be used when there is added salt present. Experimental data was obtained in three cosolvent systems consisting of methanol–water and ethanol–water. Also, the effect of different acids on the solubility of a weak base is reported. A pronounced effect on the solubility by the addition of salt is explained in terms of the Ksp.

N-Acetyl-beta-D-glucosaminidase (EC 3.2.1.30) activity was measured fluorimetrically in: (a) lymphocytes from 20 normal donors, (b) enriched B and T lymphocyte populations prepared by E rosette sedimentation from 8 normal subjects, (c) lymphocytes from 15 untreated B cell chronic lymphocytic leukaemic patients. The pH profiles and optima (4.7) were similar in all preparations. Normal B lymphocytes had higher activity than normal T lymphocytes and both these preparations and normal unfractionated lymphocytes had significantly higher activity than chronic lymphocytic leukaemic lymphocytes. The apparent Michaelis constants were similar in normal unfractionated, B enriched and T enriched lymphocytes, whereas a reduced affinity for the enzyme was observed in the leukaemic lymphocytes. The difference in enzyme content between normal and chronic lymphocytic leukaemic lymphocytes cannot therefore be explained on the basis of a high B cell percentage in patients with chronic lymphocytic leukaemia.

The pH profiles of β-galactosidase, having tyr replaced by m-fluorotyrosine, were compared to those of normal enzyme. The inflection point on the alkaline side was lowered about 1.5 pH units in the fluoro-enzyme, corresponding to the difference in the phenolic pKa values of m-fluorotyrosine and tyr. When glycosidic bond breakage was rate-limiting, the Vm at pH 7.0 was higher for the fluoro-enzyme. When hydrolysis was rate-limiting or when acceptors which made transgalactosylis rate-limiting were used, the Vm was lower for the fluoro-enzyme. This shows that a tyr in β-galactosidase is a general-acid catalyst in the glycosidic bond breaking reaction and a tyr (probably the same one) is a general-base catalyst in the hydrolytic reaction.

A reanalysis of the diatom-inferred pH profile from a sediment core collected in 1980 from Kejimkujik Lake has been made in the light of improved techniques and ecological information. Using Index B calibrated for lakes in Atlantic Canada, the lake pH was 4.6 to 4.7 before anthropogenic disturbances in the watershed began ca. 1850. These disturbances had little immediate impact on pH, but minima of pH 4.5 were evident in the period 1916 to 1950. A diatom-inferred pH of 4.75 in the surface sediments compares with a measured pH of 4.96 in 1980. Disturbances in the watershed since ca. 1850 resulted in a large increase in a form of Asterionella ralfsii, a diatom associated with colored, humic water. A decline in this diatom since 1950 and an increase in taxa less typical of humic water is consistent with the hypothesis of loss of organic matter accompanying acidification from acidic precipitation.

The kinetics of the trypsin-catalyzed hydrolysis of two specific amide substrates is discussed on the assumption of an acylenzyme intermediate. The limiting process is the acylation step. The acylation rate constant exhibits a bell-shaped pH profile. The pK values have been determined and compared with previous results from our laboratory. The activation by substrate previously observed during deacylation is not detected in the acylation step. The enzymatic and non-enzymatic (hydroxyl ion-catalysed) rate constants hav been compared according to the “five factors” proposed by Bender .

The N-acetyl-β-d-hexosaminidases A and B were extracted from the liver tissue of a patient who died from juvenile GM2-gangliosidosis at the age of\(14{1 \mathord{\left/ {\vphantom {1 2}} \right. \kern-\nulldelimiterspace} 2}\) years. The enzymes were separated from each other and studied in comparison to enzyme preparations from normal tissue. The kinetic parameters studied (pH profiles, Michaelis constants, heat lability) and the substrate specificity of the B-enzyme appeared normal. The activity of the A-enzyme against two artificial substrates was lowered in the pathological tissue to 53% and 41% of the control and to 27% of the control against the storage compound trihexosylceramide (ceramide-glucose-galactose-N-acetylgalactosamine). Furthermore, using the main storage compound ganglioside GM2 (ceramide-glucose-galactose-[N-acetylneuraminic acid]-N-acetylgalactosamine) as substrate, no N-acetyl-β-d-galactosaminidase activity was found in the extracts from pathological liver tissue.

Hog pancreatic α-amylase contains 2 -SH groups and 1 tightly bound Ca2+ per enzyme molecule. We have prepared derivatives of this enzyme by using 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), iodoacetamidonaphthol, and Hg2+. These derivatives retain enzymatic activity thereby demonstrating that the -SH groups of this enzyme are not required for enzymatic activity. The pH profile of these derivatives is the same as that of the native enzyme although the specific activity at each pH is depressed to a varying extent, depending on the derivative. The reaction with DTNB and iodoacetamidonaphthol proceeds only after prior removal of the tightly bound Ca2+. The Hg2+ derivatives, however, may be formed without prior removal of the Ca2+. Upon reacting the amylase with one mole of Hg2+ an -S-Hg-S- bridge is formed. This leads us to conclude that the 2 -SH groups lie very close to each other. These derivatives may be useful for an X-ray crystallographic study of this enzyme.

The methods used for the assay of lipoxidase have been compared. The pH profile of the enzyme, as measured by two of the methods, was found to be considerably altered by changes in the ionic strength of the reaction medium.

Raman spectra of aqueous solutions of 5′-AMP and 5′-GMP have been recorded as a function of pH. Band intensities and frequencies have been monitored to determine bands which may be considered as diagnostic for the concentration and the p K of the solution species. Quantitative band intensity measurements indicate only a selected number of bands can be considered as diagnostic of the base or the secondary phosphate proton dissociation. The utility of the pH profiles derived from specific band intensity variations for 5′-AMP is discussed in terms of the effect of acidification on solution characteristics of polyadenylic acid.