pH-stat Assay Research Articles

Anti-halitosis activity of Syzygium aromaticum essential oil against Streptococcus sanguinis

Background: Halitosis (oral malodour) is known as an indicator for oral disease. Halitosis is caused by the accumulation of volatile sulfur compounds (VSCs) produced by oral microbial activities, including Streptococcus sanguinis. Bacteria producing sulphur compounds may produce biofilm promotion of caries formation. Caries formation is caused by the acid produced by bacteria may lead to tooth demineralization in low pH condition. Syzygium aromaticum or clove belongs to one of tropical spices in Indonesia and is also known as the queen of spice due to its unique taste and aroma. Essential oil extracted from the flower and leaf of S. aromaticum has been used for traditional medicines for preventing inflammation and infection caused by microorganisms. Methods & Materials: The aim of this study was to test whether Syzygium aromaticum essential oils (SAEO) at various concentrations (20-100 μg/mL) has anti-halitosis activity through several systems, including prevention the formation of S. sanguinis biofilm, eradicating the established S. sanguinis biofilm, inhibiting production of VSCs using optical density assay, and reducing acid production using pH-stat assay in vitro. SAEO was isolated using solvent extraction and identified using pyrolysis gas chromatography-mass spectrometry (py/GC-MS). Results: The py-GC/MS chromatogram showed that SAEO consisted of eugenol (22.10%) and aceteugenol (13.31%). For biofilm system, SAEO at lowest dose (20 μg/mL) effectively inhibited S. sanguinis biofilm formation up to 60% and killed the established S. sanguinis biofilm up to 52%. Furthermore, SAEO at 60 μg/mL also showed a significant inhibition of VSCs production up to 58%. S. sanguinis was able to produce acid in 20 minutes, and SAEO effectively reduced acid production rate by increasing terminal pH. At 60 μg/mL, SAEO increased the terminal pH from 5.66 to 6.3 with inhibition of acid production up to 54%. Conclusion: These results suggest that SAEO may be applied for combating bad breath in management of oral diseases.

Prediction of apparent protein digestibility by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for Siberian sturgeon (Acipenser baeri, Brandt 1869)

In the present study in vivo apparent protein digestibility (APD) and in vitro degree of protein hydrolysis (DPH; pH-stat assay) of numerous feed ingredients (n = 10) were investigated in juvenile Siberian sturgeon (290 ± 22 g). The ingredients included: fish meal (FM; Clopeonella), meat and bone meal (MBM), poultry by-product meal (PBM), spray dried blood meal (BM), feather meal (FeM), solvent extract soybean meal (SBM), canola meal (CM), corn gluten meal (CGM), wheat gluten meal (WGM), and bakers' yeast (BY). APD values of the ingredients varied from 60 to 92.9% (feather meal and fish meal, respectively). APDs lower than 70% were found in blood meal (68.5%), soybean meal (66.2%), bakers' yeast (66%), canola meal (61%) and feather meal (60%). Also, the ingredients indicated APD values higher than 70%; corn gluten meal (74.3%), wheat gluten meal (76.9%), poultry by-product meal (83.1%) and meat and bone meal (85.6%). Higher DPH values (> 5%) were observed for fish meal, poultry by-product meal and wheat gluten meal; mid-range DPH values (3–5%) for corn gluten meal, meat and bone meal, soy bean meal, blood meal, bakers' yeast; and lower DPH values (<2%) for canola meal and feather meal. Linear regressions between in vivo APD and in vitro degree of DPH with enzyme extracts in feed ingredients for Siberian sturgeon resulted in a considerable correlation (R2 = 0.89–0.99). Also, linear regressions between APD and DPH suggested a close relationship between peptide bond breakage by pyloric caeca and intestine digestive enzymes and the ADP. As a result, this may be a useful tool to provide important nutritional information.

Does the commonly used pH-stat method with back titration really quantify the enzymatic digestibility of lipid drug delivery systems? A case study on solid lipid nanoparticles (SLN)

Enzymatic digestion of lipid drug carriers is very important. Commonly, pancreatin induced formation of fatty acids is monitored by the pH-stat method, which provides a fast, but unspecific readout. However, according to the literature, the pKa values of long chain fatty acids are strongly dependent on the local environment and might vary between 4.2 and 10.15. The high pKa values would lead to an incomplete detection of the lipid digestion and false results. In order to investigate these issues in more detail, we produced cetyl palmitate solid lipid nanoparticles (CP-SLN) stabilized with poloxamer 188 or polysorbate 80. The digestion of CP-SLN was investigated by two different and independent readouts. A HPTLC assay was used in addition to the pH-stat method (with or without back titration). An incomplete digestion of CP-SLN was observed with all methods. Partial digestion of polysorbate 80 contributed to the formation of fatty acids. Depending on the investigated system and the experimental conditions (FaSSIF or FeSSIF) the results of both readout methods were comparable or not. For example, in FeSSIF conditions, the values detected by HPTLC were roughly twice as high as the pH-stat results. Our findings on solid lipids agree with data from Helbig et al. on lipid emulsions, where a gas chromatography method detected much higher values than the pH-stat assay (Food Hydrocoll. 28 (2012) 10–19).The results of our pH-stat experiments with back titration at different pH values showed increased values for fatty acids from pH 7.5 to pH 10. The values obtained by back titration at high pH values (pH 9 or higher) did exceed the digestion values measured by HPTLC. Therefore, we conclude that the pH-stat method might give the same results as more specific reference methods, but it might also both under- (without back titration) or overestimate (with back titration) the enzymatic digestion of lipid drug delivery systems.A further outcome of our study was the proof that lipase is the main enzyme involved in the digestion of the solid wax cetyl palmitate, because CP-SLN loaded with the inhibitor orlistat were not digestible and gave similar values as the corresponding control samples.In summary, our experimental results confirm the theoretical considerations (based on published pKa values) that the pH-stat method will not detect all fatty acids quantitatively. The very strong impact of the local environment on the pKa value of long chain fatty acids is a serious limitation and might lead to misleading interpretations.

Species specific in vitro protein digestion (pH-stat) for fish: method development and application for juvenile rainbow trout (Oncorhynchus mykiss), cobia (Rachycentron canadum), and Nile tilapia (Oreochromis niloticus)

Abstract Aqua feed manufacture requires flexible formulations and effective methods to screen suitable feed ingredients. In vitro digestion may assist in the characterization and quality control of protein in feedstuffs for fish species once standardized species-specific digestive enzyme extracts are available. This study aimed to develop a species-specific in vitro enzymatic method to assess protein digestion in fish under the pH-stat concept. Two carnivorous (rainbow trout, Oncorhynchus mykiss , and cobia, Rachycentron canadum ) and one omnivorous (Nile tilapia, Oreochromis niloticus ) fish species were used as models. Crude digestive enzyme extracts were recovered from stomach and pyloric caeca or intestine of individuals of different weight groups, feeding status, and farming systems. The hydrolytic capacity of the species-specific enzyme extracts was standardized on purified protein substrates and measured as degree of protein hydrolysis (DH) in the pH-stat assay. A group of twenty-four feed ingredients, including fish meals and by-products of plant and animal origin, was assessed for DH using the recovered enzymes from stomach and pyloric caeca/intestine. Ingredients were hydrolyzed with fish (i) stomach extract, (ii) pyloric caeca/intestine extract or (iii) stomach enzymes followed by pyloric caeca/intestine extract. Among plant by-products, cotton seed meal presented the highest DH with stomach plus pyloric caeca/intestine enzymes, followed by soy protein concentrate and soybean meals. Blood meals were the land animal by-product with higher DH outputs compared to poultry by-product meals and feather meals. No significant difference was observed among the DHs of fish meals. The significance of measuring the DH with stomach enzyme extract is still not well understood but, overall, the pre-hydrolysis of feedstuffs with stomach enzymes increased pyloric caeca/intestine DH value. For cage and pond farmed Nile tilapia, ingredient DHs followed the same trend, describing a significant correlation and a high determination coefficient regression. Routine use of the method may yet depend on the prompt availability of more practical sources of enzymes. The determination of the degree of protein hydrolysis by the in vitro pH-stat with species-specific enzymes has shown to be a precise method that may be a useful tool to rank feed ingredients, and also an accessory method in the quality control of feedstuffs.

Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity

Acrylodan-labeled rat-intestinal fatty acid binding protein, ADIFAB, binds both of lysophosphatidylcholines (LPC) and FA. Binding displaces Acrylodan and its fluorescence peak shifts from 432 to 505 nm. A fluorescence assay that relies on this shift is presented for quantitating LPC, FA, and phospholipase A(2) (PLA(2)) activity in phospholipid bilayers in absolute units of μM/min/mg of enzyme. This is a development over an earlier assay that took into account only FA binding. Activities of bee venom PLA(2) on dipalmitoylphosphatidylcholine (DPPC) and dioleylphosphatidylcholine (DOPC) bilayers were measured. Standard pH-Stat assays validated the present assay. Products increase linearly with time for about one minute in DOPC and five minutes in DPPC corresponding to completion of 5 to 8% hydrolysis in DOPC and 20% in DPPC. Membrane polarity and microviscosity measured using electron spin resonance (ESR) exhibited discontinuities at compositions that mimicked similar percentages of hydrolysis products in the respective bilayers. The observed hydrolysis rate decrease following the initial linear period thus correlates to changes in membrane polarity. The ability of the assay to yield actual product concentrations, reveal structure in the reaction progress curves, and interpretation in light of the ESR data bring insight into the shape of the reaction curve.

In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 2. In vitro protein digestibility of common and alternative feed ingredients

Using enzyme fractions isolated from the pyloric caeca of farmed Atlantic cod, the in vitro degree of protein hydrolysis (DH) of numerous conventional and novel feed ingredients were measured by a closed-system pH-Stat assay. Regression equations describing the relationship between in vivo apparent protein digestibility (ADC) and in vitro protein DH were used to predict in vitro protein ADC. The equations resulted in good correlation (< 4 percentage points difference) between ‘measured’ and ‘predicted’ protein ADC in the majority of cases ( r = 0.90-0.99; R 2 = 0.88-0.99), while some ingredients were either over- or under-estimated (6–7 percentage points) which appears related to high ash or chitin content ( r = 0.75; R 2 = 0.61) and may indicate the need for an acid pre-hydrolysis phase and full account of non-protein nitrogen (NPN) content. The ‘predicted’ in vitro protein ADC were above 95% for wheat gluten meal, soy protein concentrate, soy protein isolate and whole krill meal; relatively high (85-95%) for soybean meal, white lupin meal, herring meal, anchovy meal, canola protein concentrate, pea protein concentrate and poultry by-product meal; mid-range (75-85%) for crab meal, shrimp meal and canola meal; and low (< 75%) for hydrolyzed feather meal and flaxseed meal. Further research is needed on the development of a two-stage hydrolysis assay for gadoids involving an acid (gastric) pre-digestion step prior to this assay to further increase agreement between in vivo protein ADC and in vitro protein DH.

In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 1. Development of the method

The method described here involves the extraction and partial purification of an enzyme fraction from the dissected pyloric caeca of commercially farmed Atlantic cod, Gadus morhua (1 kg fish) and the development of a pH-Stat method to predict protein digestibility. The various extraction and partial purification steps successfully concentrated the alkaline serine protease enzymes, trypsin (> 4-fold) and chymotrypsin (> 12-fold). It was found that the enzyme fractions produced in the manner described in this study were completely stable for up to 8 months when stored at − 20 °C and at least 10 months when stored − 80 °C after which significant loss of enzyme activity can occur, although the degree of protein hydrolysis (DH) of casein was unaffected after 12 months. It is recommended that enzyme fractions produced in a similar manner should be stored at − 80 °C and used within 8–10 months. The most suitable substrate concentration [S] to use for closed-system in vitro pH-Stat DH assays was established using a standard purified protein source (vitamin-free casein) with four [S] (0.25, 0.5, 0.75 and 1 mg N/mL protein suspension solution). No significant differences (P > 0.05) were found in the DH values between the [S] tested. The DH curve for casein at a [S] of 0.5 mg N/mL showed a rapid increase initially before leveling off at maximum DH (26%) which was achieved within a moderate duration of the assay (5–6 h). The closed-system pH-Stat assay with a [S] of 0.5 mg N/mL and minimum assay duration of 8 h is recommended for further investigation of conventional and novel feed ingredients for gadoid diets.

Normal Mucus Release Requires Both Ca2+‐mediated Exocytosis and cAMP Mediated HCO3‐ Transport

Mounting evidence indicates that the formation of normal mucus depends critically on the environment into which it is discharged. We applied histology, Ussing chamber pH-stat assays, and mucus collection methods to examine the links between mucin release and ion transport. In wild type mouse (C57BL/6) small intestine, the Ca2+ mediated agonist (carbachol) did not stimulate sustained HCO3− secretion, but induced mucin exocytosis (histologically) and a short circuit current (Isc) increase (possibly due to Cl− secretion). In contrast, cAMP mediated agonists (vasoactive intestinal peptide, isoproterenol) induced both HCO3− secretion and Isc increase, but little, if any, exocytosis. cAMP mediated agonists combined with carbachol discharged more mucus into the luminal perfusate than carbachol alone, but the cAMP enhancing effect was absent without HCO3−. In CF (ΔF508/ΔF508) mouse small intestine, neither Ca2+ nor cAMP mediated agonists induced significant HCO3− secretion, Isc increase or significant mucus discharge. These results indicate that normal mucus release requires Ca2+ mediated exocytosis, possibly Cl− secretion, and apparently post-exocytotic cAMP-mediated HCO3− secretion to affect normal mucin discharge. Concept: Mucus discharge requires Ca2+ mediated exocytosis + cAMP mediated HCO3− secretion. We thank the CFRI, CF Foundation, NIH-RO1 HL084042 and Dr. Hui Dong.

Defective cAMP‐dependent HCO3‐ Secretion Leads to Abnormal Mucus

Cystic fibrosis (CF) is a hereditary, fatal disease caused by a unique pathology that appears to begin with thick mucus that obstructs the lumina of hollow organs. In the present study, PAS‐AB Staining, morphometry, pH stat and mucus collection assay methods were used to investigate the formation of abnormal mucus in CF mouse small intestine. We found that in the wild type mouse small intestine, carbachol (100 μM, an intracellular Ca2+ mediated activator) induced goblet cell mucus release, but did not stimulate sustained HCO3− secretion. Whereas, forskolin (10 μM) + IBMX (100 μM) applied to activate intracellular cAMP‐mediated pathways did not have any detectable effect on goblet cell mucus release, but did induce sustained HCO3− secretion. With HCO3− in the basolateral solution, PGE2 (1 μM) + 5‐HT (10 μM), cAMP and Ca2+mediated activators, induced more mucus release from goblet cells in the presence than in the absence of HCO3−. In CF mouse small intestine, PGE2 (1 μM) + 5‐HT (10 μM) also stimulated goblet cell mucus release, but there was no significant difference in mucus releasing goblet cells with or without HCO3− in the basolateral solution. cAMP activated HCO3− secretion was missing in CF intestine. These results suggest that a defect in cAMP‐dependent HCO3− secretion, rather than in mucus release, leads to the abnormal mucus observed in CF mouse.Supported by Elizabeth Nash Memorial Fellowship

Prediction of culture performance of juvenile Litopenaeus vannamei by in vitro (pH-stat) degree of feed protein hydrolysis with species-specific enzymes

Rapid in vitro methods for measuring digestibility may be useful in analysing aqua feeds if the extent and limits of their application are clearly defined. The pH-stat protein digestibility routine with shrimp hepatopancreas enzymes was previously related to apparent protein digestibility with juvenile Litopenaeus vannamei fed diets containing different protein ingredients. The potential of the method to predict culture performance of shrimp fed six commercial feeds (T3, T4, T5, T6, T7 and T8) with 350 g kg−1 declared crude-protein content was assessed. The consistency of results obtained using hepatopancreas enzyme extracts from either pond or clear water-raised shrimp was further verified in terms of reproducibility and possible diet history effects upon in vitro outputs. Shrimps were previously acclimated and then maintained over 56 days (initial mean weight 3.28 g) on each diet in 500-L tanks at 114 ind m−2, clear water closed system with continuous renewal and mechanical filtering (50 μm), with four replicates per treatment. Feeds were offered four times daily (six days a week) delivered in trays at feeding rates ranging from 4.0% to 7.0% of stocked shrimp biomass. Feed was accessible to shrimp 4 h daily for 1-h feeding period after which uneaten feed was recovered. Growth and survival were determined every 14 days from a sample of 16 individuals per tank. Water quality was monitored daily (pH, temperature and salinity) and managed by water back flushing filter cleaning every 7–10 days. Feeds were analysed for crude protein, gross energy, amino acids and pepsin digestibility. In vitro pH-stat degree of protein hydrolysis (DH%) was determined for each feed using hepatopancreas enzyme extracts from experimental (clear water) or pond-raised shrimp. Feeds resulted in significant differences in shrimp performance (P < 0.05) as seen by the differences in growth rates (0.56–0.98 g week−1), final weight and feed conversion ratio (FCR). Shrimp performance and in vitro DH% with pond-raised shrimp enzymes showed significant correlation (P < 0.05) for yield (R2 = 0.72), growth rates (R2 = 0.72–0.80) and FCR (R2 = −0.67). Other feed attributes (protein : energy ratio, amino acids, true protein, non-protein nitrogen contents and in vitro pepsin digestibility) showed none or limited correlation with shrimp culture performance. Additional correlations were found between growth rates and methionine (R2 = 0.73), FCR and histidine (R2 = −0.60), and DH% and methionine or methionine+cystine feed contents (R2 = 0.67–0.92). pH-stat assays with shrimp enzymes generated reproducible DH% results with either pond (CV ≤ 6.5%) or clear water (CV ≤ 8.5%) hepatopancreas enzyme sources. Moreover, correlations between shrimp growth rates and feed DH% were significant regardless of the enzyme origin (pond or clear water-raised shrimp) and showed consistent R2 values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.

New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases

A Photobacterium strain, M37, showing lipolytic activity, was previously isolated from an intertidal flat of the Yellow Sea in Korea and identified as Photobacterium lipolyticum sp. nov. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,023 bp) corresponded to a protein of 340 amino acid residues with a molecular weight of 38,026. No sequence similarity was found with any known bacterial lipases/esterases; instead, the most similar enzymes were several filamentous fungal lipases. Although the similarity was very low (less than 16%), there were many conserved regions over the entire sequence and N-terminal oxyanion hole (RG) region, a signature sequence of filamentous fungal lipases. The novel protein M37 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The soluble protein exhibited lipase activity in a pH-stat assay using an olive oil emulsion. The M37 lipase also displayed a maximum activity at 25 degrees C and maintained its activity at a low temperature range (5-25 degrees C) with an activation energy (E(a)) of 2.07 kcal/mol. Accordingly, these results indicate that the M37 lipase from P. lipolyticum sp. nov. is a new cold-adapted enzyme.

Critical Evaluation of a Specific ELISA and Two Enzymatic Assays of Pancreatic Lipases in Human Sera

Background and Aims: Human pancreatic lipases (HPL) include the classical HPL, and two related proteins known as pancreatic lipase-related proteins 1 and 2 (HPLRP1 and 2). The aim of this study was to develop an ELISA for specifically quantifying the classical-HPL level in sera of patients with and without pancreatic disorders. Methods: The specific activity of various human (including classical-HPL) and microbial lipases was measured using Lipa Vitros and potentiometric (pH-stat) assays. A double sandwich ELISA was also set up, using an anti-classical-HPL polyclonal antibody and a biotinylated monoclonal antibody (mAb 146-40) specific to the classical-HPL. Sera (n = 53) were collected from patients with and without pancreatic disorders. The lipase concentration was deduced from the measured lipolytic activity and compared with the corresponding classical-HPL concentration, measured with the ELISA. Results: Both the purified HPLRP2 and 3 lipases of microbial origin were found to have a significant and unexpected lipolytic activity under the standard Lipa Vitros assay, whereas the ELISA test developed in the present study was found to be specific for the classical-HPL, due to the absence of cross-reactivity between mAb 146-40, HPLRP1 and HPLRP2. The efficiency of the ELISA was assessed in terms of its reproducibility and accuracy. The lower detection limit of classical-HPL was found to be 0.03 µg/l. A good correlation was found to exist between the lipase concentrations obtained in the ELISA, pH-stat and Lipa Vitros tests, in both the control and pathological groups. Conclusion: This is the first time a specific method of measuring classical-HPL in human serum has been proposed. Using this ELISA, we established with the 53 sera selected in the present study, that the Lipa Vitros assay as well as the pH-stat assay were mostly detecting classical pancreatic lipase. However, it is possible that other lipases such as HPLRP2 or lipases of microbial origin, present in some pathological sera, may well interfere with the Lipa Vitros assay.

Might the kinetic behavior of hormone-sensitive lipase reflect the absence of the lid domain?

Hormone-sensitive lipase (HSL) is thought to contribute importantly to the mobilization of fatty acids from the triacylglycerols (TAGs) stored in adipocytes, providing the main source of energy in mammals. To investigate the HSL substrate specificity more closely, we systematically assessed the lipolytic activity of recombinant human HSL on solutions and emulsions of various vinyl esters and TAG substrates, using the pH-stat assay technique. Recombinant human HSL activity on solutions of partly soluble vinyl esters or TAG was found to range from 35 to 90% of the maximum activity measured with the same substrates in the emulsified state. The possible existence of a lipid-water interface due to the formation of small aggregates of vinyl esters or TAG in solution may account for the HSL activity observed below the solubility limit of the substrate. Recombinant human HSL also hydrolyzes insoluble medium- and long-chain acylglycerols such as trioctanoylglycerol, dioleoylglycerol, and olive oil, and can therefore be classified as a true lipase. Preincubation of the recombinant HSL with a serine esterase inhibitor such as diethyl p-nitrophenyl phosphate in 1:100 molar excess leads to complete HSL inhibition within 15 min. This result indicates that the catalytic serine of HSL is highly reactive and that it is readily accessible. Similar behavior was also observed with lipases with no lid domain covering their active site, or with a deletion in the lid domain. The 3-D structure of HSL, which still remains to be determined, may therefore lack the lid domain known to exist in various other lipases.

Chloride transport across lipid bilayers and transmembrane potential induction by an oligophenoxyacetamide.

This contribution describes the discovery and properties of a synthetic, low-molecular weight compound that transports Cl- across bilayer membranes. Such compounds have potential as therapeutics for cystic fibrosis and cancer. The H+/Cl- co-transport activities of acyclic tetrabutylamides 1-6 were compared by using a pH-stat assay with synthetic EYPC liposomes. The ion transport activity of the most active compound, trimer 3, was an order of magnitude greater than that of calix[4]arene tetrabutylamide C1 a macrocycle known to function as a synthetic ion channel. Trimer 3 has an unprecedented function for a synthetic compound, as it induces a stable potential in liposomes experiencing a transmembrane Cl-/SO42- gradient. Data from both pH-stat and 35Cl NMR experiments indicate that 3 co-transports H+/Cl-. Although 3 transports both Cl- and H+ the overall process is not electrically silent. Thus, trimer 3 induces a stable potential in LUVs due to a transmembrane anionic gradient. The ability of trimer 3 to transport Cl-, to maintain a transmembrane potential, along with its high activity at uM concentrations, its low molecular weight, and its simple preparation, make this compound a valuable lead in drug development for diseases caused by Cl- transport malfunction.

Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 microm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.

In vitro digestibility of dietary protein sources for white shrimp ( Penaeus vannamei)

In vitro methods to evaluate protein digestibility are rapid and allow close observation of the peptide bonds cleaved while using only small amounts of raw materials. The pH-stat assay using an enzyme preparation from shrimp hepatopancreas, recently developed by us was used to evaluate the degree of hydrolysis (DH) and estimated protein quality in menhaden, anchovy, white fish, tuna waste, soybean protein, and langostilla meals at 23, 25, and 27°C. Data were obtained by the pH-stat method and were compared with those obtained by the pH-drop method and in vivo assays. The arginine content of the meals was compared with shrimp growth and DH by the pH-stat method. The test showed no significant differences between DH obtained at 23, 25, and 27°C. The pH-stat method predicted protein digestibility better than the pH-drop method. A significant correlation between DH and apparent protein digestibility (APD) was observed. Although a significant correlation between the pH-drop method and APD was observed, the value was low. Arginine showed a significant correlation with shrimp growth but not with APD. The relationship between DH by pH-stat at 25 and 27°C and growth, although significant, had a low correlation. The equations obtained from DH had a very high predictive ability. The standard error in percentage was low. The pH-stat assay has a potential for the estimation of growth and digestibility of shrimp feed.

Stability Of A Commercial Lipase From Mucor Jav Anicus: Kinetic Modelling Of Ph And Temperature Dependencies

The present communication reports experimental and modelling work pertaining to the independent roles of pH and temperature on deactivation of a crude lipase from Mucor juvanicus. Experimental data of lipolytic activities were generated by a classic pH-stat assay on a triolein emulsion following incubation at several pH values for a fixed time, or at several temperatures for various times; postulated models were then fitted by nonlinear fitting to such data. The pH-dependence data were best fit by assumption of three forms of enzyme with increasing states of protonation, with pKa values of 6.2 and 11.3, respectively, where only the intermediate form is stable within the time frame considered. The thermal-dependence data were best fit by assumption of parallel steps of deactivation and rearrangement, with activation energies of 228.8 and 221.7 kJ mol-1, respectively.

PH-stat method to predict protein digestibility in white shrimp ( Penaeus vannamei)

In aquaculture, feeding trials are the most dependable method of measuring nutritive value of feeds. However, these methods are time consuming, expensive, and the results may be affected by environmental factors. In vitro methods to evaluate protein digestibility are rapid and allow close observation of how far bonds are cleaved using only small amounts of raw materials. We developed a pH-stat assay using an enzyme fraction from shrimp hepatopancreas. We used the assay to evaluate the degree of hydrolysis (DH) and estimate protein quality in menhaden, anchovy, white fish, tuna waste, soybean protein, and langostilla meals. Data obtained by the pH-stat method were compared with those obtained by chemical analysis and an in vivo apparent protein digestibility (APD) assay. The pH-stat method predicted the protein quality better than chemical analysis. The APD of the experimental diets ranged from 64–91%. A significant correlation ( P < 0.05) between DH and APD was observed ( r 2 = 0.77 for enzymes from shrimp hepatopancreas and r 2 = 0.71 for a commercial enzyme mixture). The pH-stat method has the potential for estimating the digestibility of alternative protein sources for shrimp feeds.

Presence of Residual Pectin Methylesterase Activity in Thermally Stabilized Industrial Fruit Preparations

Industrial fruit products were investigated for their content of pectin methylesterase (PME) residual activity by a new procedure that allows selective concentration by affinity chromatography of the active form of the enzyme, when present. The technique utilizes a protein inhibitor, purified from kiwifruit, covalently bound to a Sepharose resin. This inhibitor, in fact, binds with high affinity the native active form of PME that can be subsequently recovered by dissociating the complex at alkaline pH with high ionic strength. In this way the enzyme is recovered at a concentration suitable for pH-stat assay. We examined fruit products from several industrial preparations of peach, pear, apple, apricot and pineapple. The analysed fruit purees showed PME activities between 10 −3and 10 −5U/mL, whereas the nectars did not show any detectable activity although cloud loss phenomenon was often observed. The diced fruit samples showed activities between 10 −2and 10 −5U/g. The highest values were found in samples of chi grape (10 −2U/g) and pineapple (10 −3U/g). A dependence of the intensity of cloud loss on the levels of PME residual activity was found.

A comparison of calcium binding in Callinectes sapidus premolt and postmolt cuticle homogenates: implications for regulation of biomineralization.

Cuticle tissue homogenates (CTHs) from Callinectes sapidus premolt cuticle bound approximately 367% more Ca2+ ions than did those from the postmolt cuticle. The pH-stat assay which was used to compare in vitro CaCO3 nucleation times confirmed that the premolt CTHs had greater inhibitory activity than did the postmolt CTHs. This inhibitory activity was indicated by CaCO3 nucleation times in excess of control values. Premolt nucleation times exceeded those of postmolt samples by approximately 340%. A positive correlation was observed between Ca2+ binding and calcification inhibitory activity for both premolt and postmolt CTHs. Heat pretreatment of CTHs at 70 degrees C for a 24-hr period had no significant effect on their Ca2+ binding. However, this heat pretreatment decreased their calcification inhibitory activity. Pretreatment of CTHs with Ca2+ diminished their calcification inhibitory activity. These results are consistent with a mechanism for inhibition of biocalcification by these proteins which involves their initial reversible binding to nascent calcite nuclei growth steps and kinks, rather than their in vivo interaction with free Ca2+ ions in solution.