Stability Of Alkaline Phosphatase Research Articles

Влияние малых доз ионизирующего излучения при облучении in vitro на показатели крови

Abstract. The purpose is the determination of the degree of influence of small absorbed doses of ionizing radiation on biochemical and hematological parameters during irradiation of peripheral blood samples of cattle in vitro. For the work, blood samples were taken from lactating cows kept under the influence of the background absorbed dose in the Krasnoyarsk Territory, followed by in vitro irradiation at doses of 5, 50, 500 mGy. Methods. Peripheral blood was taken from blood vessels from the tail vein into vacuum tubes with a coagulation activator, in vitro irradiation of samples was carried out on an installation equipped with a Cs-137 source, biochemical studies of blood serum were carried out using a PE-5400UF spectrophotometer. Hematological blood parameters were determined according to generally accepted methods. Results. Ionizing radiation in vitro in doses of 5 mGy, 50 mGy and 500 mGy of peripheral blood samples of cattle affects hematological parameters in different directions, which is characterized by wave-like fluctuations in hemoglobin values, a decrease in the content of erythrocytes and a decrease in the erythrocyte sedimentation rate. It was found that a single irradiation of peripheral blood samples of cows at a dose of 5 mGy reduced the concentration of beta-globulins and creatinine and did not affect the content of total protein and protein fractions. When exposed to ionizing radiation at a dose of 50 mGy, the content of total protein, the level of beta-globulins, creatinine decreased in blood samples, and the content of albumins and alpha-, gamma-globulins did not change. When irradiated at a dose of 500 mGy, the relative content of alpha-globulins, the concentration of ALT and AST decreased, the content of creatinine, total protein, albumins and beta-, gamma-globulins did not change. The stability of alkaline phosphatase, albumins and gamma globulins to ionizing effects at doses of 5 mGy, 50 mGy and 500 mGy has been established. Scientific novelty. The linear dependence of the concentration of alpha-globulins, AST and ALT on the absorbed dose was revealed, the numerical values of the approximation coefficients describing the decrease in concentration and biochemical parameters were determined. The inverse direct linear relationship between creatinine stability and absorbed dose has been established. Practical significance is the established digital values of the approximation coefficients of alpha globulins, AST and ALT can be used in the reconstruction of radiation dose values in the range of 5–50 mGy.

Effect of Varied Concentrations of Ethylhene Glycol as Pooled Serum Preservative on the Stability of Alkaline Phosphatase (ALP) Ezyme

Quality controls aim to ensure the quality, accuracy, and thoroughness of an examination. Internal quality assurance is carried out by the laboratory itself using control materials, both commercial control materials and homemade control materials. This controlled material is then used to ensure the quality of an inspection. The stability of the control material is strongly influenced by the storage temperature where the recommended temperature is -20? which will be stable for up to 6 months and 2-8? which is stable for 6 days. In the manufacture of homemade control materials, a stable preservative, antifreeze, and antibacterial is needed but still maintains the integrity of the group serum. The preservative used is ethylene glycol. This study aims to determine the optimal concentration of ethylene glycol in stabilizing alkaline phosphatase levels in refrigerator temperature storage (4-8ºC) for 60 days. In this study, tests were conducted on pooled sera preserved with ethylene glycol in varying concentrations, namely 7.5%; 10%; 12.5%; 15%; and 17.5% and observed the stability of the Alkaline Phosphatase enzyme in pooled sera for 60 days with an interval of 10 days with a storage temperature of 4-8?. The study was conducted in July-August 2021 in the BLUD laboratory of RSUD dr. Ben Mboi, Manggarai. The results showed that the stability of alkaline phosphatase persisted until day 30, began to decrease on day 40, and continued to decrease until day 60 in each different concentration variation. different, namely 7.5%; 10%; 12.5%; 15%; and 17.5% had the same stability up to 30 days. The recommendation from the author is to observe at different temperatures so that the stability of Alkaline phosphatase can be known.

Dephosphorylation of pyridoxal 5′‐phosphate‐derived Schiff bases in the presence of bovine alkaline phosphatase

AbstractThe present paper reports on the study of the dephosphorylation of pyridoxal 5′‐phosphate and four derived hydrazones (containing the residues of pyrazine, 2‐furan, 2‐thiophene, 3‐pyridine carboxylic acids) induced by bovine alkaline phosphatase from intestinal mucosa at 298.2 K and pH 10 (0.05 m Tris–HCl buffer). We observed and discussed characteristic changes in the UV–vis and fluorescent spectra of substrates. Michaelis–Menten parameters of the enzymatic dephosphorylation are calculated. The stability of alkaline phosphatase in the presence of hydrazones is confirmed. The dephosphorylation of the Zn(II) complex with pyridoxal 5′‐phosphate‐derived hydrazone is analyzed.

Ages at a Crime Scene: Simultaneous Estimation of the Time since Deposition and Age of Its Originator.

Blood is a major contributor of evidence in investigations involving violent crimes because of the unique composition of proteins and low molecular weight compounds present in the circulatory system, which often serve as biomarkers in clinical diagnostics. It was recently shown that biomarkers present in blood can also identify characteristics of the originator, such as ethnicity and biological sex. A biocatalytic assay for on-site forensic investigations was developed to simultaneously identify the age range of the blood sample originator and the time since deposition (TSD) of the blood spot. For these two characteristics to be identified, the levels of alkaline phosphatase (ALP), a marker commonly used in clinical diagnostics corresponding to old and young originators, were monitored after deposition for up to 48 h to mimic a crime scene setting. ALP was chosen as the biomarker due to its age-dependent nature. The biocatalytic assay was used to determine the age range of the originator using human serum samples. By means of statistical tools for evaluation and the physiological levels of ALP in healthy people, the applicability of this assay in forensic science was shown for the simultaneous determination of the age of the originator and the TSD of the blood spot. The stability of ALP in serum allows for the differentiation between old and young originators up to 2 days after the sample was left under mimicked crime scene conditions.

Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen–thawed stallion spermatozoa

Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen–thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is seminal plasma, whereas sperm extracts contribution is very low. In addition, we found that AP activity at physiological pH (7.0) is significantly lower than that observed at pH 8.0, including the optimal AP pH (pH 10.0). Alkaline phosphatase did not exert any effect on sperm–oocyte interaction assessed by heterologous oocyte binding assay. Additionally, we observed a thermal stability of seminal plasma AP, concluding that it is similar to that of bone isoforms. Positive correlations were found between seminal plasma AP activity and sperm concentration, whereas a negative correlation was present between both spermatozoa extracts and seminal plasma AP activity and seminal plasma protein content. A significant decrease in sperm extract AP activity was found in frozen–thawed samples compared with freshly ejaculated ones (n = 21), concomitantly with the decrease in sperm quality parameters. The positive correlation between seminal plasma AP activity measured at pH 10 and viability of frozen–thawed spermatozoa suggests that seminal plasma AP activity could be used as an additional predictive parameter for stallion sperm freezability. In conclusion, we provide some insights into AP activity in both seminal plasma and sperm extracts and describe a decrease in AP after freezing and thawing.

On‐going Studies on the Structural Stability of E. coli Alkaline Phosphatase by an Undergraduate Biochemistry Laboratory Course

Our biochemistry laboratory course engages in a guided‐inquiry exercise in which we study the high degree of structural stability of E. coli alkaline phosphatase (AP) and the roles of two disulfide bonds toward thermal and chemical denaturation. Recently we have compared the denaturation of the enzyme using guanidinium hydrochloride and urea, in the absence and presence of the reducing agent TCEP, using CD and fluorescence spectroscopic techniques. We have also investigated the effect of temperature on the chemical denaturation processes. We also have continued to determine the nature of a chaperonin‐like protein(s) found in the periplasmic lysates that contributes to the dramatic thermal stability of AP, relative to the highly purified commercial enzyme using tandem mass spectrometry. Being a portion of an undergraduate biochemistry laboratory course, these investigations represent data obtained from individual laboratory groups who make oral presentations at the end of their research, providing a more real world approach to the educational experience. This work is supported, in part, by a grant from NSF (DUE 0837398).

Continuing Studies on the Structural Stability of E. coli Alkaline Phosphatase by an Undergraduate Biochemistry Laboratory Course

On‐going investigations into the roles of the two disulfide bonds in E. coli alkaline phosphatase (AP) on structural stability and limited proteolytic degradation have been incorporated into a guided inquiry portion of an undergraduate biochemistry laboratory course. We studied the effects of reducing agents (DTT and TCEP) on the thermal and chemical (urea and guanidinium hydrochloride) denaturation of AP using a combination of tryptophan fluorescence and CD spectroscopy, also investigating the roles of Hofmeister series ions during thermal and chemical denaturation. We also present evidence for potential chaperonin‐like proteins found in the periplasmic lysates that contribute to the dramatic thermal stability of AP, relative to the highly purified commercial enzyme and used tandem mass spectrometry to identify likely stabilizing proteins. Being a portion of an undergraduate biochemistry laboratory course, these investigations represent data obtained from individual laboratory groups, that make oral presentations at the end of their research, providing a more real world approach to the educational experience. This work is supported, in part, by a grant from NSF (DUE 0837398).

Hofmeister effects on activity and stability of alkaline phosphatase

We have studied the effects on alkaline phosphatase of adding high concentrations (normally 1.0 M) of simple salts. It is necessary to allow for significant effects of salts on the extinction coefficient of the reaction product, and on the apparent pH of the buffer. Both activity and stability of the enzyme correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones–Dole viscosity B coefficients ( B + for cations and B − for anions). The catalytic activity or V max/ K m of the enzyme showed a bell-shaped relationship with the ( B − − B + ) values of the salts present, being optimal with salts (such as NaCl, KCl, and KNO 3) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is believed to be enzyme-specific and relates to the impact of both cations and anions on the enzyme's surface pH, active site, and catalytic mechanism. Anions play a more predominant role than cations in affecting enzyme stability. The rate of irreversible thermal inactivation is strongly reduced by addition of kosmotropic anions like SO 4 2− (half-life increased from 8 to 580 min at 60 °C). This effect is general and the mechanism probably involves the ability of the ions to affect the water solvation layer around the enzyme molecule and to interact with both the surface and internal structure of the enzyme.

Effects of replacing active site residues in a cold‐active alkaline phosphatase with those found in its mesophilic counterpart fromEscherichia coli

Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold-adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal-binding sites in the active site of APs, and all three metal ions participate in catalysis. The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs. In contrast, two of the residues that bind the third metal ion (numbered 153 and 328 in Escherichia coli AP) are different in various APs. This may explain their different catalytic efficiencies, as the Mg2+ most often present there is important for both structural stability and the reaction mechanism. We have mutated these key residues to the corresponding residues in E. coli AP to obtain the double mutant Asp116/Lys274, and both single mutants. All these mutants displayed reduced substrate affinity and lower overall reaction rates. The Lys274 and Asp116/Lys274 mutants also displayed an increase in global heat stability, which may be due to the formation of a stabilizing salt bridge. Overall, the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold-active Vibrio AP both locally and globally, and this influences kinetic properties.

Activity and stability of alkaline phosphatase (ALP) immobilized onto magnetic nanoparticles (Fe 3O 4)

Direct binding of alkaline phosphatase (ALP) on magnetic nanoparticles (Fe 3O 4) in the presence of carbodiimide (CDI) using two different methods is described. The activity and stability of immobilized ALP with both shaking and sonication method were compared. The results indicated the ALP binding efficiency to be in the range of 80–100% with both the immobilization techniques. The activities retained were in the range of 20–38% with shaking method and 30–43% with sonication method, respectively. The activities of the immobilized ALP preparations were found to be stable compared to the free (unbound) ALP for at least 16-week storage period. Moreover, ALP immobilized on magnetic nanoparticles was successfully used for dephosphorylation of plasmid DNA before it was used for ligation reaction. The use of immobilized ALP for plasmid dephosphorylation allows easy manipulation, reduces the procedural time, and also avoids exposure of reaction mixture to high temperature.

The activity and stability of alkaline phosphatase in solutions of water and the fused salt ethylammonium nitrate

The fused salt ethylamonium nitrate has several properties which resemble those of the biologically important solvent water. In order to shed light on the role of solvent in determining protein structure we have examined the influence of ethylammonium nitrate on the activity and stability of the enzyme alkaline phosphatase. Significantly, although reduced enzymatic activity was observed in ethylammonium nitrate solutions up to 60% (v/v) in water, the enzyme was stable to brief exposure to solutions as high as 80% (v/v) in the fused salt.

The stability of alkaline phosphatase in sodium dodecyl sulfate

While sodium dodecyl sulfate (SDS) is a most effectieve detergent for solubilizing membrane proteins, this detergent is also an effective protein denaturant [l-4] . At appropriate detergent concentrations and after equilibrium dialysis, the SDS-protein complexes formed contain approximately 1.4 g of SDS bound/g protein [ 1,2] . By raising the ionic strength and the SDS concentrion, the amount of detergent bound can be increased to about 2 g/g protein [3]. However with most enzymes which have been studied activity is completely lost after treatment with SDS. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) was released from milk fat globule membrane (MFGM) in a soluble, active form by treatment of the membrane with SDS. Enzyme activity was recovered after electrophoresis in polyacrylamide gels containing SDS. Four other mammalian alkaline phosphatases and the enzyme from E. coli retained activity over a wide range of detergent concentrations.

Factors affecting the activity and stability of alkaline phosphatase in a marine pseudomonad.

Conditions optimum for the assay of alkaline phosphatase of marine pseudomonad B-16 (ATCC 19855) and for maintaining the activity of the enzyme have been determined. The pH for optimal activity of the cell-bound enzyme was 9.0, whereas that for the enzyme after its release from the cells exceeded 9.4. Release was effected by first washing the cells in 0.5 M NaCl and then suspending them in 0.5 M sucrose. In the absence of salts, the activity of the cell-bound enzyme decreased rapidly at 25 C and less rapidly at 4 C. This loss of activity could be arrested but not restored by adding Mg(2+). In the presence of Na(+), activity of the cell-bound enzyme dropped to about 50% of that prevailing initially, but in this case adding Mg(2+) restored enzyme activity completely. The activity of the enzyme after its release from the cells into 0.5 M sucrose was approximately 50% of that of the equivalent amount of enzyme in the original cells. This activity was relatively stable at both 25 and 4 C. Adding Mg(2+) to the released enzyme restored its activity to that of the cell-bound form. The synthesis of alkaline phosphatase by the cells was not affected by adding 50 mM inorganic phosphate to the growth medium. The K(m) of the released enzyme for p-nitrophenyl phosphate was found to be 6.1 x 10(-5) M.

Studies on the heat stability of alkaline phosphatase in serum

Studies on the heat stability of alkaline phosphatase in serum