Enzymes incubation

Tissue Mixed with a proteolytic enzyme incubated at 65 °C headspace gas analyzed GC/ECD NR NR Ramsey and Flanagan 1982... 

Cambella and Antia [385] determined phosphonates in seawater by fractionation of the total phosphorus. The seawater sample was divided into two aliquots. The first was analysed for total phosphorus by the nitrate oxidation method capable of breaking down phosphonates, phosphate esters, nucleotides, and polyphosphates. The second aliquot was added to a suspension of bacterial (Escherichia coli) alkaline phosphatase enzyme, incubated for 2h at 37 °C and subjected to hot acid hydrolysis for 1 h. The resultant hot acid-enzyme sample was assayed for molybdate reactive phosphate which was estimated as the sum of enzyme hydrolysable phosphate and acid hydrolysable... 

Enzyme Incubation conditions Immobilized Units (%) Active Units (%)... 

The mutation at Lys-258 was carried out to replace Lys with Ala.16,39 This mutant lost activity by more than 106-fold compared to the wild-type enzyme. Other amino acids, such as Met and Arg, were also introduced into this position.16 It was found that enzymic activity in these mutants diminished significantly. In contrast, spectroscopic studies showed that Lys-258 mutants incubated with Asp or /J-hydroxy-Asp exhibited spectral changes almost identical with those of the wild-type enzyme incubated with a pseudosubstrate, a-Me-Asp. a-Me-Asp is known to give a Michaelis complex in which the catalytic process is seized prior to the a-proton abstraction step because of the absence of the a-proton in a-Me-Asp. This suggested that in the mutants the reaction was stopped at the step just prior to the a-proton removal. Both activity and spectrum studies supported the catalytic role of Lys-258. 

Starting the stopwatch at zero time, rapidly pipet 2.0 mL of the Sample Preparation into the equilibrated substrate. Mix, and replace the tubes in the water bath. Add 2 mL of Tris Buffer (instead of the Sample Preparation) to the substrate blank. After exactly 30 min, add 10 mL of TCA Solution to each enzyme incubation and to the substrate blank to stop the reaction. Heat the tubes in the water bath for an additional 30 min to allow the protein to coagulate completely. 

Sample Preparation Using Glycine-Hydrochloric Acid Buffer, prepare a solution of the sample enzyme preparation so that 2 mL of the final dilution will give a corrected absorbance of enzyme incubation filtrate at 275 nm (AA, as defined in the Procedure) between 0.200 and 0.500. Weigh the enzyme preparation, quantitatively transfer it to a glass mortar, and triturate with Glycine-Hydrochloric Acid Buffer. Quantitatively transfer the mixture to an appropriately sized volumetric flask, dilute to volume with Glycine-Hydrochloric Acid Buffer, and mix. 

In experiments in which sodium borohydride was added to the enzyme incubation mixture of the apiose/xylose synthase, no reduction products could be found that would arise from epimerization at C-3 of the 4-ketose intermediate.44 In the synthesis of apiose, ring contraction therefore seems to take place with the L-threo-pentos-4-ulose 27 (see Scheme 11), and not with the L-erythro intermediate, which would be analogous to the ring contraction in the biosynthesis of L-dihydro-streptose. 

The reaction mixture contained angiotensin I, a phosphate buffer at pH 8.0, Nad (chloride is required for activity), and the enzyme. Incubations were at 37°C for 30 minutes and were terminated by treatment with a boiling water bath for 5 minutes. Centrifugation was used to remove precipitated protein. Fluorescamine in acetone was added to the supernatant solution, and samples were injected for analysis. The enzymatically formed fragments were separated on HPLC as shown in Figures 9.24 and 9.25 the rate of His-Leu formation is shown in Figure 9.26. 

Figure 9.60 HPLC for the measurement of ferrochelatase activity in human leukocytes with mesoporphyrin and Zn2" as substrates, (a) Enzyme incubation mixture, (b) Blank incubation with boiled leukocytes. Column ODS-Hypersil (250 mm X 5.0 mm i.d.) eluent, 88% (v/v) methanol in 1 M ammonium acetate, pH 5.16. Flow rate, 1.5 mL/ min fluorescence detection, excitation at 403 nm and emission at 574 nm. Peaks 1, Zn-deuteroporphyrin (internal standard) 2, Zn-mesoporphyrin 3, mesoporphyrin. (From Guo et al., 1991.)...

Figure 9.87 Elutions of the nicotinate phosphoribosyltransferase (N-PRTase) assay solution through a /xBondapak C18 column after various enzyme incubation times. The incubation mixture contained 5 mM MgCl2,100 y,M nicotinate, 75 tiM ATP, 30 /iM PRibPP, and 25 yu.L of 4 mg/mL N-PRTase in 50 mM Tris-HQ (pH 8). Elution conditions 5 yuL sample injection volumes, 0.7 mL/min flow rate, 25 m M (NH))P04 (pH 8) elution buffer, 25°C. (From Hanna and Sloan, 1980.)...

The incubation mixture contained in a final volume of 2 mL 50 mM caco-dylic acid (pH titrated to 6.5 with 10 M KOH), 150 mM KC1, and 10 mM ATP. The reaction was started by adding 100 fiL of diluted enzyme. The mixture was incubated at 25°C, and samples 500 /u,L of the enzyme incubation mixture were drawn after 10, 20, and 30 and added to 250 /u,L of 6% (w/v) trichloroacetic acid. After centrifugation, 500 fiL of the supernate was added to the same volume of 0.5 M tri-n-octylamine in Freon. The mixture was recentrifuged, and the upper layer was used for HPLC analysis. 

Figure 4.4 Residual activity as effected by dissolution of alkaline protease AL 89 in hydrophilic solvents (M. Kristensen and LH. Pedersen, unpublished). Enzyme incubated in anhydrous DMF (squares) or DMSO (triangles) in the absence (filled symbols) or presence (open symbols) of 0.2 M sucrose.

Fig. 1. The effect of hsp70 on the refolding of cAAT and pmAAT. Refolding of acid unfolded cAAT or pmAAT was performed by rapid dilution of the denatured enz3rmes in the refolding buffer to a final protein concentration of 1.8 pM. When present, hsp70 (1.8 pM) was added to the refolding buffer before initiation of the refolding reaction. After incubation for 120 min at 10 C, the transaminase activity recovered was measured as indicated under Methods. Reactivation data are expressed relative to that of the native enzyme incubated under identical conditions.

The basal activity of aminotransferase fell by 50% during consumption of the Bt-deficient diet. The stimulation occurring with addition of PLP to the enzyme incubation mixtures rose in this period from 200% (twofold stimulation) to 400% (fourfold). The basal activity of the aminotransferase, and probably of ail enzymes of the body, varies from subject to subject. It may even vary with repeated enzyme assays using the same sample of red blood cells. Thus, the basal activity is not used to assess vitamin Be, status. The percentage stimulation is relatively constant in normal subjects and is thus a more useful indicator of B status. It should be noted that the storaffe of ivti blood teiis can lead to gradual release of the cofactor from the enzyme thus, an artefactual diagnosis of Bh deficiency is possible. 

Dephosphorylation may be carried out using bacterial alkaline phosphomonoesterase (Heppell et al. 1962). Typical conditions are 20 fig desalted oligonucleotide mixture in 2 ml 1 M ammonium bicarbonate with 10 fig enzyme incubated for 1 hr at 37°C (De Wachter and Piers 1967). This removes 3 -terminal phosphates. Brownlee and Sanger (1967) carried out combined T1 ribonuclease and bacterial alkaline phosphatase digestion of the low molecular weight rRNA using enzyme/substrate ratios of 1/20 and 1/5 respectively in 0.01 M Tris pH 8.0 at 37°C for 1 hr. This produces fragments like XpXp...XpG where X represents any nucleoside except G. 

Usually removal of the cell wall is reaUzed by treatment of plant material (leaves, tissue cultures, suspended cells, etc.) with a designed cocktail of enzymes such as cellulases, pectinases and/or hemicellulases, in an incubation medium of the appropriate osmolality to avoid cell burst. In addition, the physiological status of the source plant material also influences the release of viable protoplasts, and several other factors affect protoplasts release, including the extent of cell wall thickening, temperature, duration of enzyme incubation, pH of the enzyme solution, gentle agitation, and nature of the osmoticum [40]. 

Urine, blood, TDG Enzyme incubation, derivatization Negative ion CI GC-MS 1 ng/mL Black and Read (1988)... 

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