Tris-HCl buffer

The ionic species of the mobile phase will also affect the separation. This is shown in Table 4.3 by the difference in resolution values for magnesium chloride buffer compared to sodium sulfate buffer. In addition, calibration curves for proteins in potassium phosphate buffers are shallower than those generated in sodium phosphate buffers. The slope of the curve in Sorenson buffer (containing both Na and ) is midway between the slopes generated with either cation alone (1). Table 4.4 illustrates the impact of different buffer conditions on mass recovery for six sample proteins. In this case, the mass recovery of proteins (1,4) is higher with sodium or potassium phosphate buffers (pH 6.9) than with Tris-HCl buffers (pH 7.8). 

Poly(L-malate) decomposes spontaneously to L-ma-late by ester hydrolysis [2,4,5]. Hydrolytic degradation of the polymer sodium salt at pH 7.0 and 37°C results in a random cleavage of the polymer, the molecular mass decreasing by 50% after a period of 10 h [2]. The rate of hydrolysis is accelerated in acidic and alkaline solutions. This was first noted by changes in the activity of the polymer to inhibit DNA polymerase a of P. polycephalum [4]. The explanation of this phenomenon was that the degradation was slowest between pH 5-9 (Fig. 2) as would be expected if it were acid/base-catalyzed. In choosing a buffer, one should be aware of specific buffer catalysis. We found that the polymer was more stable in phosphate buffer than in Tris/HCl-buffer. 

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

Quantum yield and luciferase activity The quantum yield of coelenterazine in the luminescence reaction catalyzed by Oplophorus luciferase was 0.34 when measured in 15 mM Tris-HCl buffer, pH 8.3, containing 0.05 M NaCl at 22°C (Shimomura et al., 1978). The specific activity of pure luciferase in the presence of a large excess of coelenterazine (0.9pg/ml) in the same buffer at 23°C was 1.75 x 1015 photons s 1 mg-1 (Shimomura et al., 1978). Based on these data and the molecular weight of luciferase (106,000), the turnover number of luciferase is calculated at 55/min. 

Fig. 4.5.5 Effect of pH on the luminescence of coelenterazine catalyzed by Periphylla luciferases A, B and C, and on the stability of the luciferases. The effect on light intensity (solid lines) was measured in 3 ml of 50 mM phosphate buffers, pH 4.1-7.25, and 50 mM Tris-HCl buffers, pH 7.1-9.7, all containing 1 M NaCl, 0.025% BSA, and 0.3 pM coelenterazine. To measure the stability (dotted lines), a luciferase sample (5 pi) was left standing for 30 min at room temperature in 0.1 ml of a buffer solution containing 1 M NaCl and 0.025% BSA and having a pH to be tested, and then luciferase activity in 10 pi of the solution was measured in 3 ml of 20 mM Tris-HCl, pH 7.8, containing 1M NaCl, 0.05% BSA, and 0.3 pM coelenterazine at 24°C. The amounts of luciferases used for measuring each point were luciferase A, 150 LU luciferases B and C, 170 LU. One LU = 5.5 x 108 quanta/s. From Shimomura etal., 2001.

Purification of Pholas luciferase (Michelson, 1978). Acetone powder of the light organs is extracted with 10 mM Tris-HCl buffer, pH 7.5, and the luciferase extracted is chromatographed on a column of DEAE Sephadex A-50 (elution by NaCl gradient from 0.1 M to 0.6 M). Two peaks of proteins are eluted, first luciferase, followed by a stable complex of luciferase and inactivated pholasin. The fractions of each peak are combined, and centrifuged in 40% cesium chloride... 

Fig. 6.2.4 Change in the absorption spectrum of pholasin (14.5 p,M) caused by the luminescence reaction catalyzed by Pholas luciferase (1.1 p.M). The curve shown is the differential spectrum between a cell containing the mixture of pholasin and Pholas luciferase (0.9 ml in the sample light path) and two cells containing separate solutions of pholasin and the luciferase at the same concentrations (in the reference light path), all in 0.1 M Tris-HCl buffer, pH 8.5, containing 0.5 M NaCl. Four additions of ascorbate (3 iM) were made to the sample mixture to accelerate the reaction. The spectrum was recorded after 120 min with a correction for the base line. From Henry and Monny, 1977, with permission from the American Chemical Society.

Fig. 6.3.4 Luminescence spectrum of the Watasenia bioluminescence reaction measured with a crude extract of light organs that contain particulate matters, in chilled 0.1 M Tris-HCl buffer, pH 8.26, containing 1.5 mM ATP. From Tsuji, 2002, with permission from Elsevier.

Assay of photoprotein. The activity of the photoprotein was measured in 1ml of 20 mM Tris-HCl buffer, pH 8.0, containing 0.6 M NaCl at room temperature. The intensity and total amount of light emitted were recorded. The luminescence intensity is markedly intensified by adding 5 il of catalase solution (crystalline bovine liver catalase 1.5 mg/ml) and 10 pi of 3% H2O2. 

Assay of luciferin. A buffer solution of 20-50 mM Tris-HCl (pH 7.2 1.9 ml) containing 20 mM magnesium acetate and luciferase (typically 0.1 ml of the luciferase stock solution) is injected into a vial containing luciferin sample (1-10 pi) and 20 mM KCN (0.1 ml made daily in 0.1 M Tris-HCl buffer, pH 7.2), and the resulting light emission is measured in terms of total light. 

The concentrated luciferase solution is dialyzed overnight against 4 liters of 1 mM Tris-HCl buffer, pH 8.5, containing, 0.1 mM EDTA and 3 mM DTT. Then luciferase is further purified on a column of DEAE-BioGel A (1 x 25 cm, Bio-Rad) by elution with a linear increase of NaCl from 0 to 100 mM in the same buffer as that used in dialysis. The purified luciferase had a specific activity (based on initial maximum intensity) of approximately 8.5 x 1014 quanta sec 1mD1Aj810. 

Fig. 9.4 Time course of the chemiluminescence reaction of (NH SO t -activated panal at pH values 4.5, 5.0, 5.5, and 6.0, in 3 ml of 10 mM acetate buffer in the presence of lOmg of CTAB, 20 pi of 0.1 M FeSC>4, and 20 pi of 10% H2O2 and at pH 8.0, in 3 ml of 50 mM Tris-HCl buffer containing 0.18 mM EDTA, 10 mg of CTAB, lOmg of NaHCC>3, 20pi of 0.1 M FeSC>4, and 20pi of 10% H2O2. All at 25°C. From Shimomura, 1989, with permission from the American Society for Photobiology.

Assay of luminescence activity. A methanolic solution of the activation product (5-50 xl) is mixed with 3 ml of 50 mM Tris-HCl buffer, pH 7.8, containing 0.18 mM EDTA and about 5 mg of CTAB. After leaving the solution for a few minutes, 15 (rl of 50 mM FeS04 and 30 il of-10% H2O2 are added in this order. The light emission begins when H2O2 is added. 

Fig. 9.7 Bioluminescence of Panellus stipticus fruiting body (1) fluorescence of PM-2 in the presence of CTAB upon excitation at 440 nm (2) chemiluminescence of PM-2 in the presence of CTAB (3) chemiluminescence of PM-2 in the presence of 3-(dodecyldimethylammonio)propanesulfonate (SB3-12) (4) and chemiluminescence of the hot-water treatment product of PM-1 in the presence of SB3-12 (5). Curves 2-5 were measured in 2 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 0.18 mM EDTA. Chemiluminescence was elicited by the addition of 5 p.1 of 50 mM FeSC>4 and 10 xl of 10% H2O2. From Shimomura, 1991b, with permission from Oxford University Press.

Assay of Ophiopsila photoprotein. The luminescence reaction is initiated by the injection of 0.1 ml of 1% H2O2 into 3 ml of Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl and 10-100 pi of a sample solution, and the total light emission is measured. When assaying the... 

Fig. 10.2.2 Influence of the concentrations of ATP, Mg2-1- and Ca2+ on the maximum luminescence intensity of the photoprotein of the millipede Luminodestnus. The luminescence reaction was started by mixing a solution of the photoprotein (A280 0.3, 10 pi) with 2 ml of 10mM Tris-HCl buffer, pH 8.3, containing either 1 mM MgCb plus various concentrations of ATP or 0.05 mM ATP plus various concentrations Mg2+ or Ca2+. From Shimomura, 1981, with permission from the Federation of the European Biochemical Societies.

Fig. 10.2.4 Influence of temperature on the peak light intensity and total light of Luminodesmus photoprotein, when 25 pi of a solution of the photoprotein was injected into 2ml of lOmM Tris-HCl buffer, pH 8.3, containing 0.1 mM ATP and 1 mM MgCl2, that had been equilibrated at various temperatures.

In order to solubilize the bound luciferase, the pellet is homogenized with 20-30 volumes of cold 10 mM Tris-HCl buffer, pH 7.5, containing 1M NaCl, and the activity of the homogenate is measured. The homogenate is centrifuged and the activity of supernatant is also measured. A close agreement between the two measurements indicates that 1 M NaCl has solubilized the bound luciferase. If the two values differ significantly, a further effort of solubilization is needed (see Section C1.3). 

Contaminating CO2. The ubiquitous presence of CO2 causes a great difficulty to the experiment. Normal atmosphere contains 0.03% CO2, and a 5-6 ml portion of pure water equilibrated with atmosphere contains 0.06 pmol of CO2. However, the amount of CO2 in buffers and luciferase solutions is much greater. For example, 5 ml of freshly prepared Tris-HCl buffer, pH 7.8, contained 0.13 pmol of CO2 after 20 min degassing, and the value increased to 0.23 pmol when the same buffer was tested one week later (Shimomura et al., 1977). It is strongly advised to eliminate all nonessential CO2 sources from the experimental environment. 

Fig. 4. X-band EPR spectra of [Fe3S4]+ clusters in wild type and mutant forms of P. furiosus Fd. All spectra were recorded at 4.2 K microwave power, 1 mW microwave frequency, 9.60 GHz modulation amplitude, 0.63 mT. All samples were in 100 mM Tris-HCl buffer, pH 7.8.

The carboxylafion of indole into indole-3-carboxylate was observed by the purified indole-3-carboxylate decarboxylase as well as by the whole cells. For the carboxylafion reaction, temperatures over 30°C were not appropriate. The activities at 10, 20, and 30°C were about the same. The activity was maximal at pH 8.0 (Tris-HCl buffer, 100 mM). As shown in Fig. 10, the resting cells of A. nicotianae F11612 also catalyzed the carboxylafion of indole efficiently in the reaction mixture containing 20 mM indole, 3M KHCO3, 100mM potassium phosphate buffer (pH 6.0) in a tightly closed reaction vessel. By 6h, 6.81 mM indole-3-carboxylic acid accumulated in the reaction mixture with a molar conversion yield of 34%. Compared to the carhoxylation of pyrrole by pyrrole-2-carboxylate decarboxylase, the lower value compared might derive from the lower solubility of indole in the reaction mixture. 

Membranes (50 pi in a total assay volume of 100 pi) were incubated with UDP-Gal (0.1 mM) and MgSO (10 mM) in 25 mM Tris-HCl buffer pH 7.5, for 10 or 60 min. Reactions were stopped by heating at 100°C for 3 min. Lupin galactan (0.1 mg) was added as a 0.1% solution, methanol was added to give a final concentration of 70% by volume, and the tubes were capped, heated at 70°C for 5 min and centrifuged (13000g 5 min). Supernatants were discarded or retained for analysis. Pellets were washed twice more with 70% methanol at 70 C and the supernatants were discarded. The final pellets were either dissolved in preparation for scintillation counting, or were suspended in water and freeze dried in preparation for analysis. 

Figure 5. Incorporation at different pH values. Tris-HCl buffers were used over the range pH 6.3 - 9.5 and MES buffers from pH 5.0 - 6.5. (10 min assays)...

PL activity was determined by monitoring A236 for the formation of unsaturated products released from 0.1% polygalacturonic acid (P-1879, Sigma), dissolved in 0.1 M Tris/HCl buffer, pH 8,0 supplemented with 0.1 mM CaCb. One unit of PL is the enzyme activity liberating 1 pmol of unsaturated oligoglacturonides from pectate per min at 25°C. Activities are given in mU / ml extract per min. 

PMT assays were performed as described by Vannier et al. [3] by adding an equal volume of an enzyme preparation to a 0.1 M Tris-HCl buffer containing 3.36 pM of [ C]SAM (1.8 GBq mmol, 740 kBq ml", NEN), 1% (WA ) BSA and 12% sucrose, with or without 0.2% pectic acceptor. The incubation was run at 28°C for 12 h. After precipitation of the reaction product in 70% ethanol, the methylated polymers were selectively extracted with 0.5% ammonium oxalate and radioactivity was measured in a Tricarb 2250 CA Packard scintillation counter. 

Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HCl buffer (pH 8.0) with ImM CaCl2, were incubated at 37 C for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms Ipmol of 4,5-unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M cm [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37°C. Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HCl buffer (pH 8.0) with 0.5 mM CaCl2. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min under the conditions of reaction. Specific viscosity (n p) is (t/to)-l, where t is the flow time (sec) of the reaction mixture and t is the flow time of the buffer. The inverse pecific viscosity (n p ) is proportional to the incubation time and the amount of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. 

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