Tris-HCl

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. 

Figure 2 Stability of /3-poly(L-malate) measured by its activity to inhibit purified DNA polymerase a of P. polyceph-alum. The relative degree of inhibition is shown (100 rel. units refer to complete inhibition). The DNA polymerase assay was carried out in the presence of 5 /tg/ml /S-poly(L-malate) as described [4]. The polymer was preincubated for 7 days at 4°C in the following buffer solutions (50 mM) KCl/HCl (—A—). Citrate (—V—). 2-(A/-Morpholino)-ethanesulfonic acid, sodium salt (—O—). Sodium phosphate (— —). N-(2-Hydroxyethyl)piperazine-N -(2-ethanesul-fonic acid), sodium salt (— — ). N,N-b s (2-Hydroxyethyl)-glycine, sodium salt (—T—). Tris/HCl (— —). 3-(Cyclo-hexylamino)-l-propanesulfonic acid, sodium salt (— —).

Luminescence reaction (Viviani et al., 2002a) The luciferin-luciferase luminescence reaction was carried out in 0.1 M Tris-HCl, pH 8.0, containing 2mM ATP and 4mM Mg2+. Mixing luciferase with luciferin and ATP resulted in an emission of light with rapid onset and a kinetically complex decay. Further additions of fresh luciferase, after the luminescence has decayed to about 10% of its maximum value, resulted in additional luminescence responses similar to the initial one (Fig. 1.15). According to the authors, the repetitive light emission occurred in consequence of the inhibition of luciferase by a reaction product, as seen in the case of the firefly system (McElroy et al., 1953). The luminescence spectrum showed a peak at 487nm (Fig. 1.16). 

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.

Fig. 3.2.4 Fluorescence spectra of F (solid lines), and the bioluminescence spectrum of F plus P, in 20 mM Tris-HCl, pH 7.6, containing 0.15 M NaCl, at 4°C. Both F and P were obtained from Meganyctiphanes norvegica. From Shimomura and Johnson, 1968a.

Fig. 3.2.7 Left panel Effects of temperature on the luminescence intensity and stability of the protein P from Meganyctiphanes. The initial light intensity was measured with F plus P in 5 ml of 20 mM Tris-HCl/0.15 M NaCl, pH 7.5, at various temperatures. In the stability test, P was kept at the indicated temperature for 10 min, then mixed with 5 ml of 25 mM Tris-HCl/1 M NaCl, pH 7.59, containing F, to measure initial light intensity. Right panel Effect of the concentration of salts on the light intensity of the luminescence of F plus P, in 25 mM Tris-HCl, pH 7.6, at near 0°C. In the case of NaCl, the light intensity decreased to about a half after 10 min. From Shi-momura and Johnson, 1967, with permission from the American Chemical Society.

Fig. 3.3.1 Luminescence spectrum of coelenterazine catalyzed by the luciferase of the decapod Oplophorus in 15 mM Tris-HCl, pH 8.3, containing 50 mM NaCl (solid line). For comparison, the luminescence catalyzed by the luciferase of the anthozoan sea pansy Renilla is shown with dashed line (in 25 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl).

Fig. 3.3.2 Influence of pH on the activity of luciferase ( ) and the quantum yield of coelenterazine (o) in the bioluminescence of Oplophorus. The measurements were made with coelenterazine (4.5 pg) and luciferase (0.02 pg) for the former, and coelenterazine (0.1 pg) and luciferase (100 pg) for the latter, in 5 ml of 10 mM buffer solutions at 24° C. The buffer solutions used sodium acetate (pH 5.0), sodium phosphate (pH 6.0-7.5), Tris-HCl (pH 7.5-9.1), and sodium carbonate (pH 9.5-10.5), all containing 50 mM NaCl. Replotted from Shimomura et al., 1978, with permission from the American Chemical Society.

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. 4.1.4 Influence of pH on the total light emission and initial light intensity of aequorin. Buffer solutions containing 0.1 mM calcium acetate, 0.1 M NaCl, and 10 mM sodium acetate (for pH < 7) or 10 mM Tris-HCl (for pH > 7) were adjusted to various pH with acetic acid or NaOH, and then 2 ml of the solution was added to 3 pi of aequorin solution containing 1 mM EDTA to elicit luminescence, at 22°C. The data shown are a revision of Fig. 9 in Shimomura et al., 1962. The half-total time is the time required to emit 50% of total light.

Fig. 4.1.11 Influence of the concentration of apoaequorin on the yield of regenerated aequorin after 12 h at 4°C (solid line), and on the initial light intensity of the apoaequorin-catalyzed luminescence of coelenterazine (dashed line). The regenerated aequorin was measured with a 10 pi portion of a reaction mixture (0.5 ml) made with 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 10 pi of methanolic 0.6 mM coelenterazine, and various amounts of apoaequorin. The luminescence activity of apoaequorin was measured in 2 ml of 10 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl, 2 mM CaCb, 2 mM 2-mercaptoethanol, 10 pi of methanolic 0.2 mM coelenterazine, and various amounts of apoaequorin. Reproduced with permission, from Shimomura and Shimomura, 1981. the Biochemical Society.

Fig. 4.3.1 Effect of pH on the total light emission of phialidin (A), and the temperature stability profiles of phialidin (minute open circles) and aequorin (solid line) (B). In A, each buffer contained 0.1 M CaCl2 plus 0.1 M Tris, glycine or sodium acetate, the pH being adjusted with NaOH or HC1. In B, the photoprotein samples in 10 mM Tris-EDTA buffer solution, pH 8.0, were maintained at a test temperature for 10 min, and immediately cooled in an ice water bath. Then total luminescence activity was measured by injecting 1ml of 0.1 M CaCl2/Tris-HCl, pH 7.0, to 10 pd of the test solution. From Levine and Ward (1982), with permission from Elsevier.

Luminescence activity. The specific luminescence activities (quanta/s emitted from 1ml of a solution of A280nm,icm 1.0) of luciferases A, B and C are in a range of 1.2 4.1 x 1016 photons/s when measured with the standard assay buffer (20 mM Tris-HCl, pFl 7.8, containing 1M NaCl, 0.05% BSA, and 0.14 xg/ml of coelenterazine, at 24°C). These are the highest specific activities of coelenterazine luciferases. 

Fig. 4.5.3 Effect of temperature on the light intensity of coelenterazine catalyzed by Periphylla luciferases A, B, C and L, in 3 ml of 20 mM Tris-HCl, pH 7.8, containing 1 M NaCl and 0.05% BSA. The luminescence reaction was started by the addition of 10 (xl of 0.1 mM methanolic coelenterazine. The amounts of luciferase used for the measurement of each point luciferase A, 170 LU luciferase B, 190 LU luciferase C, 210 LU luciferase L, 210 LU. From Shimomura et al., 2001.

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.

Fig. 5.8 Influence of pH, temperature, NaCl concentration, and the concentration of coelenterazine on the light intensity of luminescence reaction catalyzed by the luciferases of Heterocarpus sibogae, Heterocarpus ensifer, Oplophorus gracilirostris, and Ptilosarcus gruneyi. Buffer solutions used 20 mM MOPS, pH 7.0, for Ptilosarcus luciferase and 20 mM Tris-HCl, pH 8.5, for all other luciferases, all with 0.2 M NaCl, 0.05% BSA, and 0.3 p,M coelenterazine, at 23°C, with appropriate modifications in each panel. Various pH values are set by acetate, MES, HEPES, TAPS, CHES, and CAPS buffers.

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.

Fig. 6.3.6 Effects of salt concentration (left panel) and pH (right panel) on the initial light intensity emitted from the homogenate of the Symplectoteuthis oualaniensis light organ. The salt effect was tested in 50 mM Tris-HCl, pH 7.2, and the pH effect in the various buffers containing 0.5MKC1 or NaCl. From Tsuji and Leisman, 1981.

Fig. 6.3.7 Luminescence spectrum of a homogenate of the luminous organ of Symplectoteuthis oualaniensis in the presence of 0.5 M KC1 (from Tsuji and Leisman, 1981). A homogenate suspension (1 ml) and 1MKC1 (1 ml), both made with 50 mM Tris-HCl, pH 7.6, containing 1 mM dithioerythritol, were mixed and the spectrum was measured 6 min after mixing. Note that the luminescence of the photoprotein symplectin isolated from the luminous organs showed a maximum at 470—480 nm (Takahashi and Isobe, 1993, 1994).

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. 

Fig. 7.1.5 Fluorescence spectra of purified Chaetopterus photoprotein (CPA) in 10 mM ammonium acetate, pH 6.7 (solid lines), and the bioluminescence spectrum of the luminous slime of Chaetopterus in 10 mM Tris-HCl, pH 7.2 (dashed line). Note that the luminescence spectrum of Chaetopterus photoprotein in 2 ml of 10 mM Tris-HCl, pH 7.2, containing 0.5 M NaCl, 5 pi of old dioxane and 2 pi of 10 mM FeSC>4 (Amax 453-455 nm) matched exactly with the fluorescence emission spectrum of the photoprotein. No significant change was observed in the fluorescence spectrum after the luminescence reaction.

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. 

Assay of scintillon activity. A small volume (5-50 xl) of a scintillon sample is mixed with 1ml of 50 mM Tris-HCl, pH 8.0, containing 10 mM EDTA and 1 mM DTT. To this mixture, 1 ml of 0.2 M sodium citrate, pH 5.2 (or 30 mM acetic acid) is injected and the light emission is measured. 

The solution is dialyzed against the same buffer using a hollow fiber assembly, and then added onto a column of Affi-Gel Blue (50-100 mesh, 2 x 15 cm, Bio-Rad) prepared with the same buffer. The column is washed with the same buffer. Then luciferase is eluted with 50 mM Tris-HCl, pH 8.5, containing 5mM EDTA, 3 mM DTT, and 0.5 M NaCl (Hastings and Dunlap, 1986, state that it may be preferable to omit the Affi-Gel step because of difficulties encountered). 

The eluted luciferase is precipitated with ammonium sulfate. The precipitate is dissolved in 1 mM Tris-HCl, pH 8, containing 0.1 mM EDTA, 3 mM DTT and 0.1 M NaCl, and chromatographed on a column of Sephacryl S-300 (2.6 x 97 cm) using the same buffer. Luciferase is eluted in two peaks, corresponding to the molecular weights of about 420,000 (an aggregate) and 130,000, in a ratio of about 8 1. The fractions of these two peaks are pooled separately the Mr 420,000 luciferase is concentrated by either ultrafiltration or precipitation with ammonium sulfate. 

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. 

Purified LBP is obtained from the crude LBP separated in the gel filtration of the 35 kDa luciferase on Sephadex G-100 (see Fig. 8.2). The fractions of crude LBP are combined and the protein is precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a small volume of lOmM Tris-HCl/5 mM 2-mercaptoethanol, pH 8, and a small amount of luciferin is added as a tracer. Then, the crude LBP is purified on a column of Sephadex G-200 (Hastings and Dunlap, 1986). The fractions of LBP are identified by luminescence produced by the addition of luciferase at pH 6.3 the luminescence due to the tracer luciferin is proportional to the amount of LBP in each fraction. 

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