Chapter 8 Exploratory Data Analysis of Grapevine Data
8.1 Data Analysis Methods
One of the advantages of DTNB over other sulphydryl reagents is that the kinetics of its reaction with haemoglobin sulphydryl groups can be monitored on a simple UV-Visible spectrophotometer (Neer et al., 1968). Furthermore, DTNB is sensitive to the electrostatic environments that characterize the reactivity of the CysF9[93]β sulphydryl group (Neer, 1970; Amiconi et al., 1971; Okonjo et al., 1995).
We restricted our DTNB experiments to pH 9.0 and below because it is known that above pH 9.0 complications arise on account of the increased rate of hydrolysis of disulphide bonds (Robyt et al., 1971). The reactions of DTNB with both the stripped human oxyhaemoglobin and inositol-bound haemogloin solutions, were monitored at 412 nm on a Varian Cary UV-Visible spectrophotometer under pseudo-first order conditions. This was achieved by reacting each of the haemoglobin samples with at least a sixty – fold excess of DTNB per sulphydryl group.
A 10 µmol (haem) dm-3 (5 µmol dm-3 reactive sulphydryl groups) solution of a haemoglobin derivative in a chosen buffer was allowed to equilibrate at 25°C in a thermostated water bath. The spectrophotometer was set by inputing all the necessary parameters: wavelength, temperature and times (start and end). A 3 cm3 aliquot of this haemoglobin solution was then transferred into a 1 cm x 1 cm cuvette. (For experiments carried out in the presence of inositol-P6, a calculated volume of the 0.01 mol dm-3 inositol-P6 that would result in a fiinal concentration of 10 µmol dm-3 was added to the 10 µmol (haem) dm-3 solution of haemoglobin before equilibration). The cuvette was placed in the cell compartment of the spectrophotometer which was also thermostated at 25°C. The reaction was initiated by quickly mixing the haemoglobin with a calculated volume of the stock 29.07 mmol dm-3 DTNB solution in 95%
ethanol that would give a final DTNB concentration of 300 – 600 µmol dm-3. The reaction timing was initiated simultaneously by pressing the auto start button of the spectrophotometer. The kinetic traces (change in absorbance or transmittance as a function of time) were displayed on a computer screen and stored on the computer.
Each kinetic run was repeated at least two additional times under identical experimental conditions.
86
The data were analyzed with two softwares. The first was a 1990 update of DISCRETE, a computer programme for the analysis of multiple exponential signals (Provencher, 1976a; 1976b). The other was a SigmaPlot® Systat software. The two softwares gave similar results. The analyses gave a single kinetic phase for stripped haemoglobin and for haemoglobin in the presence of inositol-P6. The pseudo-first order rate constants calculated from different kinetic traces, kobs, were plotted against the DTNB concentration. The resulting linear graph gave a slope, kF, the apparent second-order forward rate constant, and a positive intercept.
3.6.2 Kinetics of the reaction of 5,5'-dithiobis(2-nitrobenzoate), DTNB, with the sulphydryl groups of human deoxyhaemoglobin A. (The experiments described in this section were carried out at the Department of Biochemistry and Molecular Biology, University of Parma, Italy).
The kinetics of the reaction of 5,5'-dithiobis(2-nitrobenzoate), DTNB, with the sulphydryl groups of human deoxyhaemoglobin A was monitored at 412 nm.
Immediately after deoxygenation was complete, the sealed, specialized cuvette containing ≈ 10 µmol (haem) dm-3 solution of human deoxyhaemoglobin A was transferred from the thermostated room into the cell compartment of the Cary 400 spectrophotometer thermostated at 25°C with a Crioterm® water bath. A spectrum of the haemoglobin solution was taken between 300 and 700 nm. The disappearance of the characteristic peaks for human oxyhaemoglobin A at 541, 577, 415 & 344 nm, and the appearance of the characteristic peaks for human deoxyhaemoglobin A at 555 &
430 nm, confirmed complete deoxygenation (Antonini and Brunori, 1969; Brunori et al., 1968; Figures 4.18 & 4.19). About 10 cm3 of 29.07 mmol dm-3 DTNB in 95%
ethanol was similarly deoxygenated by allowing humidified nitrogen gas to pass over the solution for about 30 minutes in a small, septum-sealed, amber bottle.
Prior to the initiation of the reaction, the deoxyhaemoglobin solution in the cell compartment was re-connected to a continuous flow of humidified nitrogen gas. The spectrophotometer was set by inputing all the necessary parameters: wavelength, temperature and times (start and end). A calculated volume (mm3) of deoxygenated 29.07 mmol dm-3 DTNB solution that would give a final concentration between 100 and 400 µmol dm-3 of DTNB was introduced into the sealed cuvette via a gastight microsyringe. The kinetics of the reaction was immediatley initiated by gently but
87
quickly shaking the mixture in the cuvette and pressing the start button on the spectrophotometer.
The kinetic traces (change in absorbance or transmittance as a function of time) were displayed on a computer screen and stored on the computer. Each kinetic run could only be repeated once under identical experimental condition due to the length of time required for deoxygenation and because only three such specialised cuvettes were available for use. For the same reason we could not conduct a full pH-dependence study of the reaction. However, we were able to monitor the kinetics of the reaction at pH 8 at several concentrations of DTNB. The data were analyzed with the SigmaPlot® Systat software.
3.6.3 Kinetics of reaction of 5,5'-dithiobis(2-nitrobenzoate) with the sulphydryl groups of cat haemoglobin.
The reaction of DTNB with liganded haemoglobin derivatives of the domestic cat was monitored at 412 nm in a manner similar to that of human oxyhaemoglobin A.
The only difference was that the reaction was monitored on a Cecil BioQuest® UV-Visible spectrophotometer and there was no need for deoxygenation to be carried out.
Also, the normal 1 cm by 1 cm cuvettes were used, and as such, we were able to repeat each kinetic run more than once.
88 3.7 EQUILIBRIUM STUDIES
3.7.1 Determination of equilibrium constants for the reaction of DTNB with sulphydryl groups of human deoxyhaemogobin A. (The experiments described in this section were carried out at the Department of Biochemistry and Molecular Biology, University of Parma, Italy).
A 50 µmol (haem) dm-3 (25 µmol dm-3 in reactive sulphydryl groups) oxyhaemoglobin solution was prepared in a phosphate buffer of desired pH and ionic strength 50 mmol dm-3. A 1 cm3 aliquot of this solution was measured into each of the three available, specially fabricated, 0.2 cm × 1 cm cuvettes. The solution was deoxygenated as previously described. After complete deoxygenation was confirmed from the spectrum, a known volume of a deoxygenated 29 mmol dm-3 DTNB solution was added with a gastight microsyringe into the deoxyhaemoglobin solution in each cuvette. The mixtures were shaken gently and allowed to equilibrate for between 6 and 8 hours under a continuous nitrogen flow. All the deoxygenation and equilibration processes were carried out at 25°C in a CARRÉ refrigerant plant thermostated room.
After equilibration, the spectrum of each solution was taken from 300 – 700 nm on the Varian Cary 400Scan UV/Visible spectrophotometer which was connected to an external computer recording unit equiped with a Cary® WinUV software. Owing to the limited number of the specially fabricated cuvettes available for it was difficult to obtain sufficient data points to determine the equilibrium constant (Kequ) at a single wavelength. It therefore became necessary to employ other means to obtain more data points from the experiments performed at each pH. Eyer et al. (2003) have determined the molar absorption coefficients of 5-thio-2-nitrobenzoate (TNB) at four other wavelengths apart from 412 nm. Therefore, by assuming molar absorption coefficients of 13.8, 14.0, 11.0, 8.0 and 4.0 (× 103 mol-1dm3 cm-1) at 405, 412, 436, 450 and 470 nm respectively, the equilibrium constant was calculated for the content in each cuvette from the change in absorbance at each of the listed wavelengths, using a programme written on a MicroMath Scientist software (Appendix G1). The differences in absorbance at specific wavelengths between the spectra before and after equilibration were noted and imputed into a programme written on the MicroMath Scientist Software for the analysis of equilibrium data (Appendix G1, page 278).
This procedure was repeated in phosphate buffers pH 5.6 – 7.8 and borate buffers pH 8.0 - 9.0 (ionic strength = 50 mmol dm-3), and with 2.9 mmol dm-3 DTNB.
89
3.7.2 Determination of equilibrium constants for the reaction of DTNB with