Selection of optimal probe and conditions

Table 4.1 The B-A transition parameters obtained from CD and absorbance difference spectra

4.1.4 Selection of optimal probe and conditions

The B-A-transition of DNA was studied by CD and absorption difference spectra and the spectral changes were characterized for different types of samples at different conditions (Table 4.1). With ethanol, an increase in salt concentration by four times induced the B-A transition at about 2% lower ethanol content than at standard salt concentration. TFE induces the B-A transition at 5% lower alcohol content than ethanol.

Comparison of poly [d(A-T)] and natural DNA transition curves revealed that the width of transition in natural DNA is almost 5 times broader than that of poly [d(A-T)] (Table 4.1). Because of its narrow transition width, poly [d(A-T)] is taken as the suitable probe for the initial studies on the B-A transition.

In order to have an explicit understanding about a process, it is necessary to have some awareness about the potential side reactions, which might interfere with the process under investigation. Either the stopped flow technique or the electric field jump technique seems to be the optimal method to follow the kinetics of the B-A transition. Two potential side reactions, which might couple with B-A transition, are helix-coil transition and aggregation. The absorbance difference spectra of poly [d(A-T)] for B-A transition showed two clear isobestic points indicating that only two conformations are involved in the process. This shows the absence of aggregation of DNA under the present experimental condition. The superposition of spectral changes of denaturation with that of B-A transition can be avoided by measuring the transition at wavelengths where the spectral changes of denaturation is minimal compared to that of B-A transition. Therefore, to determine the optimal wavelength to follow the B-A transition, the denaturation spectra of poly [d(A-T)] and natural DNA should be studied.

4.1.4.1 Denaturation spectra

In order to find an optimal wavelength to study the B-A transition, the denaturation spectra of poly [d(A-T)] (fig 4.9) and natural DNA were recorded. From the spectrum it is clear that denaturation of poly [d(A-T)] does not have any considerable amplitude at 280 nm. The denaturation spectra of natural DNA showed that at the wavelengths of interest that is at 248 nm and 265 nm, denaturation has some considerable amplitude. So for poly [d(A-T)] 280 nm is taken as the ideal wavelength for following the B-A transition whereas for natural DNA the reaction is followed at both wavelengths.

200 225 250 275 300 325 350 0.00

0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

25°C

20°C

15°C

10°C

∆Α

λ [nm]

Figure 4.9 Absorption difference spectrum of 60% EtOH (v/v)- poly [d(A-T)] at various temperature. (20 µM poly[d(A-T)], 100 µM NaCl, 100 µM Cacodylate, pH 7, 20 µM EDTA).

Absorption spectrum at 0°C was subtracted from spectrum at each temperature and plotted.

4.1.4.2 Melting temperature measurements

Melting temperatures of poly [d(A-T)] at various percentages of ethanol were determined in order to find out the optimal working temperature in the B-A transition range. From figure 4.10 it is clear that above 55% ethanol (v/v), the melting temperature of poly [d(A-T)] shows an increase with increasing ethanol percentage.

This indicates that the double helix is stabilized in the presence of ethanol. B-A transition occurs at ~ 70% of ethanol. In this range the melting temperature of poly [d(A-T)] is around 18°C. Therefore 8°C was taken as the optimal working temperature for poly [d(A-T)].

0 10 20 30 40 50 60 70 80 10

12 14 16 18 20 22 24 26 28

t [°C]

% EtOH

Figure 4.10 Melting temperature of poly [d(A-T)] as a function of ethanol (% in v/v units, 15 µM poly[d(A-T)]). Starting ion concentration: (125 µM NaCl, 125 µM Cacodylate pH 7, 25 µM EDTA).

4.2 Stopped flow measurement

The B-A transition of DNA can be induced by addition of ethanol to a DNA solution and the transition can be easily studied by following the absorbance changes at 248 nm or at 286 nm. Therefore, stopped flow technique was selected as the first method to study the kinetics of this transition. Solutions were taken in such a way that a DNA concentration, which corresponds to an absorbance of 1, is obtained in the mixing chamber. Experiments were conducted such that the proportion of ethanol was changed from 60%, where the DNA is in equilibrium B-form, to 80%, where it is in equilibrium A-form. Figure 4.11 shows the transient up to 0.1 seconds, which clearly shows the absence of any process occurring under this condition.

Experiments were repeated under the same conditions and the transients recorded up to 20 seconds did not reveal any change in the light intensity. Further stopped flow experiments were performed at ethanol percentages outside the B-A transition range in which the ethanol proportion was changed from 0% to 50% and from 20% to 60%

ethanol. The transients did not reveal any indication for the occurrence of any physical or chemical processes.

0.00 0.02 0.04 0.06 0.08 0.10

0.00 0.02 0.04 0.06 0.08

∆I [V]

t [s]

Figure 4.11 Transients observed at 248 nm after mixing of solutions in the stopped flow experiment at 19°C. Contents in Syringe A. (60% EtOH (v/v)-1.818 mM sonicated Salmon sperm DNA, 150 µM NaCl, 150 µM Cacodylate pH 7, 30 µM EDTA) Contents in Syringe B. EtOH.

Absorbance changes at 248 nm were recorded for DNA samples as a function of time, where ethanol percentages was changed from 0% to 50%, 60%, 70%

and 80% in a Cary spectrophotometer with quick mixing of the sample. The recording of absorbance was started within 20 seconds after mixing. DNA samples with 50%

and 60% of ethanol which are outside the B-A transition range, showed an increase of absorbance of approximately 0.004 in 6 minutes whereas samples at 70% and 80%

ethanol showed a decrease in absorbance of approximately 0.002 in 6 minutes. These small changes in absorbance might be due to settling of air bubbles or dust particles.

These experiments revealed the absence of any physical or chemical process occurring in this time range.

It is known that high ethanol percentages induce aggregation of DNA and this is reflected by an increase in absorbance. Stopped flow experiments were conducted in such a way that ethanol percentages were increased from 70% to 85%

and also from 80% to 90%. Under these conditions, aggregation of DNA is expected

to occur (Potaman et al., 1980). At both these percentages of ethanol, a decrease in the light intensity was observed in stopped flow experiments. Figure 4.12 shows the transient observed after mixing 80% EtOH-DNA solution with pure EtOH, which results in a final ethanol content of 90% in solution. At 248nm, the B-A transition is reflected by a decrease in absorbance, whereas aggregation by an increase in absorbance. The nature of the transients and the ethanol percentages at which this change is observed confirms the assignment of the observed decrease in light intensity to aggregation.

0 10 20 30 40 50

0.0 0.2 0.4 0.6 0.8 1.0 1.2

∆I [V]

t[s]

Figure 4.12 Transients observed at 248 nm after mixing of solutions in the stopped flow experiment at 19°C. Contents in Syringe A. (80% EtOH (v/v)-1.818 mM sonicated Salmon sperm DNA, 150 µM NaCl, 150 µM Cacodylate pH 7, 30 µM EDTA) Contents in Syringe B. EtOH.

The stopped flow experiments did not reveal any spectral change, which can be attributed to the B-A transition. This confirms that the B-A transition occurs faster than millisecond time scale.