Unpublished Work (C) 2005 Nima Golzy

Hydrolysis of Cytosine to Uracil

Conducted: 01 to 15 November 2005

Nima Golzy (F08-2)

Jordan and Ahmad

Chem 4A: Tue 10-2

Matt Strader, GSI

Hydrolysis of Cytosine to Uracil

Abstract:

            Spectrophotometry of solutions undergoing a simultaneous reaction, such as the hydrolysis of cytosine to uracil, can reveal changes in concentration over time. Using this information, first-order kinetics can be used to determine rate constants by plotting points and fitting least-squares regression lines. Rates at different pHs and different temperatures can be collected to measure the effects of solution acidity and environment temperature on reaction rates. For pH 3 and 9, rates of approximately 1.3 × 10^-6 s^-1 and 4.3 × 10^-6 s^-1 were found, respectively. Using Arrhenius curves, extrapolation of reaction rates was made to include lower temperatures. Using this information it was found that at body temperature (37 °C) the rate of hydrolysis drops by four orders of magnitude, enough to explain why the human genome does not destroy through its own hydrolysis.

Introduction:

            Of the several causes of deoxyribonucleic acid (DNA) damage, the hydrolysis of cytidine to uridine ranks as one of the most important pathways to genome destruction (Cohen). For laboratory purposes, this reaction is best described using pseudo-first order kinetics. According to the lab manual, the rates of hydrolysis for cytosine, cytidine, and cytidine monophosphate (CMP) are close enough to be considered similar, therefore cytosine was used in this experiment.

            Just as any other reaction, this experiment was sensitive to temperature and pH, thus these two parameters were carefully controlled. To ensure the reaction occurred within a convenient time frame, the cytosine solutions were kept in an oven to hydrolyze. To measure the effect of pH on cytosine’s hydrolysis, this experiment was performed by several groups, each using solutions at different pH levels.

            Monitoring the rate of the reaction required the use of a spectrophotometer, which was preferred over other analysis methods because of the simplicity of the formulas needed, the universal nature of the procedure, and the precise results procured. Spectrophotometers are instruments that pass focused light of specific wavelengths through solutions to detect the intensity of light that is able to get through the solution (Cohen). In many different situations, water contains different minerals and metals, thus before spectrophotometry of solutions was performed, a distilled water blank was be prepared and tested with the device. The resulting value was an arithmetic deviation that should have been subtracted from all subsequent measurements read from the device, however, the spectrophotometer used stored this deviation in memory and performed the correction automatically.

            Spectrophotometers are necessary because concentration change over time is required to monitor the rate kinetics of cytosine’s hydrolysis to uracil. There are four factors involved in spectrophotometry: molar absorptivity, pathlength, concentration, and absorbance. Spectrometry is a method of determining the concentration of a solution by measuring how much light is absorbed by the solute, thus it is only logical for concentration and absorbance to be directly proportional. Pathlength is the length of solution that the light must pass through before being measured for final intensity; the more solute particles the light encounters the less intense it will be upon emerging from the solution. Therefore, one would expect absorbance to also be directly proportional to pathlength:

A ∝ bc (or) A = kbc.

            The variable A represents absorbance, b represents pathlength (in centimeters), c represents the solution’s concentration (in molarity), and k is the proportionality constant. This constant, however, varies from solute to solute, thus it can be considered a unique property characteristic to the dissolved substance. The term ascribed to this constant is molar absorptivity and each type of solute has its own value. Finally, we get the Beer-Lambert Law (commonly called Beer’s Law):

A = εbc.

            In this experiment, the pathlength for all solutions was 1.000 cm, so the variable b was ignored when constructing a calibration curve. The molar absorptivities of cytosine and uracil–logically–remained constant, so instead of generating error by calculating this value for each solution, it was assumed that molar absorptivity would remain constant while transferring any error to discrepancies in absorbance. This reverts Beer’s Law back to the original assertion that absorbance is directly proportional to concentration, but it gives new meaning to the relationship. The absorbance is dependent on the molar absorptivities of the constituent solutes cytosine and uracil. To fix the discrepancy associated with molar absorptivity’s error being metaphorically put on absorbance’s shoulders, the value of the latter measurement is divided by the absorbance at the isosbestic point, the wavelength at which the molar absorptivities of cytosine and uracil are the same (in this experiment it was visually determined to be ~263nm) (Cohen).

            To put it simply, without derivation or explanation, in preparing a calibration curve, spectrophotometry was performed on solutions of different time lengths. Since cytosine and uracil have maximum absorbances at different wavelengths (275nm and 259nm, respectively), two calibration curves were constructed. The absorbances at each wavelength were divided by the corresponding absorbance at 263nm in order to “normalize” the curves and reduce error. These resulting absorbances were plotted against percent concentration of either cytosine or uracil (see Data and Analysis) to construct calibration curves (Cohen).

            Moving away from the theory of Beer’s Law in relation to spectrophotometry and first-order kinetics, the issue of pH arises. The experiment was performed by several groups using solutions of different pH levels, but that did not control acidity enough. The hydrolysis of cytosine to uracil releases NH₃, a base. Therefore, a buffer was used by each group to maintain the pH level. A buffer resists changes in pH because any added acid or base is consumed by ions that are readily reactive but have a very weak effect on pH themselves. Buffers work best when the solution’s pH is approximately equal to the buffer’s pK_a value (Cohen). For the solution with pH of 3, a buffer of 0.5M phosphate was used. For the solution with pH of 9, a buffer of 0.5M carbonate was used. These specific buffers were found in the lab manual.

            Acidity is also affected by changes in temperature, so since the solutions underwent hydrolysis at 100°C, a slight correction was ultimately made for pH. Referring to the table in the lab manual (Cohen-103), it was ascertained that for the solution with pH of 3 the correction was +0.156; the solution with pH of 9 had a correction of –0.172. Although nominal deviations, these new pHs are required for precision.

Procedure:

            Using a precise automatic pipet to transfer fluids, 5.00 ml of a 1.00 × 10^-3 M cytosine solution were injected into each of two 50 ml volumetric flasks. In the first flask, to be used for the pH of three, 5.00 ml of 0.500 M phosphate buffer solution were added to the cytosine. The same was done to the second flask, to be used for the pH of nine, but carbonate was used instead of phosphate. Also to the first flask, in order to compensate for the ionic strength of the solution, 7.9 ml of 1.000 M sodium chloride was added. Furthermore, 4.9 ml of NaCl were added to the second flask. After adding these solutions, the flasks were filled to the calibration line with distilled water to make final solutions with concentrations of 1.00 × 10^-4 M. The pH levels of these two solutions were measured using a pH meter. They were appropriately close enough to the desired values of 3.00 and 9.00 (meaning within 0.1 pH units away).

            Since this experiment was meant to determine the rate constant for the hydrolysis of cytosine, the latter was placed in an oven for different periods of time to change from cytosine to uracil. This was done by obtaining ten test tubes with screw tops and filling them with the prepared solutions. Five test tubes were each filled with approximately 5 ml of pH 3 cytosine (first flask) and the other five tubes were filled with approximately 5 ml of pH 9 cytosine (second flask). For each solution type there were five test tubes, each being allowed to hydrolyze for different periods of time. The heating times were zero hours, three hours, one day, six days, and fourteen days, and the temperature at which they were kept was 100 °C.

            The ‘zero hour’ time period actually refers to about 15 minutes of submerging the test tube in boiling water to decontaminate the sample within. This was treated as zero hours and the theoretical uracil content should be zero. The other four test tubes were placed in an oven for the time periods shown.

            After acquiring 1.00 × 10^-3 M solutions of cytosine and uracil from the stock room, standard solutions were prepared according to Table 1 below. Before doing so, however, the stock solutions were diluted to exactly 1.00 × 10^-4 M to match the concentrations of the mixtures in Flasks 1 and 2. Seven standard solutions were prepared.

Table 1: Content of the standard solutions used to construct calibration curves.

            A Hewlett-Packard 8453 Diode Array Spectrophotometer was used to evaluate the absorbances of these standard solutions at two different wavelengths. Before measuring their absorbances, solutions A and G (pure cytosine and pure uracil, respectively) were analyzed to determine the wavelengths at which maximum absorption occurs. The wavelength for cytosine was 275.0 nm and the wavelength for uracil was 259.0 nm, with the isosbestic point occurring at 263.0 nm. After finding these wavelengths, a very small amount of concentrated hydrochloric acid (about 0.2 ml per 4.0 ml of solution) was added to each standard to ensure ionization. Finally, the seven standard solutions were run through the spectrophotometer in a UV-cuvet after having run a distilled water blank. The spectrophotometer was set to measure absorbance at three fixed wavelengths: 259, 263, and 275 nm.

            After two weeks of hydrolysis and waiting, the experimental solutions in the ten test tubes were brought out for analysis. Unfortunately, the caps on two of the test tubes were not screwed on tightly enough, resulting in complete evaporation of prepared experimental solutions. Therefore, it should be noted that all further calculations and references to the experimental solutions will be missing the 6-day and 14-day solutions for the pH of 9. Similar to the absorption spectrum analysis of the standard solutions, the experimental solutions had 0.2 ml of concentrated HCl added before spectrophotometry was performed. Each of the experimental solutions was run at 259, 263, and 275 nm.

Data and Analysis:

            Two calibration curves were constructed so as to calculate the concentration of cytosine remaining after hydrolysis. The first curve was dependent on the wavelength 275 nm and was called the cytosine curve. The second curve was calculated using the wavelength of 259 nm and was called the uracil curve. The curves were made by plotting the absorbance of each standard solution against the percent concentration of cytosine or uracil, but remembering the correction for error, the absorbances were divided by the isosbestic absorbance before being plotted. The following curves express the concepts of Beer’s Law in that absorbance and concentration are directly proportional.

Table 2: Raw data for standard solutions

Table 3: Absorbances divided by isosbestic points and corresponding concentrations

            The calibration curve for cytosine’s wavelength has the equation:

(A-275 / A-263) = 0.0092 (%C) + 0.4429.

            The calibration curve for uracil’s wavelength has the equation:

(A-259 / A-263) = 0.0025 (%U) + 0.8016.

            Both of these least-squares regression lines resulted in correlation coefficients greater than 0.999, so they were assumed to be quite precise. To use these calibration curves to calculate how much cytosine was present in the experimental solutions, several things had to be remembered. When plugging the experimental absorbances into the calibration curves, they were first divided by the corresponding absorbance at 263 nm to somewhat normalize the data, per the explanation in the Intro. When determining the concentrations of cytosine using the second calibration curve, it was assumed that the percent concentrations of cytosine were equal to 1 minus the percent concentrations of uracil. Moreover, percent concentrations were multiplied by the experimental solutions’ concentration of 1.00 × 10^-4 M to attain the actual cytosine concentrations. The raw data attained from spectrophotometry are shown below in Table 4.

Table 4: Raw data for experimental solutions

            The calculations for determining the percent cytosine of “3 - 1 day” are shown as follows. The absorbance of the solution with pH of 3 and hydrolysis time 1 day at 275 nm was divided by the absorbance of that solution at 263 nm. This value was then plugged into the left side of the cytosine calibration curve and solved for %C, which represents percent concentration. This value was then multiplied by 1.00 × 10^-4 M to obtain the actual concentration of cytosine in the solution. From these steps, it was calculated that the concentration of cytosine under a pH of three and after one day at 100 °C was 9.16543 × 10^-5 M. This however, was attained using the cytosine calibration curve, though the same process was utilized using uracil’s calibration curve.

Abs 275 / Abs 263 = 1.23780 / 0.93243 = 1.28612

1.28612 = 0.0092 (%C) + 0.4429

%C = 91.65435

0.9165435 × 1.00 × 10^-4 M = 9.16543 × 10^-5 M

Table 5: Concentration of cytosine under a pH of 3 over time

Table 6: Concentration of cytosine under a pH of 9 over time

            The rate constants for cytosine’s hydrolysis were determined by plotting the natural log of [Cytosine]_t over [Cytosine]₀ against the variable time, t. Notice was made to convert hours and days into seconds. There are be two rate constants per pH level, one from each calibration curve. Once this plot was made, a least-squares regression line was fitted to the data and the slope was obtained. The negative of this slope came to be the rate constant.

Table 7: Data used to determine rate constant for pH of three

Table 8: Data used to determine rate constant for pH of nine

            In a solution of pH 3, cytosine hydrolyzes with a rate constant of 1.3830 × 10^-6 s^-1 according to the cytosine calibration curve. According to the uracil calibration curve, it hydrolyzes with a rate constant of 1.2648 × 10^-6 s^-1 at pH 3. In a solution of pH 9, cytosine hydrolyzes with a rate constant of 4.1650 × 10^-6 s^-1 according to the cytosine calibration curve. According to the uracil curve, it hydrolyzes with a rate constant of 4.4245 × 10^-6 s^-1 at pH 9.

Table 9: Rate constants and half-times for the hydrolysis of cytosine

            A pH rate profile was constructed after determining the rate constants for cytosine’s hydrolysis. The rate profile is a method of measuring the effect of acidity on reaction kinetics. To do this, the natural log of the rate constants were plotted against pH. Qualitative analysis indicates that ignoring the four outliers with high y values, the rate of hydrolysis is relatively high

at lower pH levels. Once a pH of 5 is established, the rate drops significantly, becoming about one-third of what it was before. Starting from a pH of 8, the rate constant starts to rise again. The absolute minimum where hydrolysis of cytosine is at its lowest occurs at a pH of 7, the most neutral level.

            Next, an Arrhenius curve was constructed to extrapolate results to lower temperatures. This curve follows the Arrhenius equation found in the lab manual and is made by plotting the natural logarithm of rate constant against the inverse of temperature. The slope of the least-squares regression line is equal to negative the reaction’s activation energy divided by the universal gas constant. Since it is best to make Arrhenius curves at stable pH levels, the solutions analyzed were at a pH of 7, since the flattest portion of the pH rate profile occurred at seven.

Table 10: Raw data for Arrhenius curve

            Now adding two more data points provided by the lab manual, the curve looks like:

Table 11: Two additional points for the Arrhenius curve

            Before adding the two data points, the equation yielded a slope of -13 283. By solving for the variable E_a, it was determined that the activation energy is 110.4 kilo-Joules per mole of cytosine.

-E_a / R = -13 283

E_a = 13 283 K × 8.314 J⋅mol^-1⋅K^-1

E_a = 110 435 J = 110.4 kJ/mol

            At 37 °C, the rate of the reaction would be 3.914 × 10^-10 s^-1. At 25 °C, the rate of the reaction would be 6.970 × 10^-11 s^-1.

            After adding the two data points, the equation yielded a slope of -13 583. By solving for the variable E_a, it was determined that the activation energy is 112.9 kilo-Joules per mole of cytosine. Further calculations showed that at 37 °C, the rate of the hydrolysis of cytosine would be 3.244 × 10^-10 s^-1. At 25 °C, the rate would be 5.556 × 10^-11 s^-1.

Table 12: Activation energies and predicted rate constants at pH 7

Discussion:

            Hypothetically, one could logically assume that the rate of cytosine’s hydrolysis would be lowest at the pH and temperature of human blood. Blood averages at a pH of 7.40 (Wikipedia) and body temperature is said to be 37 °C (Cohen). Thus, it was expected that the rate of hydrolysis would be lowest around pHs of 7 and 8. Moreover, many reactions occur faster at higher temperatures, so it was predicted that the same would happen with cytosine, only “higher” would be interpreted as greater than 37 °C.

            Graph 8, the pH rate profile, shows that the rate minimizes at a pH of 7. This fits well with the hypothesis, since cytidine hydrolysis is destructive to the body’s genome. Referring to Graph 10, it is shown that the rate of reaction increases as temperature increases. Notice must be made that the higher temperature values are found on the left side of the graph since the independent axis represents inverse temperature. Therefore, the human body is under ideal conditions for halting cytidine hydrolysis at a slow rate.

            All measurements were derived from the spectrophotometer. The initial concentration of cytosine was 1.00 × 10^-4 M, which includes error of its own. The only other source of numbers was the spectrophotometer, and that gives numbers precise to the nearest ten thousandth (at least). Therefore, it is hard to calculate error. Random variation and especially unknown systematic error is evident from the discrepancies in isosbestic point. This point should have been the same in all spectrophotometry runs, but it was not. Preparations were probably not performed as accurately as they should have been. For the most part, though, the procedure did not allow much room for error. Lastly, the fact that several groups were doing the experiment and results were compiled suggests variations in the way solutions were prepared and tested. Though each group had the same lab manual, each group used different personal methods to carry the experiment out. Multi-group experiments often lead to confusion as to whether everyone did their job correctly. Fortunately, results show that laboratory members were relatively accurate.

            An important application of these results lies in the human body. As stated in the introduction, cytosine’s hydrolysis is destructive to the human genome. Therefore, it is reasonable to calculate the number of cytosines hydrolyzes per day. It should be noted that cytosine in double-stranded DNA changes into uracil at a rate 200 times slower than that of single-stranded DNA. Assuming that there are approximately two billion base pairs in the human genome and also assuming that base pairs GC and AU appear in equal quantities, it can be claimed that there are approximately one billion cytosines in the human genome. At a regular body temperature of 37 °C and a normal body pH of 7, the rate of hydrolysis of isolated cytosine is 3.244 × 10^-10 s^-1. Using integrated rate laws for first-order kinetics, it was determined that in the course of one day, about 28 028 isolated cytosines convert to uracil. However, dividing by 200 results in approximately 140 cytosines hydrolyzing into uracil while in DNA per day. Compared to one billion cytosines being within the human body, 140 seems like a very trivial number. In fact, scraping one’s skin on a sharp surface might peel away more cytosines. One hundred forty cytosines per day can be seen as 0.001 622 cytosines per second. The human body is tempered quite ideally for DNA, since different pH levels and temperatures could potentially raise the number of lost cytosines by several-fold.

            Certain organisms called hyperthermophiles are able to grow above 80 °C, living in temperatures as hot as 113 °C. Assuming hyperthermophiles live in pH levels of about 7, the rate constant of hydrolysis can be calculated from Graph 10. Using a temperature of 110 °C, the hydrolytic rate for the cytosine of an organism would be 1.3740 × 10^-6 s^-1. This value is significantly larger than the same rate for humans by four orders of magnitude. To see how this affects the absolute number of cytosines lost, it will be assumed that a hypothetical hyperthermophile also contains one billion cytosines. Given that the growth cycle of this organism is about five hours, each growth cycle results in the hydrolysis of 122 143 cytosines. Moreover, each day a hyperthermophile has 559 689 cytosines hydrolyzed into uracil.

            This number is much greater than that of humans, most evidently due to a different temperature. For humans, the hydrolytic rate for cytosine is 3.244 × 10^-10 s^-1. For hyperthermophiles, the same rate is 1.3740 × 10^-6 s^-1, about four orders of magnitude greater than the rate for humans. For an organism to live at such an extreme temperature, certain adaptations must be made. Enzymes must be hardy enough to withstand these temperatures since proteins often become denatured and unfunctional at extremely high temperatures. Assuming that structural difficulties are overcome, a hyperthermophile will probably be able to fix the cytosine problem by having a very effective uracil-DNA glycosolase enzyme. This is the enzyme responsible for removing the incorrect uracil and making room for replacing it with a cytosine (Cohen). After all, reactions occur faster at higher temperatures, so although cytosine hydrolyzes very quickly, these organisms probably make up for it by having a very quick response.

Conclusion:

            The two rate constants determined using the cytosine and uracil calibration curves for solution of pH 3 were 1.3830 × 10^-6 s^-1 and 1.2648 × 10^-6 s^-1, respectively. The two rate constants determined using the same calibration curves for solution of pH 9 were 4.1650 × 10^-6 s^-1 and 4.4245 × 10^-6 s^-1. The half-life for cytosine in pH 3 was determined to be about six days whereas the half-life was just under two days for pH 9. According to the pH rate profile, it was shown that the rate of cytosine hydrolysis tends to slow down at normalized pH levels near seven while being about three to four times higher in acidic solutions and about twice as high in basic solutions. According to a plot of rate against temperature, it was shown that temperature affects rate by increasing it with a rise in temperature. This was deemed ideal for humans, as body temperature is about 37 °C and high hydrolytic rates are found at around 80 °C to 120°. Overall, it was determined that cytosine hydrolysis is not a large contributor to genome damage according to data collected. Approximately 140 cytosines are hydrolyzed to uracil per day, and assuming the enzyme uracil-DNA glycosolase does its job at all effectively, these lost cytosines are quickly replaced by new ones. Therefore, in the condition of humans, cytosine hydrolysis is not an incredibly large factor in genome destruction, though still important.

References:

            Cohen, Ronald. Laboratory Manual. Regents of the University of California. Berkeley, 2005.

            Harris, Daniel. Quantitative Chemical Analysis, 6ed. W.H. Freeman and Company. New York, 2003.

            Wikipedia Encyclopedia. “Blood.” 2005.