Bio lab graphic summary

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

Enzyme Activity and Contributing Factors The purpose of this laboratory is to understand factors impacting enzyme activity using the enzyme β-galactosidase and ONPG hydrolysis, as an example. This reaction yields a colored product that can be measured by spectrophotometry. Background:

Enzymes are protein catalysts that accelerate the rate of a reaction by reducing the activation energy, without themselves being used as a reactant in the reaction. Enzymes reduce activation energy by helping orient two reactants in a reaction or providing functional chemical groups that help form intermediates that help the reaction proceed (Figure 1). Enzymes are involved in various physiological and cellular processes, including cellular respiration, photosynthesis, digestion, and cellular signaling (e.g. phosphorylation cascades).

Figure 1. An enzyme changes the activation energy of a reaction. (Freeman et al, 2016)

A single enzyme molecule can convert molecules of substrate into product multiple times

during a given time period. The maximum reaction rate for a group of enzymes is called Vmax. Imagine that the concentration of substrate is so high that there is essentially no time when an enzyme molecule is not bound to substrate. That is, as soon as a bound substrate molecule reacts to form product, it leaves the enzyme and is instantly replaced by a fresh substrate molecule. This is what we call a "saturating" concentration of substrate—because at this concentration, the active site is always filled (saturated) with substrate. Under such conditions, the rate at which an individual enzyme molecule can turn substrate into product is the highest it can possibly be. This maximum cycling rate per molecule is called the turnover number (TON). A group of 100 enzyme molecules, each able to convert substrate to product at the same maximum cycling rate, would produce an observed Vmax that was 100-fold higher than the TON.

Enzyme activity is influenced by a number of factors, including temperature, substrate concentration, enzyme concentration, pH, or buffer composition (presence or absence of specific ions). Enzymes are proteins; which means that they must be kept in specific conditions to prevent the protein from denaturing and losing its enzymatic activity. The purpose of maintaining homeostasis in an organism’s body is to ensure proper enzyme activity at all times. Enzyme concentration described in Units (U) which reflects the activity of the enzyme under specific conditions that usually reflect standard conditions; the conditions include a set temperature, pH, buffer type, and buffer concentration.

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

Beta-Galactosidase β-galactosidase is an enzyme used by bacteria and other living organisms to hydrolyze

lactose, a disaccharide, into the monomers galactose and glucose. Both glucose and galactose can be used by the bcteria as energy sources by living cells.

An alternative substrate for this enzyme, o-nitrophenylgalactoside (ONPG), is colorless in solution. β-galactosidase speeds up the conversion of ONPG to o-nitrophenol (ONP) in the same way as it acts on lactose. The product of ONPG hydrolysis is yellow in solution, so we can measure the rate at which the product accumulates by measuring the appearance of yellow color.

Absorbance Spectroscopy

Figure 2. The Spectronic 20 by Bausch and Lomb has been a best seller for over 50 years.

Spectrophotometers measure the absorbance of light that is passed through a sample. This

is a useful, simple, quick, and relatively inexpensive means to analyze the properties of a sample. Spectrophotometers are used to estimate the number of cells in a culture, concentrations of protein or DNA in a sample, and to quantitate biological activity of a molecule in instances where the activity results in a colorimetric change in the solution.

Spectrophotometers are used to measure absorbance at a specific wavelength or absorbance across a range of wavelengths. Measuring absorbance at a specific wavelength allows determination of the concentration of a solute or the absorption coefficient of a chromophore. By recording the absorbance of light by a sample across a range of wavelengths, an absorbance spectrum is created. This spectrum may exhibit one or more peaks which are called absorbance

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

maximums (max). The location of the peaks is characteristic of certain arrangements of atoms and can be used to identify unknown molecules.

Figure 3. Absorption Spectrum of Chlorophyll a and b from plants. The characteristic peaks of the absorbance maxima for chlorophyll a and b allow identification of the presence of these molecules in a mixed solution of unknowns. [Image retrieved from http://www.chm.bris.ac.uk/motm/chlorophyll/chlorophyll_text.htm]

The Spectronic 20, by Bausch & Lomb came onto the market in 1954. It was a low-cost

bench-top machine useful for quick measurements in the visible spectrum between wavelengths of 340 and 950 nm. This model is still in use today and is commonly the instrument of choice for absorbance measurements in teaching labs (Filmore, 2007). Measurements on the spectrophotometer are taken as either transmittance or absorbance readings. Transmittance refers to the light that passes through the sample. Transmittance is defined by the equation

T=I/Io where T is transmittance, I is the intensity of light after passing through the sample, and Io is the intensity of light initially shone on the sample. Transmittance is usually reported as a percentage:

[%T=(I/Io) x 100] Absorbance (also called optical density or OD) is defined as

A=log10(1/T)

If 100% of the light is transmitted through the sample, A=log1.0=0, so no light is absorbed. There are no units for absorbance. Absorbance is generally plotted on the y-axis and the wavelength of light at which the measurement was taken or the concentration of the solution is plotted on the x-axis.

The absorbance of a sample can be correlated to the length of the light path (the diameter of a test tube or width of a cuvette holding the sample) and the solute concentration. The Beer- Lambert Law describes this relationship: A = Elc

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

where A is the absorbance, E is the absorption coefficient (absorptivity or extinction coefficient), l is the length of the light path, and c is the concentration of the light absorbing substance in the sample. The absorption coefficient for a molecule can be found in the published literature or determined experimentally. The absorption coefficient for biological molecules is the slope of the line plotted for absorbance versus concentration of the molecule in a chamber with a 1 cm path length at a specified wavelength and in a specified solvent. This plot is a standard curve that can be used to determine the concentration of a solution for which an absorbance reading has been taken. The Beer-Lambert Law is not applicable for ionizing compounds and extremely high concentrations of solutions.

Care must be taken in choosing the appropriate sample chamber for the spectrophotometer. Quartz cuvettes can be used throughout the UV and visible spectrum (~200-800 nm). Glass absorbs light in the UV range and therefore cannot be used below 340 nm. The Spectronic 20 does not measure below 340 nm, so glass test tubes will be the chamber of choice for samples when using this instrument.

Before measuring the wavelength of the sample of interest, the transmittance of light should be calibrated to 100% (zero absorbance) for a “blank” sample. The blank is a solution that contains everything except the one component of the sample solution that is to be measured. By “blanking” or “zeroing” the machine, any absorbance of light by the solution or the cuvette is subtracted from the sample. Any time a different substance is to be measured or the wavelength at which the measurements are taken is changed, the machine must be “zeroed” again.

To measure absorbance at a specific wavelength, light from a broad range tungsten-halogen bulb (340-800 nm) is passed through a prism or grating (the Spec 20 uses diffraction grating). The prism or diffraction grating is used to select for a specific wavelength of light. After passing through the prism or diffraction grating, the light is focused through a series of slits, lenses, filters, and mirrors that concentrate the light, increase the purity of the desired wavelength, and focus the light on the sample. The light next passes through the sample and the light intensity is detected by a photomultiplier tube or photodiode. The signal is amplified and converted to an electrical signal that is reported by a recorder or meter as either % transmittance and/or absorbance.

Figure 3. The inner workings of a typical single-beam spectrophotometer (the Spectronic 20 is an example of this type of equipment) [http://www.sci.sdsu.edu/TFrey/Bio750/UV-VisSpectroscopy.html]

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

The Visible Light Spectrum The visible spectrum of light has a range of wavelengths of 400-780 nm (Figure 4). Light is electromagnetic radiation, and these different wavelengths can be separated from each other by passing the light through a prism. The prism causes different wavelengths to diffract differently while passing through. The wavelength of an electromagnetic wave is the distance (in meters, centimeters, or nanometers) between two points on a wave (e.g. crests or troughs). Another useful measure of waves is their frequency, which is the number of wavelengths that cross a particular point per unit of time. The unit for frequency is herts (Hz), which is the inverse of seconds. Red light has the longest wavelength while violet light has the shortest wavelength. Since frequency and wavelength are inversely related, light with longer wavelengths has a lower frequency and light with shorter wavelengths has a higher frequency.

Figure 4. The Visible Light Spectrum. (https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/uv-vis/spectrum.htm )

The range of wavelengths that comprise visible light is what allows us to see different

colors. All colors of light together appear as white light. When white light is shone on a substance or liquid that absorbs specific wavelengths of light, it appears to have a specific color. Different materials absorb different wavelengths of light, depending on their chemical composition. Human eyes are detecting the light that is not absorbed by the material, and that is the color we use to describe the material.

Why is ONP yellow?

What color(s) of light does ONP absorb?

What wave length(s) might you choose to measure ONP? nm

What wave length(s) would you not choose to measure ONP? nm

Why?

Procedure 1. Measuring absorbance accurately with a spectrophotometer:

In order to use the spectrophotometer to measure the concentration of a substance you will take advantage of the fact that the amount of light absorbed is linearly proportional to the concentration of the substance. Therefore, you will be concerned with something called "Optical Density" (OD): a measure of how effective the substance is at blocking (being dense to) light. High

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

optical density corresponds to strong absorbance, and strong absorbance is the consequence of high concentration. In order to make accurate measurements with a spectrophotometer, there are two main factors to consider: 1. You must set the limits of possible measurements:

a. One limit corresponds to infinite absorbance. That is, none of the light is transmitted. You will use a mechanical shutter inside the spectrophotometer to block all of the light, and thus simulate infinite absorbance. This is done with the "0 Set" button ("0" in this case corresponds to 0% transmittance of light). With the "0 Set" button depressed, set the absorbance to infinity (% transmittance = 0).

b. The other limit corresponds to no absorbance. That is, all of the light is transmitted. You will use a solution that contains everything except the substance you want to measure; this is your "blank", to set the baseline absorbance to 0. Your blank should be 3 ml of buffer solution with enzyme added. Because there is no substrate present, no product will be produced. By setting absorbance to 0 for the blank, only the newly produced material will be sensed by the machine when you do a real experiment with substrate present.

2. You must minimize extraneous influences on light readings.

a. To do this, you should take the following precautions: mark the tubes so that they are always read in the same orientation, wipe the tubes before placing them in the spectrophotometer, and always close the lid of the sample chamber when taking a reading. These steps will reduce variation due to unevenness of the glass or dirt on the tubes.

b. Practice reading the Optical Density of the colored tubes provided. First, estimate the relative ODs by using your eyes as a spectrophotometer. You may be surprised at how good they are!

Rank the lettered tubes (A-E) according to decreasing "eyeball OD".

Highest eyeball OD -------------------------------------------> Lowest eyeball OD Now, use the spectrophotometer to measure the Optical Density of the same set of tubes. (the wavelength will depend on the color of the solution). Repeat measurements 3 times for the whole set; use water as a blank.

Highest OD -------------------------------------------------------------> Lowest OD

O.D. Tube A Tube B Tube C Tube D Tube E

1st trial 2nd trial 3rd trial

Are your spectrophotometer results reproducible? Do the measurements made with the spectrophotometer agree with your eyeball measurements?

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

Procedure 2: Assaying the Activity of β-galactosidase By monitoring the appearance of product, ONP (yellow color), you will examine the time

course of the enzyme catalyzed conversion of ONPG to ONP. Qualitative measurement: Here we are using a concentrated solution of substrate to make the color change easier to see.

1. Label two tubes, one as “blank” and the other one as “reaction” 2. Add 3 ml of reaction buffer to the blank tube. 3. Add 3 ml of 2 mM ONPG solution to the reaction tube. 4. Add 50 L of enzyme (2U) to each tube. 5. Cover the tubes with a piece of parafilm and mix the solutions by inverting the tubes. 6. Watch closely over several minutes – record the time it takes for the reaction to start.

When was the yellow color first visible? Compare the tube to your blank. Approximately, for how long did the mixture continue to become more yellow? Quantitative measurement:

This exercise is designed to help you understand how the rates of reactions are determined using spectrophotometric data.

1. Set the spectrophotometer to 420 nm, which is the optimum absorbance wavelength for ONP.

2. Label 3 (13x10) glass test tubes as: 1, 2, 3.  Remember to wear gloves to keep the outside of the tubes clean. You may wipe the

tubes with a KimWipe prior to introducing them in the spectrophotometer. 3. Add 3 mL of 0.2 mM ONPG to tubes 1, 2, and 3. 4. Add 50 L of β-galactosidase to the tubes. 5. Set the baseline absorbance to zero in the spectrophotometer using the blank tube.

 Use the blank tube prepared in the previous exercise. 6. Measure optical density (absorbance) for each tube in 1 minute intervals, for 10 min.

Data Analysis: Once all data has been collected, the data must be analyzed to determine if the experimental group is significantly different from the control group. A useful and common tool is the Student’s T-Test to compare two sets of data. The goal of the T-test is to determine if any differences between the two groups are due to chance or differences in the variables of the experiment. By performing a T-test, you are calculating a t value, and every t value has a corresponding P value. The P value indicates if the null hypothesis is correct. A larger P value indicates the null hypothesis is true, while a small P value indicates that the difference between the two groups is not due to chance and the null hypothesis is false. Further explanations on basic statistics and the t-test can be found online: https://www.graphpad.com/guides/prism/7/statistics/index.htm?usingstatistical_analyses_step_b y_s.htm

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

References:

1. Boyer, Rodney. Modern Experimental Biochemistry (3rd Edition) New York: Addison, Wesley, Longman Publishers, p.141-155. 2000.

2. Filmore, David. Seeing with Spectroscopy. Enterprise of the Chemical Sciences. p. 87-91. 3. Freeman, S., Quillin, K., Allison, L., Black, M., Podgorski, G., Taylor, E., and J. Carmichael.

2017. Biological Science, Sixth Edition. Pearson. 4. Chandler, V., Donovan, S., Goodwin, W., Sprague, K. and C. Stiefbold. 1998. Enzyme Kinetics.

Tested Studies for laboratory teaching, Vol 19, p. 81-97.

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

Data Sheet: Name: PS ID: In this experiment, we will be comparing replicates of the same enzymatic reaction. State your hypothesis: State the null hypothesis: 1. Absorbance data for the production of ONP.

Time O.D.420 Tube 1 O.D.420 Tube 2 O.D.420 Tube 3 Mean O.D.420

1 min

2 min 3 min

4 min

5 min

6 min

7 min

8 min

9 min

10 min

2. Use the grid below to plot your data. Label the axes appropriately.

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

3. Label the part of the curve where the rate of the reaction is highest.

4. Label the part of your curve where the rate of the reaction is lowest.

5. The highest rate is (estimate value, include units)

6. The lowest rate is (estimate value, include units)

7. Re-analyze your data—calculate the rate of the reaction for each 1 minute interval.  Rate(x) = OD/minute  Rate mean(�̅�): average of the rate for each time point for the 3 replicates

 SD – standard deviation, 𝑠 = √ ∑(𝑥−�̅�)2

n−1 , where n = 3

Time Rate Tube 1

(x1) Rate Tube 2

(x2) Rate Tube 3

(x3) Rate Mean (𝒙) S.D. (s)

1 min 2 min 3 min 4 min 5 min 6 min 7 min 8 min 9 min

10 min

8. Use the grid below to plot your data. Label the axes appropriately.

Ana Medrano, Ph.D., Donna Pattison, Ph.D., & Rita E. Sirrieh, Ph.D. BIOL 1161 Lab

9. Statistical Analysis:

Once you have calculated the mean and the standard deviation, you will calculate the standard error of the mean (SEM) using the equation:

𝑺𝑬𝑴 = 𝑺𝑫

√𝒏

Finally, you will use the GraphPad online calculator to apply the Student’s T-Test to your data (https://www.graphpad.com/quickcalcs/ttest1/ ). As mentioned above, the T-Test will tell you if there is a significant difference between your data sets. You will need to enter the mean, SEM, and n value into the calculator. Then, select a paired t-test, and click calculate. The result will provide you with the t value, df (degrees of freedom), and P value. The df is used to correlate the t value to the P value.

10. Drawing conclusions: a. Does your P value indicate that your two means are sufficiently different from one another

that the difference is probably not due to chance?

b. Do you accept or reject your null hypothesis?

c. Briefly, what is your group’s conclusion about your experiment and original question you posed about your experimental system?

11. Do all time intervals provide equally reliable measurements of the reaction rate at 0.2 mM ONPG? Why or why not?