BIOL 1010 Winter 2013 LAB # 2 ENZYMOLOGY: Activity of turnip peroxidase Introduction

Objectives:
Improve knowledge of enzyme kinetics and investigate the factors affecting enzyme activity. Learn how to make graphic representation of your data and calculate reaction rates. Learn the basic principle of spectrophotometry and gain experience using a spectrophotometer. Learn how to use a micropipettor Learn how to write hypothesis.

Enzymes:
Biochemical reactions in all living organisms are catalyzed (speed-up) by enzymes, the majority of which are protein molecules. In an enzyme-catalyzed reaction, the substrate binds to the active site of the enzyme and is converted into the reaction products in a process often requiring several chemical steps. The products are released into solution and the enzyme is ready to form another enzyme-substrate complex and to start another catalytic cycle. Enzymes are specific for their substrate and are also reaction specific. This specificity is due to the nature of enzymes active site and its overall three-dimensional structure. Any substance that changes the shape of the active site will interfere with the activity and efficiency of the enzyme. Substances that block or reduce enzymatic activity are called inhibitors; substances that enhance enzymatic activity are called activators. Environmental conditions can also cause enzymes to become inactivated due to the disruption and unfolding of their three-dimensional shapes. This unfolding is called denaturation. Therefore, factors affecting enzyme activity include temperature, pH, concentration of enzyme, concentration of substrate, concentration of products, and the presence of inhibitors or activators. Enzymatic rate: Enzyme activity is experimentally determined by measuring the rate of substrate disappearance or the rate of product appearance over time, whichever is easier to measure. Substrate disappearance or product appearance is usually determined via a colorimetric reaction that is measured using a spectrophotometer (see below). Typically, the curve of product appearance (or substrate disappearance) versus time is initially linear and reaches a plateau when the substrate(s) become limited. Enzymatic rate is simply calculated by determining the slope of the linear portion of the curve ( = absorbance of product appearance/disappearance divided by time).

As mentioned earlier, experimental factors such as the amount of enzyme or the amount of substrate(s) as well as environmental factors such as pH and temperature directly affect enzymatic rate. o It is rather intuitive to predict that an increase in the concentration of substrate(s) will increase enzyme reaction rate in a linear fashion as long as there is enough enzyme present. If the amount of enzyme becomes limited the reaction rate will reach a maximum. Likewise, an increase in the concentration of enzyme will increase reaction rate as long as enough substrate is present. o Environmental factors affecting enzyme activity include pH and temperature. Note that different enzymes will react differently to changes in these environmental factors. Effect of pH: most enzymes have an optimum pH range within which they function most efficiently. Outside this range, pH affects the three-dimensional shape of the enzyme, resulting in the unfolding of the protein, a process known as denaturation, which results in the inactivation of the protein. Effect of temperature: enzymes are also very sensitive to temperature. Enzyme activity tends to increase as the temperature increases, up to a certain temperature. At some point, however, temperature destroys the structural integrity of the enzyme which causes a sharp decline in enzyme activity as the temperature continue to increase.

In this lab, you will determine the enzymatic rate of the enzyme peroxidase for its substrate hydrogen peroxide, as well as the effect of substrate concentration and pH on enzymatic activity.

Peroxidases:
In this exercise you will extract the enzyme peroxidase from turnips. Peroxidases are common in plant and animal cells. Cells using molecular oxygen in their metabolism generate small amounts of hydrogen peroxide (H2O2) which is very toxic. Peroxidases (and other enzymes) speed up the conversion of H2O2 to water in order to prevent cells damage. The reaction catalyzed by peroxidase 2

enzymes is an oxidation/reduction reaction and therefore takes place in the presence of a reducing agent; the reaction can be symbolized as follows:

RH2 + H2O2 + Peroxidase

R + 2 H2O + Peroxidase

- RH2 is the reducing agent (causes another substance to be reduced, in this case hydrogen peroxide) and acts as hydrogens and electrons donor - R is the oxidized form of the molecule In our experiment, guaiacol is used as the reducing agent and peroxidase activity is assessed by following the formation of oxidized product (tetraguaiacol) which is brown in color. Overall the catalyzed reaction can be symbolized as follows:

4 guaiacol + 2 H2O2

tetraguaiacol + 8 H2O
(Brown)

The activity of the enzyme can be determined by following how much product is produced, therefore how intense the brown color is. The appearance of a colored product and therefore enzyme activity can be measured using a spectrophotometer.

Spectrophotometry:
Visible light is an electromagnetic energy made of various wavelengths ranging from 380 nm to 750 nm. We perceive these wavelengths as different colors. As light meets matter, the light can be reflected, transmitted or absorbed. Substances that absorb visible light are called pigments. Proteins and nucleic acids absorb light in the ultraviolet range. A molecule absorbs wavelength of light in a characteristic pattern. In general you can make a good guess about a solution's absorbance pattern by looking at the color of the solution: the color of a solution is the color that is the most reflected or transmitted and therefore is the color NOT absorbed. For example, a solution of chlorophyll is green because it reflects and transmits green light, while absorbing all the other colors. However, this does not mean that all greens are identical, or that the absorbance of other colors is necessarily complete.

A spectrophotometer is made of the following components: Light source: tungsten filament lamp that produces white light. Prism: separates the white light beam into the different wavelengths or colors of light. Narrow slit: used to select one particular wavelength of light that passes through the sample (incident light, IL). Sample holder: keeps the sample you are measuring in the proper orientation. Light detector: photosensitive cell that measures the light that passes through the sample (transmitted light, TL). The energy received at the photocell is converted into a voltage fluctuation, which is then displayed on a meter scale.

When light passes through a solution some of it will strike molecules and be absorbed, thus the amount of light coming out on the other side is less than the amount that entered. Note that there are two ways of looking at this: you can either think about how much light makes it through the solution (transmittance) or how much light gets absorbed by the molecules (absorbance). Clearly the two are related, however, they are measured differently: Transmittance is expressed as the percentage of light that passes through (% Transmittance or %T): TL % T T x 100 x 100 IL Absorbance (A) is expressed as:

A - Log (T) 2 - Log (%T)

So, if transmittance is 100%, then A=0. If transmittance is 1%, then A=2. When light passes through a solution, there are three sources of absorbance: the container (usually a clear glass test-tube or cuvette), the solvent which could be water or a buffer, and the dissolved molecules. In most cases it is only the amount of dissolved molecules that you want to measure. To ensure that the container and solvent do not interfere with your readings we use a 'blank'. A blank is a tube which contains only the solvent without the dissolved molecules you want to measure. It is

used as a reference point. By defining the amount of light that passes through the blank as 100% transmission or A = 0, any reduction in the transmission or any increase in absorbance after the light passes through the sample tube is due to the dissolved molecules of interest. Two factors determine the amount of light absorbed: the length of the light path through the solution. the concentration of molecules in the solution.

The length of the light path is always the same since we always use the same size of tubes or cuvettes. The effect of concentration is usually what you want to determine. When plotting the relationship between % Transmittance and Absorbance versus concentration, it is observed that absorbance rises linearly with concentration, while transmittance decreases in an exponential fashion (see picture below).

The linear relationship between absorption and concentration is described mathematically by the Lambert-Beer law:

A = cl
With: - A: absorbance - : extinction coefficient (in L mol-1 cm-1); is a measure of the amount of light absorbed per unit of concentration and is the inverse of the slope of the line. - c: concentration (mol L-1) - l: light path, in cm (usually = 1 cm) It is this linear relationship between absorbance and concentration that makes absorbance useful in the lab; this linear relationship means that if a solution shows twice the absorbance of another, then its concentration is twice as high. Note that the Law is not obeyed at high concentrations of solute; at high solute concentration, absorbance versus concentration is not linear and the law is not valid anymore. 5

Micropipettors:
Micropipettors are used to accurately measure small volumes of solution typically from 0.5 L to 1000L (= 0.0005 mL to 1 mL). They consist of a barrel, which houses a spring loaded-piston attached to a plunger on the top of the pipettor. There are four types of pipettors depending on the volume of sample you want to measure. P2 is used to measure volumes between 0.5-2 L P20 is used to measure volumes between 2-20 L P200 is used to measure volumes between 20-200 L P1000 is used to measure volumes between 100-1000 L.

Micropipettors must be used in conjunction with plastic disposable tips. The liquid is actually drawn into the tip, not into the barrel. There are three types of tips depending on the size of the pipettor; P2 uses the small tips; P20 and P200 uses the medium size tips; and P1000 uses the larger tips. A window at the front of the barrel indicates the selected volume of liquid that the pipettors will measure. The volume can be adjusted by turning the adjusting ring. The volume is read using the three numbers in the window as follows: Pipettors used: P2 2 0 0 Volume measured: 2.0 L P20 2 0 0 20 L P200 2 0 0 200.0 L P1000 1 0 0 1000.0 L

These volumes correspond to the maximum volumes each of the pipettors can measure. The micropipettors are research grade instruments and therefore are very expensive. Please use them appropriately and handle them with care. Do not drop them. Do not exceed the minimum and maximum volumes.

Please watch the video (link below) before coming to class to learn important information about proper pipetting techniques. Please note that there will be some questions in the quiz on information mentioned in the video. http://www.youtube.com/watch?v=uEy_NGDfo_8&feature=related

Writing hypothesis:
Performing laboratory experiments requires proper planning and preparation. It first starts with the observation of a phenomenon involving two events that seems somehow connected. For example, you notice that in November many trees undergo color changes in their leaves and that the average daily temperatures are dropping. Are these two events connected? How? The next part is to formulate a possible explanation on how these two events are related. This is what we called a HYPOTHESIS. The main problem students encountered when writing a hypothesis is that they merely state a prediction or a conclusion. For example, when writing something like "Trees will change color when it gets cold."; you are only making a prediction. Or if you write, "Ultraviolet light causes skin cancer.", this sounds more like a conclusion. In other word, these statements fail to include the predicted cause and effect that connect the two events and make up a proper hypothesis.

To avoid such mistakes, it is best to write a hypothesis as a formalized hypothesis. A formalized hypothesis is a if-then type of sentence. For example, If leaf color change is related to temperature , then exposing plants to low temperatures will result in changes in leaf color. Note that not all If-then sentences are hypothesis. For example, "If I play the lottery, then I will get rich." This is a simple prediction. A formalized hypothesis will instead look like this: if the frequency of winning is related to frequency of buying lottery tickets, then increasing the frequency of buying lottery tickets will also increase my chances of winning.

So what are the ingredients for a good hypothesis?

Let consider the following hypothesis: "If smoking causes lung cancer, then individuals who smoke have a higher frequency of developing the disease." 1. Your statement must contain two variable; an independent variable (the one you can control; in red) and dependant variable (the one you will measure/observed; in blue). 2. Make sure that your statement contains a subject group defining who or what the scientist will be studying (e.g. persons smoking). 7

3. Include a treatment to which the subject group will be exposed to in the experiment (e.g. exposure to smoke or smoking). 4. Finally mention how the outcome will be measured (e.g. frequency of people developing of the disease). Remember that your statement must be testable to constitute a hypothesis. The ultimate value of a formalized hypothesis is it forces you to think about what results we should look for in an experiment before performing the experiment. The normal course of action is to first stated a hypothesis, and then design an experimental procedure to test whether your statement is correct or not. In this experiment, you will be given an experimental procedure and you will be asked to write a formalized hypothesis based on the design of the experiment you will be performing.