Enzyme Activity and Spectrophotometry

Enzyme Activity and Biological Processes

Introduction to Enzymes

• Enzymes are used to study biological processes.
• Today's focus: enzyme activity, specifically the breakdown of starch.
• Starch is the substrate of the enzyme alpha amylase.
• Alpha amylase is present in saliva with an ideal pH around 7.
• Function: to break down starch into sugars for energy use.

Experiment Overview

• Testing the reaction rate of alpha amylase.
• Spectrophotometers will be used, building on previous knowledge.
• Future experiments: effect of pH or temperature on enzyme activity (Wednesday).
• Temperature is more complex than pH due to timing and control issues.

The Role of Proteins

• Proteins are crucial for bodily processes and survival including speeding up reactions like breaking down molecules.
• Proteins provide support and communication to cells.
• Examples: actin filaments, microtubules in the cytoskeleton, and antibodies to fight infections.

Protein Structure

• Made up of amino acids.
• Primary structure: linear sequence of amino acids.
• Ribosomes synthesize polypeptides (another word for proteins).
• Secondary structure: amino acids form alpha helices or beta sheets, depending on amino acid properties.
• Approximately 20 natural amino acids exist.
• Tertiary structure: further folding of the protein.
• Quaternary structure: some proteins need multiple proteins to function (e.g., hemoglobin).

Enzymes: Proteins and RNAs

• Enzymes are molecules (proteins and RNAs) that catalyze reactions.
• Proteins have cavities where substrates bind to be broken down or joined.
• Examples:
  • Ribosome (protein and RNA).
  • Telomerase, which prevents chromosomes from shortening during cell division.

Enzymatic Reactions and Energy

• Enzymatic reactions can be represented in energy diagrams with the Y axis as free energy and the X axis as the reaction's progress.
• Spontaneous reactions have higher free energy in reactants than products.
• Reactions need energy to start; enzymes speed up the process.
• Transition state: equilibrium state between reactants and products, hard to isolate.
• Enzymes lower the energy required to reach the transition state.
• Energy of activation: energy input needed to reach the transition state; lowered by enzymes.
• \Delta G (free energy released/absorbed) is not changed by the enzyme.

Enzyme-Substrate Complex

• Enzyme needs to bind the substrate in its active site, forming an enzyme-substrate complex.
• Small molecular rearrangements occur for better fit.
• Enzyme forms an enzyme-product complex before releasing the product.
• Enzymatic reaction rate is affected by enzyme-substrate affinity, pH, and temperature.
• Extreme pH or temperature can denature or destroy enzymes.
• Example Questions:
  • What pH do enzymes in the stomach (e.g., pepsin) work best at? (pH 2-3)
  • What about enzymes that work in extreme heat?

Optimal pH and Temperature

• Optimal pH varies; pepsin works best at pH 2, trypsin at pH 8.
• Optimal temperature: human enzymes work best at 37 degrees Celsius.
• Heat-loving bacteria enzymes can work at 75 degrees Celsius.
• Taq polymerase is used in PCR and can survive up to 80 degrees Celsius.

Starch Breakdown and Alpha Amylase

• Studying the breakdown of starch by alpha amylase.
• -ase suffix usually indicates an enzyme.
• Amylase uses water to break down starch into multiple sugars (hydrolysis).
• Plants make starch, while humans make glycogen.
• Hydrolyzing starch provides a source of energy for metabolic reactions like ATP production.

Measuring Enzyme Activity

• Measuring changes in the starch absorbent to indirectly measure enzyme activity, using the iodine test.
• Iodine turns starch darker, lighter color indicates enzyme activity.
• Lower absorbance equals higher enzyme activity.
• The longer the enzyme interacts with starch, the more it breaks it down, lowering absorbance.
• Spectrophotometer used at a wavelength of 580nm.

Important Procedures and Reminders

• Time reactions carefully when checking enzyme activity.
• Add enzyme last to start the reaction.
• Start timers immediately after adding the enzyme.
• Add Lugol's iodine solution only when the timer stops, as it stops/slows the enzyme reaction.
• Plotting results and calculating the slope of the graph yields the reaction rate.
• Reaction rate units: something per unit time (e.g., molecules per minute).
• Select two time points on the graph before it plateaus to calculate the slope; slope = \frac{y2 - y1}{x2 - x1}
• In this experiment, the slope is negative because the graph goes down.

Experiment-Specific Procedures

• Create a blank without starch for the spectrophotometer.
• Make a starch stock solution: Combining 2.5 grams per liter of starch, you will add 1 ml of that to 9 ml of water. This produces 10 mls of a 0.25 grams per liter starch concentration to use for your experiments.
• Create two controls:
  • Negative control: no amylase to ensure reagents are working correctly.
  • Positive control: with amylase, incubate for five minutes at room temperature, then add iodine.
• Test tubes in table three: same values for each tube; vary incubation time (1, 2, 4, 6, 8, 10 minutes).
• Option to use a stopwatch to time all tubes simultaneously or time each tube individually.
• Incubation times are the x values for the graph; absorbance values are the y values.

Micropipette Practicum Information

• Reading a micropipette: read the numbers carefully to measure the correct volumes.
• Smaller micropipettes have a line in the center that represents the point.