Biology 173 - Intro Bio Lab Unit 1; Enzyme Action, Gut Microbiome, & Basic PCR

Lecture 1: Intro

Scientific Method:

  • Explore/Observe → Ask a Question → Conduct Research → For a Hypothesis → Experiment → Analyze data and Form Conclusions 

    • → Do results support hypothesis?

      • Yes → Report Results

      • No → Go back to hypothesis, retesting

Observations

  • Qualitative: recording descriptions, describing, thinking of things

  • Quantitative: numerical measurement, organized into tables and graphs

  • Hypothesis: possible explanation that leads to a testable prediction and motivates experiments

    •  More broad

    • Based on observations and assumptions

    • NOT an “educated guess:, “if-then” statement, or “if I do X, Y will occur”

    • Must be falsifiable; must make specific predictions that can be critically tested by experiments or observational studies

  • Prediction: a description of an expected outcome in the test (experimental) group and in each control group

    • Confirming a prediction supports (but does not “prove”) that hypothesis

    • Negating a prediction suggests that it may be wrong

  • Ex. 

    • Observation: When you turn it on, your desk lamp doesn’t work

    • Question: Why doesn’t your desk lamp work?

    • Hypothesis 1: Bulb is burnt out

    • Hypothesis 2: Bulb is not plugged in; it’s loose

    • Some hypotheses are untestable; supernatural and religious explanations are outside the bounds of this course

  • Ex. If a plant receives fertilizer, then it will grow to be bigger than a plant that doesn’t receive fertilizer.

    • This is NOT a hypothesis; hypothesis might be “Plant growth is affected by fertilizer.”

Forming and Testing Hypotheses

Controlled Experiment

  • A single variable is changed in the experimental group and compared to the control group

  • Independent variable: manipulated/changed by researchers

  • Dependent variable: affected in response to the independent variable

  • Controls: makes sure other factors aren’t influencing the experiment → is the independent variable really the thing influencing the results?

    • Provides a baseline value for comparison

    • Makes sure everything is working properly

  • Negative Control: variable is not changed; expect no effect

    • Ex. Placebo, Saline

  • Positive Control: variable with a known effect; expect a specific effect

    • Another cancer drug that you know works

Dilutions:

  • Ex. Making a solution that is ⅓ its original concentration; how much solution + how much water?

    • ⅓ concentration → 1 out of 3 parts

    • 1 part substance + 2 parts water = 3 total parts

    • 100µ substance + 200µ water = 300µ

  • Ex. 60% = 60/100 = 60 parts out of 100 parts

    • 60µ substance + 40µ water = 100µ total

Stepwise/Serial Dilutions:

  • Step-wise series of dilutions, where the dilution factor states the same for each step

  • Each new dilution sues the PREVIOUS solution as the source; add H2O to dilute

Spectrophotometer:

  • Instrument that measure light absorbance of samples

  • Absorbance: light absorbed by a sample

  • Transmittance: amount of light passing through a sample

    • Product is proportional to the amount of light that passes through

Ecology I: Species Interactions

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Lecture 2: Enzyme Action, Part I

Bioluminescence = a spark of energy

Enzymes & Metabolism:

  • Metabolism = the totality of an organism’s chemical reactions

  • A metabolic pathway begins with a specific molecule and ends with a product 

  • Catalyst = chemical agent that speeds up a reaction without being consumed in the reaction

  • Enzyme = catalytic protein

  • In catalysis, enzymes or other catalysts speed up specific reactions by lowering the Ea (activation energy) barrier

    • We’re talking about Gibbs free energy, we want to be at a lower energy level

  • Enzymes hasten spontaneous reactions that would occur eventually

The Activation Energy Barrier

  • It takes energy to break bonds, enzyme gives you the push to break the bonds and form new ones to get to a lower energy state

  • Reaction is exergonic, and occurs spontaneously, but the rate is slow due to Ea

    • Energy is released from the system

    • You end up at a lower energy level

    • Exergonic reaction = spontaneous because it’s easier to go down than go up

The Catalytic Cycle

  • Usually can catalyze the forward or reverse reactions

  • How does the active site lower the activation energy barrier?

    • The enzyme:

      • Orients the substrates correctly

      • Strains the bonds

      • Provides a favorable environment

  • Ex. Lucy & Chocolate Factory:

    • Substrate: chocolate + wrapper

    • Enzyme: lucy & ethel

    • Active site: their hands

    • Product: wrapped chocolate

    • How to measure the rate of the reaction?

    • Experiment 1: Measuring product formation over time

      • Count total number of wrapped chocolates each minute for ten minutes

      • [ ] = reate

      • [Enzyme] is constant (2 units)

      • [Substrate] is the speed of the conveyor belt delivering chocolates

      • Reaction Rate: Take the slope of the graph of product formed over time

        • slope = # of wrapped chocolates produced per minute

Enzyme Kinetics:

  • Enzyme kinetics = study of enzyme mechanisms through reaction rates under various conditions

    • When an enzyme is saturated, it can’t handle any more substrate

  • Think of enzyme catalysis in two parts

    • 1. find time - time to bind next sub

    • 2. processing time - time to convert substrate into product

  • High [substrate] decreases find time but does not affect process time

How to Measure Enzyme Function?

  • Determine the rate of enzyme reaction

    • Amount of substrate converted to product per unit time

    • Follow the increase in [product] or decrease in [substrate]

  • Spectrophotometry – measure light absorbance

  • Absorbance is proportional to [compound]

  • What affects reaction rate?

    • [substrate] & [enzyme]

      • Substrate concentration increase would increase reaction speed but only up to a certain point 

    • Temperature, pH

    • Reaction order

    • Efficiency of enzyme

    • Factors that affect enzyme shape

  • Polyphenoloxidase (= our “model system”)

    • Oxidizes polyphenolics to quinones

      • Orthoquinone breaks down into other compounds

      • Some of those are colored (absorb strongly at 525nm)

      • Increase in absorbance over time tells us rate (= slope)

  • Polyphenyloxidase = responsible for the reaction that turns food brown

    • Non-essential – the gene can be deleted from the plant and it looks normal/grows normally

    • Found in a lot of plants (i.e., avocado, mango, apple, potato, walnuts, and many more)

    • What Does Polyphenoloxidase Do?

      • Presumably, it is beneficial to the plant in some way

      • Causes browning/bruising in damaged part of plant

    • Is it a defense mechanism?

      • May deter herbivory (of at least some organisms). Deters the whole plant from being consumed 

      • If so, how would you test that?

Enzyme Action Lab

  • Substrate: Catechol

  • Product: Orthoquinone (brown)

  • How is it measured? 

    • Absorbance vs time

  • How do we find the reaction rate?

    • Slope of the absorption vs time graph

  • Factors influencing enzymatic activity

    • Enzyme concentration (Enzyme 1)

    • Substrate concentration (Enzyme 2)

    • Temperature

    • Inhibitors, cofactors

    • Salt concentration

Lab 2:

  • Part I- varying amounts of enzyme

    • Organize group, see effect of VOLUME of enzyme

  • Part II- varying [enzyme]

    • Improve on design of Part I

    • Dilute enzyme to see impact on rate

  • Difference between Volume & Concentration

    • How much of something/how much space it takes up

    • Concentration: amount/weight PER volume

Lecture 3: Enzyme Action, Part II

Enzyme Kinetics

  • Vmax = maximum rate of reaction

    • All active sites are full (active saturation)

  • KM = affinity of the enzyme for the substrate

    • The lower  KM = greater affinity 

    • [substrate concentration] at which the reaction rate is half-maximal 

  • Higher Vmax is better, lower KM = “better”

  • We want enzymes that are efficient; don’t have to use a lot of substrate to get the reaction to go 

    • Vmax  &  KM = quantitative measures of enzyme efficiency 

How to Measure Initial Reaction Rate?

  • Abs vs. time graph

    • Initial rate = slope over first 0-2 minutes of reaction

  • Rate vs. [substrate] graph

    • Use to determine Vmax & KM

    • Dependent on [enzyme], temp, pH, inhibitors, etc.

Optimal Conditions for Enzyme Activity

  • Each enzyme has an optimal environment in which it can function; favors the most active shape

  • Competitive inhibitors: binds to the active site

    • Competes with substrate

  • Noncompetitive inhibitors: binds to another part of the enzyme and changes its shape

    • Doesn’t compete based on same site, but prevents other things from binding

    • Shuts down the enzyme, not affected by amount of substrate

  • Non-specific inhibitors: affects ALL enzymes in the system

    • Something like a poison, completely denatures all enzymes

  • Percent Inhibition = (rate without inhibitor) - (rate with inhibitor)(rate without inhibitor)  x 100

  • Competitive Inhibitors - % inhibition varies with different substrate concentrations

    • Increasing substrate reduces % inhibition

    • Vmax stays the same

  • Noncompetitive inhibitors: % inhibition is constant throughout the substrate concentrations

    • Noncompetitive inhibitors lower the rate of vmax

Cofactors

  • Non-protein enzyme helpers

  • Might be inorganic (metal in ionic form)

    • Chelating agents can pull metal ions from solution, and inhibit enzyme function

Lecture 4: Enzyme Action, Part 3

Plant Defense

  • Organisms can control their reactions by regulating enzyme activity

  • Physical Defenses:

    • Thorns, spines, hairs

    • Thick bark, rind or cuticle, hard shell

    • Mineral crystals, silica, calcium oxalate

  • Chemical Defenses:

    • Toxins, irritants, foul taste or smell

      • Think of caffeine, capsaicin, poison ivy oils

    • Ironically, some may be consumed because of these compounds

  • Animal Partners:

    • Ants given nectar to live on plant and attack predators

  • All of these defenses have associated costs to the plant

Plant Hierarchical Organization

  • Plants evolved three basic organs: roots, stems, and leaves

  • Organized into a root system and a shoot system

  • Plant response to environment depends on those two systems

Last week:

  • The rate doesn’t double until about 12

  • The graph levels off because the enzymes are saturated

Microbiome 0 Activity:

  • The rate doesn’t double until about 12

  • The graph levels off because the enzymes are saturated

Competitive vs Noncompetitive Inhibitors:

  • Competitive:

    • Competes with substrate for reaction site

    • Lowers rate of true reaction

      • Less effective at high substrate

      • Fraction of active sites are occupied by inhibitor

    • % inhibition changes with increasing substrate concentration

    • V max will ultimately be just as high as without inhibitor

    • K M will be ___________, because it takes a higher [substrate] to get the same rate of product formation (or same proportion of active sites filled by substrate)

Noncompetitive Inhibitors

  • Changes shape of active site, crippling enzyme

  • % inhibition constant with increasing [substrate]

  • Increasing [substrate] speeds up reaction somewhat, but V max is ultimately lower, active site no longer efficient

  • _______________________K M , depending on whether substrate binding/processing is affected

  • Km: to find Km:

    • look at where vmax is:

    • Look for half of vmax

    • Look at where the substrate concentration is for this half value

Lecture 5: Guest Lecture

Plant Defense

Lecture 6: Enzyme Recap

Part IV:

  • Results of running the reaction at 0C, 20C (room temp), 45C, and 65C

  • You have to prewarm and precool the enzyme at substrate: pretreatments

    • Water has a high specific heat, so it doesn’t change temperature quickly

  • Results:

    • 45, 20, 0, 65 → fastest to slowest temperatures

  • Warmth helps the reaction run a little faster; high heats denature the enzyme

Part V:

  • Results of freezing and thawing the enzyme vs boiling it and letting it cool

  • Control = never-frozen, never-boiled

  • Results:

    • Control ≈ frozen, boiled

  • The boiled enzyme had a cloudiness meant that the protein unfurled, which made the vial cloudy and increased absorbance

    • This is why absorbance doesn’t matter → look at reaction rates

    • Boiled enzyme was denatured

Part VI:

  • Results of using potassium arsenite

    • Use 100µl of arsenite with varying substrate concentration

  • Percent inhibition should be close at each concentration

  • Arsenite inhibits very little or not at all; this is unexpected but the true result

    • This might be confused for non-competitive, but it is NOT

  • There is no inhibition happening; arsenite is not an inhibitor

  • Learned that there are no - SH groups in PPO (enzyme)

Part VII:

  • Results of checking whether para-hydroxybenzoic acid is in inhibitor

  • PHBA has a chemical structure similar to catechol

    • PHBA show partial inhibition

    • % inhibition at high substrate concentration is less than % inhibition at low substrate concentration

  • PHBA is a competitive inhibitor

Part VIII:

  • Results of using polyphenoloxidase have a metal ion cofactor (copper or iron)

    • Cofactor required for reaction to occur

  • Incube enzyme with different chelating agents (compound which tightly binds metal ions)

  • Chelators will strip the metal ions from the active site of the enzyme

    • PTU reacts with copper

    • Potassium cyanide reacts with both copper and iron

  • The enzyme has a copper cofactor for sure

    • You don’t know if the enzyme for sure has an iron cofactor

  • Need to test a chelator that only draws out iron to improve this experiment 

Part IX:

  • The effect of increasing salt

  • % inhibition is not truly appropriate, as it isn’t actually an inhibitor

    • Inhibitor has to bind to the enzyme

  • Salt denatures the enzyme

  • Should have pretreated your enzyme to allow for denaturation

  • As the concentration of salt increases, reaction rate decreases (to a certain point)

    • How is this biologically relevant?

      • We need salt to live; major ecosystems on the planet that require saltwater

      • The saltiness of the water decreases because the glaciers are melting

Gut Microbiome:

  • Most of the human GI tract microbes are in the colon

  • Four rates dictate composition of colonic microbiota

  • Gut microbiome is ALL microbes in the gut environment

    • Large intestine/colon

  • Food for the colonic microbiota = Fiber

  • What happens to fiber?

    • Fermentation

    • 1.) Hydrogen and Carbon Dioxide are common byproducts

    • 2.) methane producing microbes are present in ~20% of people

    • 1º Degraders  take fiber, and turn it into intermediate products like acetate

    • Butyrate producers take that and turn into butyrate 

  • Gasses produced by microbes in the large intestine (hydrogen (H2 ) and methane (CH4)) can be detected in breath

    • Gasses produced during microbial fermentation of fiber in colon (gold color)

    • Diffuse into blood stream

    • Released from blood into lungs

    • Exhaled in breath

  • We take a big breath for breath samples because we want what’s at the bottom of the breath