BIOS 113 Practicum 2 Study Guide Spring 2025

Enzyme

Usually proteins, but not always, that increase the rate of chemical reactions without being consumed. Speed up reaction by lowering activation energy; bind to the substrate (starting molecule/reactant) and correctly orient them so they can react to form products

Activation Energy

energy required to initiate a chemical reaction, which enzymes lower

Catechol Oxidase

enzyme used to transform catechol (reactant) into Benzoquinone (product)

Active Site

physical location on enzyme that binds the substrate, specific to chemical species so enzymes can only catalyze one (or few) type of chemical reaction 

Michaelis-Menten Curve

Graph plotting enzyme velocity (mole product produced per unit time) against substrate concentration (M); Y-axis shows velocity, X-axis shows substrate concentration

Vmax

theoretical maximum velocity of an enzyme

Km

substrate concentration at which the enzyme velocity proceeds half of the Vmax

Inhibitor

Nonreactive molecules that prevent enzymes from reacting with their substrates

Enzyme activity affected by

temperature and pH affect the enzyme activity

Inhibitor used in enzyme lab

PTU (phenylthiourea)

Effect of pH on enzyme activity

pH affects ability of enzyme to bind to substrate, decreased enzyme activity or denaturation

Competitive Inhibitor

Binds to the active site of an enzyme, blocking substrate access (Vmax stays, Km increases)

Non-competitive Inhibitor

Binds elsewhere on the enzyme, altering its shape, affecting activity (Vmax decreases, Km stays), can occur with or without presence of substrate

Uncompetitive Inhibitor

Binds only to the enzyme-substrate complex, decreasing both Vmax and Km

Aerobic cell respiration

Cellular respiration using oxygen; 1 glucose (reactant) & oxygen (reactant) -→ 36 ATP & H2O & CO2

Anaerobic cell respiration

Cellular respiration without oxygen; 1 glucose (reactant) → 2 ATP & ethanol (product in plants) or lactic acid (product in animals)

Glycolysis (step 1)

Glucose is broken down into 2 pyruvate molecules (in the cytoplasm)

Pyruvate oxidation (step 2)

each pyruvate is transported into the mitochondria and converted into acetyl-CoA

Citric acid cycle (step 3)

Acetyl-CoA enters this cycle to produce NADH, FADH₂, and ATP

ETC/electron transport chain (step 4)

Uses NADH and FADH₂ to produce a large amount of ATP

CO2 Production in Fermentation

CO2 is collected over water in fermentation tube and volume of gas produced over time reflects the rate of fermentation

Mitosis

Cell division process involving prophase, metaphase, anaphase, and telophase

Synapsis

connection of homologous chromosomes in preparation for crossing over (meiosis)

Crossing Over

genetic material exchange between non-sister chromatids

Karyotyping

Organizing chromosomes by size and banding patterns to determine chromosomal abnormalities

DCPIP

A dye used to visualize the speed of reaction in mitochondrial respiration experiments; blue in solution, clear when reduced

FAD

replaced with DCPIP, oxidizer, is reduced

Succinate

reducer, is oxidized: target reaction is oxidation of succinate to fumarate in Citric Acid cycle.  

Point Mutation in hemoglobin gene (sickle-cell mutation)

Sickle-cell anemia is caused by a single nucleotide substitution (point mutation) in the beta-globin gene (HBB); This mutation changes the amino acid, leading to abnormal hemoglobin known as Hemoglobin S (HbS); This structural change causes red blood cells to become rigid and sickle-shaped under low oxygen, leading to various complications.

Restriction Enzyme

An enzyme that cuts DNA at specific recognition sites to analyze variations.

Gel Electrophoresis

Technique used to separate DNA fragments by size

Ethidium Bromide Staining

A method to stain DNA for visualization under UV light.

Punnett Square

A grid used to predict the genetic outcomes of a cross.

Catechol Oxidase experiment

catechol conducts browning reaction in fruits and vegetables; can be sped up and measured like an enzyme

Control reaction (without enzyme)

reaction without enzyme confirms that the substrate doesn’t convert into product on its own

Control reaction (without substrate)

reaction without substrate ensures that the enzyme or buffer doesn’t affect absorbance

Control reaction (without inhibitor)

reaction without inhibitor establishes maximum expected enzyme activity (positive control)

Effect of temperature on reaction velocity

lower temperature = slow enzyme activity; higher temperature = enzyme begins to denature, reduction velocity reaches its maximum

Optimum temperature

every enzyme has an optimum temperature where the reaction velocity reaches its maximum

Fermentation pathways

ethanol (plants), lactic acid (animals)

steps in cellular respiration

glycolysis, pyruvate oxidation, Krebs cycle (citric acid cycle), and ETC

Yeast fermentation

measuring CO2 production; Glucose → Ethanol + CO2 + Energy (ATP)

Mitochondrial respiration

Using DCPIP to substitute FAD in the Krebs cycle

Temperature and substrate effects on respiration rate (cellular respiration/fermentation lab)

low temp = slow; optimal temp = maximum rate; high temp = enzyme begins to denature

Mitosis - interphase (model 1)

Loose chromatin, no visible chromosomes; The cell is just doing its normal functions and replicating DNA

Mitosis - prophase (model 2)

Chromosomes have condensed and are now visible; Nuclear envelope starts to break down, spindles form

Mitosis - metaphase (model 3)

Chromosomes are lined up in the center (metaphase plate); Spindle fibers clearly attached to centromeres

Mitosis - anaphase (model 4)

Sister chromatids are being pulled apart toward opposite poles

Mitosis - telophase (model 5)

Chromatids are at the poles; Two nuclear envelopes start reforming

Mitosis - early cytokinesis (model 6)

Cell begins to pinch in (cleavage furrow forming)

Mitosis - late cytokinesis (model 7)

Almost completely split, two nuclei clearly separated

Mitosis - two daughter cells (model 8)

Full division complete; Two identical cells side by side

Meiosis I and II

Reductional division (Meiosis I) and equational division (Meiosis II)

Meiosis I - Reductional division

reduces chromosome number by half, separating homologous chromosomes, from 2n to n

Meiosis I - prophase 1

chromosomes condense, homologous chromosomes pair up, crossing over

Meiosis I - metaphase 1

paired chromosomes line up along center

Meiosis I - anaphase 1

homologous chromosomes pulled apart

Meiosis I - telophase and cytokinesis

2 haploid cells form, each with 1/2 original chromosome number

Meiosis II - Equational division

separates sister chromatids, result in 2 haploid cells and divide again, ending in 4 haploid cells

Meiosis II - prophase 2

chromosomes condense again in both cells

Meiosis II - metaphase 2

chromosomes line up in center

Meiosis II - anaphase 2

sister chromatids pulled apart to opposite poles

Meiosis II - telophase and cytokinesis

each cells splits again, 4 haploid cells

Chiasmata

X shaped sites where crossing over occurs

Homologous chromosomes

2 chromosomes with same genes but maybe different alleles

Nondisjunction (meiosis)

failure of homologous chromosomes or sister chromatids to separate during anaphase

Human traits

Inheritance patterns of cilantro taste (OR6A2): cilantro aversion gene, no mendelian inheritance, complex/polygenic inheritance (on more than one gene)

PTC tasting (TAS2R38)

mendelian inheritance, dominant

Hitchhiker’s thumb

often described as mendelian recessive, might not be mendelian, likely complex/polygenic inheritance (on more than one gene)

Genetic crosses

Understanding Mendelian inheritance for human traits: we can do punnett squares

Corn dihybrid cross

Black vs. yellow kernels, starchy vs. sweet

9:3:3:1 ratio

Expected phenotypic ratio in F2 generation (second generation)

Genotyping for Sickle-Cell Anemia via Electrophoresis

DNA isolated, restriction enzyme cuts normal and mutant DNA differently, DNA fragments are separated on an agarose gel by size (gel electrophoresis)

Polymerase Chain Reaction (PCR)

used to amplify the region of the beta-globin gene that contains the mutation

PCR amplification of the beta-globin gene

Polymerase Chain Reaction (PCR) used; Primers are designed to flank the mutation site, producing enough DNA to be analyzed further; The resulting PCR product will be used for restriction enzyme digestion

Restriction enzyme digestion: Recognizing the sickle-cell allele

typically done through blood tests, particularly hemoglobin electrophoresis or genetic testing; A restriction enzyme (e.g., DdeI) is used to distinguish between the normal and sickle-cell alleles; The sickle-cell mutation disrupts a restriction site, so the enzyme will not cut the mutated (sickle-cell) allele; The normal allele is cut by the enzyme, producing smaller fragments

Gel electrophoresis process

1) After digestion, the DNA fragments are separated by agarose gel electrophoresis; 2) Smaller fragments migrate faster through the gel, while larger fragments move more slowly; 3) This separation allows us to visualize the DNA fragments corresponding to normal, carrier, or sickle genotypes; 4) UV transilluminator and Ethidium Bromide staining: viewing DNA bands under UV light; 5) DNA is stained with Ethidium Bromide (is known to cause mutagens/ damage DNA and pose health risks, so we used flashblue stain in our experiment), which binds to DNA and fluoresces under UV light (we used regular light because our stain doesn’t require it); 6) After electrophoresis, the gel is placed on a UV light, making the DNA bands visible; 7) The pattern of bands indicates the genotype of the individual

Normal (AA)

two fragments; Restriction enzyme cuts both normal alleles at the recognition site

Carrier (AS)

three fragments; One normal allele is cut; one sickle allele is not cut

Sickle-cell (SS)

one fragment; Both alleles are mutated; enzyme does not recognize the altered site, no cut