flashcard 16

Question

Provide an example of enzyme inhibition used therapeutically.

Answer 1

Disulfiram inhibits aldehyde dehydrogenase, causing accumulation of acetaldehyde after alcohol intake, producing unpleasant effects that discourage alcohol consumption.

Answer 2

Because the prodrug requires metabolic conversion (e.g., by CYP2C19) to its active form; inhibition prevents that conversion, rendering the drug ineffective (e.g., omeprazole inhibiting clopidogrel activation).

Answer 3

Ethnicity (genetic polymorphisms), age (enzyme activity declines with age), pregnancy (increased cardiac output, GFR), and disease states (e.g., liver or kidney impairment).

Answer 4

Polymorphisms in metabolic enzymes (e.g., CYP2D6 poor metabolizers) and plasma cholinesterase deficiency affecting succinylcholine metabolism or slow/fast acetylators affecting isoniazid clearance.

Answer 5

Separating components of a mixture based on their differential affinities for a stationary phase (solid or liquid) and a mobile phase (liquid or gas) passing over or through the stationary phase.

Answer 6

Gas chromatography (GC), liquid chromatography (LC/HPLC), and thin-layer chromatography (TLC).

Answer 7

Ion-exchange chromatography and affinity chromatography (including hydrophobic interaction chromatography and chromatofocusing).

Answer 8

Detecting blood or accelerants in arson investigations (GC), monitoring pesticide residues in food (LC-MS), and verifying authenticity of products (TLC or HPLC fingerprinting).

Answer 9

Mobile phase: inert carrier gas (e.g., helium); stationary phase: thin liquid film coated on the inside of a capillary column or packed in a column.

Answer 10

A separation technique using a supercritical fluid (e.g., CO₂ above its critical temperature and pressure) as the mobile phase, combining gas-like diffusivity with liquid-like solvation to separate small molecules.

Answer 11

UV/Vis detection measures absorbance at a single selected wavelength; DAD captures absorbance across a range of wavelengths simultaneously, allowing spectral profiling but at higher cost.

Answer 12

Time-of-flight (TOF): separates ions by flight time in a field-free region; Triple quadrupole: uses three quadrupoles for precursor ion selection, collision-induced fragmentation, and product ion analysis; Orbitrap: traps ions in an electrostatic field, measures oscillation frequencies for high-resolution mass spectra.

Answer 13

Column dimensions (length/width), particle size of packing (smaller particles increase resolution), adsorbent activity, column temperature (higher temperature accelerates elution but can affect selectivity), packing quality, and solvent quality (low viscosity improves efficiency).

Answer 14

1) Extraction (lysis, homogenization, solubilization), 2) Fractionation (precipitation, centrifugation), 3) Purification strategies (chromatography: ion exchange, size exclusion, affinity, HIC, chromatofocusing), 4) Concentration (ultrafiltration, lyophilization), 5) Yield assessment and analysis (SDS-PAGE, Western blot).

Answer 15

Proteins bind to oppositely charged groups on the resin based on their net charge at a given pH; elution is achieved by increasing ionic strength or changing pH to weaken protein-resin interactions (gradient elution).

Answer 16

Also called gel filtration, it separates proteins by size: large molecules bypass resin pores and elute early (void volume), while smaller molecules enter pores and elute later, with retention volume related to molecular weight.

Answer 17

A specific ligand immobilized on a matrix, often via a spacer arm to reduce steric hindrance; the target protein binds selectively to the ligand, then is eluted by changing buffer conditions or adding free ligand (step or gradient elution).

Answer 18

HIC uses a hydrophobic resin under high-salt conditions to strengthen hydrophobic interactions; decreasing salt concentration weakens these interactions and elutes proteins in order of increasing hydrophobicity.

Answer 19

A form of ion-exchange chromatography where proteins are separated based on isoelectric point by creating an in-column pH gradient; proteins elute when the local pH equals their pI, enabling high-resolution separation.

Answer 20

Dialysis removes small contaminants (salts, free ligands) by diffusion through a semipermeable membrane; desalting columns or spin filters rapidly exchange buffer to remove unwanted small solutes.

Answer 21

SDS denatures proteins and coats them with uniform negative charge (≈1.4 SDS molecules per amino acid), masking intrinsic charge and unfolding them into rodlike shapes so migration through the polyacrylamide gel depends primarily on molecular weight; smaller proteins migrate faster.

Answer 22

IEF separates proteins by isoelectric point: a pH gradient is established in a gel, and each protein migrates until it reaches the position where the gel pH equals its pI (net zero charge), allowing precise determination of pI.

Answer 23

A technique combining IEF (first dimension, separation by pI) and SDS-PAGE (second dimension, separation by molecular weight) to resolve complex protein mixtures into individual spots for proteomic analysis.

Answer 24

Proteins separated by SDS-PAGE are transferred to a membrane, blocked, then probed with a primary antibody specific to the target protein followed by an enzyme- or fluorophore-conjugated secondary antibody; detection (chemiluminescence or fluorescence) reveals presence and relative abundance.

Answer 25

High sensitivity and specificity (down to picogram levels), high throughput (96-well format), quantitative results, and ability to test various sample types (serum, plasma, urine, saliva).

Answer 26

Temporary readouts due to enzyme/substrate kinetics requiring timely measurement, limited antigen information (only indicates presence or concentration, not structure), and potential cross-reactivity if antibodies are not highly specific.

Answer 27

Manual Sanger: four separate PCR reactions with single ddNTPs, run on large slab polyacrylamide gels, slow, labor-intensive; Automated capillary: all ddNTPs mixed with unique fluorescent labels in one reaction, separated in narrow capillaries by automated machines with laser detection, faster, higher resolution, and automated readout.

Answer 28

Absorption spectroscopy measures the decrease in light intensity at specific wavelengths as light passes through a sample; absorbance (A) is proportional to concentration (c) and path length (l) according to Beer–Lambert law (A = ε·c·l).

Answer 29

Light scattering (turbidity), reflection, refraction, matrix effects, high absorbance saturating the detector, and chemical interactions altering the absorptivity ε.

Answer 30

NADH has a strong absorbance peak at 340 nm (ε ≈ 6,220 M⁻¹·cm⁻¹), whereas NAD⁺ does not. Monitoring the change in absorbance at 340 nm allows quantification of NADH formation or consumption over time.

Answer 31

Oxyhemoglobin and deoxyhemoglobin have distinct absorbance spectra: the Soret band of oxyhemoglobin peaks near 415 nm with characteristic visible peaks, while deoxyhemoglobin’s peaks are shifted; comparing absorbance at specific λ max values differentiates them.

Answer 32

Photoluminescence is light emission from a molecule after absorption of photons; it encompasses fluorescence (prompt emission with lifetimes of 10⁻¹⁰–10⁻⁸ s) and phosphorescence (delayed emission from a triplet state with lifetimes of 10⁻³–10² s).

Answer 33

The quantum yield is the fraction of excited molecules that return to the ground state via fluorescence rather than nonradiative pathways.

Answer 34

The quantum yield is the fraction of excited molecules that return to the ground state via fluorescence rather than nonradiative pathways, ranging from 0 (no fluorescence) to 1 (all excited molecules fluoresce).

Answer 35

Excitation spectrum: fix emission wavelength and scan excitation wavelengths, monitoring emitted intensity; Emission spectrum: fix excitation wavelength and scan emission wavelengths, recording intensity. Both spectra are corrected for lamp intensity and detector response.

Answer 36

Higher sensitivity (detecting much lower analyte concentrations), greater selectivity (through distinct excitation/emission wavelengths), ability to probe molecular environments (e.g., polarity), and utility in imaging techniques like fluorescence microscopy.

Answer 37

The Stokes shift is the difference between the peak excitation wavelength and the peak emission wavelength of a fluorophore, arising because excitation energy partially dissipates nonradiatively before emission, causing emission at longer wavelengths.

Answer 38

FRET is a distance-dependent energy transfer from an excited donor fluorophore to an acceptor molecule (fluorophore or quencher) within ~1–10 nm, requiring overlap between donor emission and acceptor absorption; FRET efficiency reports on molecular proximity.

Answer 39

FRAP measures molecular diffusion: a region of a fluorescently labeled sample is photobleached, then recovery of fluorescence (due to diffusion of unbleached fluorophores into the region) is monitored to quantify mobility or membrane fluidity.

Answer 40

Bioluminescence is light emission from a biochemical reaction without external excitation; luciferase is commonly used to catalyze oxidation of luciferin (with ATP and O₂) to produce light, employed as a reporter for gene expression.

Answer 41

The promoter of interest is cloned upstream of the luciferase gene; when cellular factors activate the promoter, luciferase is expressed. Adding luciferin substrate produces light proportional to promoter activity, measured by a luminometer.

Answer 42

Chemiluminescence is light emission from a chemical reaction; luminol reacts with hydrogen peroxide (catalyzed by iron in hemoglobin) to form an excited intermediate that emits blue light upon returning to the ground state, revealing trace blood at crime scenes.

Answer 43

Agarose gels (0.5–2% agarose) for separating large DNA/RNA fragments in their native form; polyacrylamide gels (SDS-PAGE) for high-resolution separation of denatured proteins or small nucleic acids; capillary electrophoresis for high-resolution, rapid separation (e.g., DNA sequencing, small metabolites).

Answer 44

Capillary electrophoresis uses narrow, buffer-filled capillaries and an electric field to separate analytes with high efficiency and speed, detecting fluorescently labeled fragments automatically; slab gels are larger, manual, and require staining or imaging.

Answer 45

After photon absorption (excitation to a higher energy state), molecules relax back to the ground state via internal conversion (nonradiative) or emission: fluorescence (singlet excited → singlet ground, fast) or phosphorescence (triplet excited → singlet ground, slow due to spin-forbidden transition).

Answer 46

The excitation spectrum plots fluorescence intensity while varying excitation wavelength at a fixed emission wavelength (similar to absorbance). The emission spectrum plots intensity of emitted light while varying emission wavelength at a fixed excitation wavelength.

Answer 47

Turbidity causes light scattering, which diverts excitation and emission photons out of the detection path, reducing measured fluorescence intensity and distorting spectral data.

Answer 48

Circular dichroism measures differential absorption of left- versus right-circularly polarized light by chiral molecules, providing information on protein secondary structure (α-helix, β-sheet content).

Answer 49

By using point illumination and a pinhole to reject out-of-focus fluorescence, confocal microscopy collects sharp optical sections at various depths, enabling 3D reconstruction with high contrast and resolution.

Answer 50

Approximately 800–2,500 nm (12,500–4,000 cm⁻¹), capturing overtone and combination vibrational bands useful for compositional analysis of liquids, solids, and biological samples.

Answer 51

Water strongly absorbs red light (>600 nm); because longer red wavelengths are absorbed, only shorter (blue) wavelengths transmit through sufficiently in large bodies, making water appear blue.

Answer 52

D₂O has vibrational absorption bands shifted to longer wavelengths (lower wavenumbers) compared to H₂O; measuring absorption around 2,000 nm can differentiate heavy water from normal water.

Answer 53

It requires well-ordered protein crystals, and many proteins are difficult or impossible to crystallize, precluding structural determination by X-ray methods.

Answer 54

NMR yields chemical shift and coupling data for NMR-active nuclei (¹H, ¹³C, ¹⁵N, ¹⁹F, ³¹P), allowing determination of atomic-level structures in solution, measurement of interatomic distances, and monitoring dynamics (folding, ligand interactions).

Answer 55

NMR analyses proteins in solution, enabling observation of conformational changes, folding/unfolding, and protein–ligand interactions in a near-physiological environment, whereas X-ray crystallography only reveals static, crystalline structures.

Answer 56

Absorbance assays measure enzyme activities and metabolite concentrations (e.g., NADH at 340 nm, bilirubin, creatinine); fluorescence assays quantify low-abundance biomarkers; chemiluminescent immunoassays detect hormones and proteins with high sensitivity.

Answer 57

Review lecture slides and practical notes for each technique (TDM, chromatography, electrophoresis, spectroscopy), understand principles and clinical applications, memorize analyte panels (LFTs, electrolytes, cardiac markers), and practice interpreting QC charts, drug interaction scenarios, and example questions.

flashcard 16

(81 cards)