qbm 4 Lecture 4 Study Notes - DNA Quantification, Protein Quantification, and Enzyme Assays

Lecture Overview

• DNA Quantification

• Protein Quantification

• Enzyme Assays

Laws of Absorption

• Molecules possess unique spectral properties due to individual energy levels associated with chemical bonds and atomic masses; these properties can identify specific compounds.

• Light interacts with electronic and vibrational modes of molecules, inducing quantization, which leads to absorption of light only at precise energy levels needed to transition from one state to another.

Relevant Absorbance Values

• 260 nm: Measurement for DNA and RNA concentration.

• 280 nm: Measurement for protein concentration.

• 340 nm: Can be used in the CDNB Assay and for NADH in Pyruvate Kinase/Lactate Dehydrogenase Assay.

• 595 nm: Used in the Bradford Assay for protein concentration.

• 600 nm: Measures bacterial growth in LB media.

• 750 nm: Applicable in the Lowry Assay for protein concentration.

• Notably, chlorophyll absorbs light at 465 nm and 665 nm, exhibiting a molar absorptivity of $10^5$ M−1 cm−1.

Transmittance

• Definition: The fraction of light that passes through a sample, calculated as $T = \frac{I}{I<em>0}$ where $I</em>0$ is incident light energy and $I$ is transmitted light after passing through the solution.

• The concentration ($c$) of the solution and path length ($b$) play key roles in determining the transmittance.

Absorbance

• Definition: Measures the amount of light absorbed by the sample, with the key property that absorbance (A) for homogeneous samples shows that each successive layer absorbs the same fraction of the incident light.

• Absorbance decreases exponentially relative to increased concentration and path length.

Beer’s Law

• Describes the relationship between concentration and absorbance:

  • $A = ebc$

  • $A = -\log(T)$

• Important terms:

  • $A$: Absorbance

  • $e$: Molar extinction coefficient (M−1 cm−1)

  • $b$: Path length (usually 1 cm)

  • $c$: Concentration (mol/L)

• Example applications include determining absorbance or optical density (e.g. OD600) using the relationships defined above.

Example Calculations

1. Absorbance from Transmittance: A sample with a transmittance of 30%:

   • $A = -\log(0.3) = 0.52$

2. Transmittance from Absorbance: A sample with an absorbance of 0.52:

   • $T = 10^{-0.52} = 0.30 (30\%)$

3. Concentration Calculation: If $e = 40,000$ and $A = 0.3$:

   • $0.3 = 40,000(1)(c) \rightarrow c = 0.0000075 = 7.5 \times 10^{-6} M$

4. Key Equations:

   • $A = -\log(T)$

   • $T = 10^{-A}$

   • $A = ebc$ (where $b = 1$ can be ignored).

DNA Quantification

• DNA absorbs light maximally at 260 nm:

  • $A_{260} = 1.0$ corresponds to 50 μg/ml double-stranded DNA (dsDNA), and 35 μg/ml for single-stranded DNA (ssDNA), the latter being higher due to greater absorption.

• Measurement techniques include using a spectrophotometer (in 10X or 100X dilution) or a Nanodrop (in 1 ml samples) without destruction of the sample.

DNA Purity Assessment

• Absorbance measurements at:

  • 230 nm: Indicates organic contaminants.

  • 260 nm: For DNA concentration measurement.

  • 280 nm: For protein measurement.

• The ratio of $A<em>{260} : A</em>{280}$ is crucial; samples are sufficiently pure if the ratio is greater than 1.5, translating to 50 ng/μl = 140.4.

RNA Quantification

• Similar to DNA quantification methodologies, RNA quantification measures absorbance at 260 nm with:

  • $A_{260} = 1.0$ indicating 40 μg/ml concentration.

• The purity is often assessed using the ratio $A<em>{260} : A</em>{280}$, typically requiring a higher threshold than 1.8.

• Techniques for cellular RNA quantification include:

  • Gel electrophoresis

  • Northern blotting

  • Reverse transcription quantitative PCR (RT-qPCR)

Protein Quantification

• Protein levels are quantified using absorbance at 280 nm:

  • Conventional methods include:

  • Amino acid analysis.

  • Chromogenic/colorimetric assays that generate standard curves with purified proteins (e.g., BSA).

    • Notable assays:

    • Bradford Assay

    • Biuret Assay

    • Lowry Assay (DC Protein Assay)

    • BCA Assay

  • Advantages: Reproducibility, ease, speed, and cost-effective.

  • Disadvantages: Estimates only, interference from various contaminants.

Absorbance Properties at 280 nm

• Absorption at 280 nm primarily due to amino acids tryptophan and tyrosine:

  • Most proteins show a maximum absorption around this wavelength.

  • Sample purity is critical since other proteins and nucleic acids absorb strongly at this wavelength.

  • Limitations exist for complex mixtures and detection threshold requires a relatively pure sample (>0.1 mg/ml).

Calculating Extinction Coefficient $e(280)$

• Formula:

  • e(280) = (#Trp \times 5500) + (#Tyr \times 1490) + (#Cys \text{ in disulfide bonds} \times 125)

Correction for DNA Contamination

• The formula for correction is:

  • $\text{Protein (mg/ml)} = (1.55 \times A<em>{280}) – (0.76 \times A</em>{260})$

Amino Acid Analysis

• The most reliable method for determining pure protein concentration through:

  • Acid hydrolysis followed by amino acid separation and quantification via HPLC.

  • Considerations:

  • Requires high purity of protein, special equipment, and significant time (2-5 days turnaround at ~$250/sample).

  • Removal of buffers is paramount.

Colorimetric Methods

• Measure color changes; increased protein leads to a more intense blue color measurable via spectrophotometer at ~595 nm.

Bradford Assay Details

• Utilizes Coomassie Brilliant Blue dye, shifting from red (465 nm) to blue (595 nm) upon protein binding.

• Interaction characteristics:

  • Strong noncovalent complexes with proteins based on charge.

  • Binds mainly to amino acids such as phenylalanine, tyrosine, tryptophan, and others.

• Incompatibilities include soluble proteins in acidic dye, and it does not detect smaller proteins (<3000 Da).

Bradford Assay Advantages and Disadvantages

• Advantages

  • Sensitive, accurate, rapid, compatible with usual buffers.

• Disadvantages

  • Nonlinear color response, variability across protein concentrations, insoluble protein analysis limitations.

Detection Limits in Assays

• Bradford Assay: detection range of 0.2 to 2 mg/ml with up to 45% variability among proteins.

• BCA Assay sensitivity measures 3.4 ng/ml detection limits with minimal variation (15%) and employs a purple color formation at 560 nm in the copper reduction reaction.

Standard Curves in Protein Quantification

• Essential for accurate quantification; plotting absorbance against known concentrations (e.g., BSA) establishes a correlation for unknown samples.

Enzyme Basics

• Enzymes lower activation energy, increase reaction rates without altering equilibrium positions.

• Highly specific for their substrates/products, crucial for metabolic regulation with regulatory activities.

• Mostly proteins, with some being RNA enzymes

Enzyme Classes

• 1/ Oxidoreductases: Involve oxidation-reduction reactions.

• 2/ Transferases: Transfer functional groups.

• 3/ Hydrolases: Catalyze hydrolysis reactions.

• 4/ Lyases: Add or remove groups to/from double bonds.

• 5/ Isomerases: Promote structural rearrangements.

• 6/ Ligases (synthetases): Join molecules using ATP.

Chemical Equilibrium in Enzyme Reactions

• Defined as the point of no net change between reactions, where both reactants and products remain constant.

• Enzymes speed the attainment of these states without shifting their positions.

Activation Energy and Transition States

• Reactions require a threshold energy level known as activation energy to transform substrates into products via unstable states (transition state).

• Enzymes stabilize this transitional form, reducing necessary activation energy.

Free Energy Measurement

• Free energy change (4G) indicates if a reaction is spontaneous (negative 4G) or requires energy (positive 4G).

• Example: Hydrolysis of ATP ($ATP + H2O <br>ightarrow ADP + Pi$) yields energy at -7.3 kcal/mol.

Enzyme Active Site Characteristics

• Active sites facilitate substrate interactions through weak binding forces, converting substrates to products. May require cofactors or coenzymes.

Example: Carbonic Anhydrase

• Catalyzes the reaction: CO2 + H2O ↔ HCO3- + H+ with remarkable efficiency (1 million reactions/second).

Enzyme Inhibitors

• Discussed inhibitors that block enzyme function can be used for therapeutic benefits.

Coenzyme A Biosynthesis

• Processes and precursors involved in generating Coenzyme A for cellular metabolism as part of critical enzymatic functions.

Key Insights on Enzyme Kinetics

• Explains first-order versus zero-order kinetics, enzyme velocity influenced by various factors, including substrate concentration and environmental conditions.

• Michaelis-Menten Model: Establishes the relationship between enzyme kinetics, substrate binding stability, and catalytic efficiency.

Enzyme Inhibition Dynamics

• Involves competitive and noncompetitive inhibitors, affecting enzyme activity and substrate interactions.

Enzyme Assay Applications

• Used to assess enzyme activity, determine kinetics, and diagnostic testing, among other applications.

Measurement and Calculation in Enzyme Assays

• Units of measurement (mmol GSH/min) and analysis techniques outlined for quantifying enzyme activity efficiently, including specific assays for glutathione-S-transferase.

Continuous Coupled Assays

• Describe coupled methods for monitoring enzymes that hydrolyze ATP, such as the Pyruvate Kinase-Lactate Dehydrogenase Assay which connects multiple enzymatic processes for throughput evaluation.