Spectrophotometry Basics

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Questions and Answers

What is the primary purpose of a monochromator in a spectrophotometer?

To separate light into distinct wavelengths (correct)

To amplify the signal from the sample

To cool the sample during analysis

To increase the intensity of emitted light

Which component is typically used to narrow down the selected wavelengths after they have been separated?

Mirrors

Lenses

Slits (correct)

Filters

What is the typical path length of modern cuvettes used in spectrophotometry?

0.5 cm

1.00 cm (correct)

1.50 cm

2.00 cm

Which of the following materials can transmit light at the shortest wavelength range?

Quartz (B) Signup and view all the answers

What design feature makes gratings preferable over prisms in monochromators?

Greater uniformity and accuracy (B) Signup and view all the answers

How are cuvettes used in spectrophotometers typically structured regarding light passage?

They are designed to be transparent for light to pass through. (D) Signup and view all the answers

What is the effect of using cheaper cuvettes made from polystyrene compared to quartz or PMMA?

They are less effective at transmitting light at shorter wavelengths. (B) Signup and view all the answers

Why is it important for the cuvette to be handled from the top during a spectrophotometry analysis?

To avoid contamination of the light passage region. (C) Signup and view all the answers

What does SDS-PAGE primarily separate based on?

Mass of polypeptide chains (D) Signup and view all the answers

What appearance indicates a pure protein sample in SDS-PAGE?

A single band (B) Signup and view all the answers

What is the role of the stationary phase in chromatography?

To interact with mobile phase molecules (D) Signup and view all the answers

What happens when a molecule interacts strongly with the stationary phase in chromatography?

It is immobilized for longer periods. (D) Signup and view all the answers

Which type of chromatography utilizes reversible electrostatic interactions?

Ion exchange chromatography (C) Signup and view all the answers

How is protein charged status affected in ion exchange chromatography?

By varying the pH and salt concentration. (C) Signup and view all the answers

What type of functional groups are present on anionic exchange resins?

Positively charged groups (C) Signup and view all the answers

What does affinity chromatography exploit for protein separation?

Specific protein-ligand interactions (C) Signup and view all the answers

What type of chromatography is also known as gel filtration?

Size exclusion chromatography (C) Signup and view all the answers

What is a characteristic of functional groups in chromatography?

They can be charge or biological mimetic molecules. (B) Signup and view all the answers

What aids the continuous movement of protein in column chromatography?

Continuous addition of buffer solution (D) Signup and view all the answers

What type of samples are collected from chromatography columns?

Fractions (D) Signup and view all the answers

Which method can be used to selectively remove charged proteins from the column?

Increasing the buffer pH. (C) Signup and view all the answers

What helps to differentiate proteins in ion exchange chromatography?

Their net charge at a specific pH (A) Signup and view all the answers

What does the slope of the line in an enzyme reaction indicate?

The rate of reaction (D) Signup and view all the answers

Which statement is true about enzyme specificity?

The active site structure determines substrate selectivity. (D) Signup and view all the answers

What does the Michaelis-Menten constant (Km) signify?

The substrate concentration at which the reaction rate is half of Vmax (B) Signup and view all the answers

What is the purpose of the Lineweaver-Burke plot?

To linearize the Michaelis-Menten equation for Km and Vmax determination (A) Signup and view all the answers

What type of enzymes are trypsin and a-chymotrypsin?

Serine proteases (D) Signup and view all the answers

What is generally the first step in DNA analysis or manipulation?

Extraction and purification of DNA (C) Signup and view all the answers

What is the main benefit of commercially prepared DNA extraction kits?

They simplify and standardize the extraction process (A) Signup and view all the answers

In Michaelis-Menten kinetics, what happens when the substrate concentration is high?

The rate of reaction becomes independent of substrate concentration. (C) Signup and view all the answers

What is the term for the interaction between an enzyme and its substrate?

Enzyme-substrate complex (D) Signup and view all the answers

How are non-DNA biological molecules removed during DNA purification?

By selectively degrading and precipitating them (B) Signup and view all the answers

What describes the graphical representation of Michaelis-Menten kinetics?

A rectangular hyperbola with an asymptote at Vmax (A) Signup and view all the answers

Why is enzyme specificity crucial in biochemical reactions?

It minimizes the production of unwanted products. (B) Signup and view all the answers

What is the initial step in an enzymatic reaction as substrate concentration increases?

The reaction rate reaches a plateau. (B) Signup and view all the answers

What property of DNA is primarily evaluated by measuring absorbance at 260 and 280 nm?

Purity (A) Signup and view all the answers

What primarily facilitates the movement of DNA through a gel during electrophoresis?

The negative charge of DNA (A), The size of the gel pores (C) Signup and view all the answers

Which ratio is considered indicative of pure DNA?

1.8-1.9 (C) Signup and view all the answers

What happens to eukaryotic genomic DNA during purification?

It fragments into small pieces. (A) Signup and view all the answers

What characteristic of smaller DNA molecules allows them to migrate faster through a gel than larger DNA molecules?

They have a greater number of pores to pass through (D) Signup and view all the answers

Which reagent is used to visualize DNA on the gel after electrophoresis?

Red safe (A) Signup and view all the answers

What is the primary role of agarose in gel electrophoresis?

To act as a sieving matrix (D) Signup and view all the answers

What is the consequence if DNA is fragmented into very small pieces?

Loss of individual genes (C) Signup and view all the answers

What is the purpose of adding tags to proteins in purification techniques?

To enable specific binding to a purification column (B) Signup and view all the answers

What is the primary purpose of using DNA ladders in electrophoresis?

To provide a reference for DNA sizes (A) Signup and view all the answers

What is the purpose of breaking open cells in the protein isolation process?

To release organelles and molecules into a mixture (C) Signup and view all the answers

In electrophoresis, negatively charged DNA molecules migrate towards which pole?

Positive anode (A) Signup and view all the answers

What is the common name for the sequence comprising six consecutive histidine residues?

His-tag (B) Signup and view all the answers

Which factor does NOT influence the migration velocity of a charged particle in electrophoresis?

The color of the sample (B) Signup and view all the answers

Which of the following is NOT a technique used for disrupting cells?

Affinity chromatography (B) Signup and view all the answers

Which ion is immobilized on the column for proteins with His-tags to bind?

Nickel (B) Signup and view all the answers

What step follows the application of the protein mixture to the column?

Wash the column with wash buffer (A) Signup and view all the answers

Which method is known for being the most selective in protein isolation?

Affinity chromatography (A) Signup and view all the answers

What is the process of transferring DNA segments between molecules referred to as?

Molecular cloning (A) Signup and view all the answers

What is the common method to evaluate the purity of an enzyme during the purification process?

Specific activity (D) Signup and view all the answers

How does changing agarose concentration affect gel electrophoresis?

It affects the size of the pores in the gel. (B) Signup and view all the answers

What is the elution buffer primarily used for?

To elute the bound protein from the column (C) Signup and view all the answers

In size exclusion chromatography, which molecules elute first?

Larger molecules (D) Signup and view all the answers

What type of analysis is commonly used to measure DNA size?

Agarose gel electrophoresis (D) Signup and view all the answers

What is measured to determine the amount of desired protein during purification?

The product formed from enzyme reactions (A) Signup and view all the answers

What is indicated by a high 260/280 absorbance ratio after measuring DNA purity?

Relatively pure DNA sample (D) Signup and view all the answers

What is the main function of the porous beads in size exclusion chromatography?

To segregate molecules based on size (A) Signup and view all the answers

Why is it important to maintain low temperatures during cell disruption?

To prevent protein degradation (D) Signup and view all the answers

What common issue is associated with the elution fraction post purification?

Contamination with ligands (C) Signup and view all the answers

What measurement changes during the purification process indicating increased purity?

Specific activity (C) Signup and view all the answers

The migration velocity of DNA in electrophoresis is proportional to which of the following?

Size of the DNA fragments (C) Signup and view all the answers

What is one major limitation of purifying DNA?

It can lead to fragmentation. (A) Signup and view all the answers

What does size exclusion chromatography help to remove from partially purified proteins?

Small molecules and salts (D) Signup and view all the answers

What results from the application of voltage in an electrophoresis setup?

Migration of DNA towards the positive pole (A) Signup and view all the answers

What does the term 'lysate' refer to in the context of cell disruption?

The mixture of cellular components obtained from cell disruption (D) Signup and view all the answers

What is the primary utility of SDS-PAGE in protein purification?

To visualize protein purity (A) Signup and view all the answers

What is the main stabilization feature of genomic DNA that is removed during purification?

Histones (C) Signup and view all the answers

How do smaller molecules behave in size exclusion chromatography?

They enter the beads and have a longer path (D) Signup and view all the answers

Why is purification of biological molecules crucial in biochemistry?

To isolate specific molecules for study (B) Signup and view all the answers

What do you need to do after eluting the protein to ensure purity?

Separate the protein from ligands by size (D) Signup and view all the answers

Which of the following methodologies does NOT rely on molecular size for separation?

Ion exchange chromatography (A) Signup and view all the answers

What is the main limitation of the biuret assay?

Requires high protein concentration (D) Signup and view all the answers

Which protein assay relies on the reduction of the folin-ciocalteu reagent?

Lowry assay (D) Signup and view all the answers

What must be considered when using the Bradford assay for protein quantitation?

Number of aromatic amino acids in the protein (C) Signup and view all the answers

What is the detection limit of the Lowry assay?

5 μg/mL (C) Signup and view all the answers

Which of the following statements about the Bradford assay is true?

It utilizes dye Coomassie Brilliant Blue G-250. (A) Signup and view all the answers

Which of the following statements is true regarding the BCA assay's compatibility with substances?

Requires high pH levels for analysis. (B), Compatible with most buffers. (D) Signup and view all the answers

Which interaction is primarily responsible for the BCA assay's less protein to protein variation?

Binding to peptide bonds. (C) Signup and view all the answers

Why is the Lowry assay not widely used today?

Long testing time (C) Signup and view all the answers

Which interaction is primarily responsible for the color change in the Bradford assay?

Ionic interactions (D) Signup and view all the answers

What is a key disadvantage of the BCA assay compared to the Bradford assay?

Longer time required to perform. (A) Signup and view all the answers

Which potential interferents can affect both the Lowry and biuret assays?

Various buffers (D) Signup and view all the answers

Which factor can significantly affect the results of the BCA assay?

Extent of basic amino acid. (D) Signup and view all the answers

What type of protein solution is the Bradford assay less effective for?

Membrane proteins in detergents (D) Signup and view all the answers

What is the effect of reducing agents in the BCA assay?

Reduce sensitivity. (D) Signup and view all the answers

What color is produced in the Lowry assay upon completion?

Intense purple (A) Signup and view all the answers

What is the main function of enzymes in biochemical reactions?

To accelerate the rate of chemical reactions. (B) Signup and view all the answers

What type of chemical interaction occurs in the biuret assay?

Binding of copper ions to peptide bonds (C) Signup and view all the answers

What is required to monitor enzyme activity in assays?

Detectable changes in substrates or products. (D) Signup and view all the answers

Which method is commonly used to monitor enzyme reactions in the laboratory?

Spectrophotometry. (D) Signup and view all the answers

What primary factor affects the sensitivity of the Lowry assay?

Presence of aromatic amino acids (A) Signup and view all the answers

Which type of substrate is ideal for enzymatic detection using color change?

Colorless substrate and colored product. (D) Signup and view all the answers

For accurate results in colorimetric assays, what is necessary to prepare alongside protein samples?

Calibration curve (A) Signup and view all the answers

What describes the graph of a reaction progress curve for an enzyme reaction where the product absorbs more light than the substrate?

It will have a positive slope. (D) Signup and view all the answers

In terms of timing, which colorimetric assay requires the longest duration to complete?

Lowry assay (C) Signup and view all the answers

Which of the following is NOT a characteristic of enzymes?

They are altered during reactions. (D) Signup and view all the answers

What is a significant advantage of the Bradford assay over others?

Low cost of reagents (B) Signup and view all the answers

What is a common issue faced in monitoring enzyme activity due to molecular characteristics?

Similar absorption properties of substrate and product. (D) Signup and view all the answers

Why is understanding enzyme properties important in biochemistry?

Most applications require knowledge of enzymes. (C) Signup and view all the answers

Which compound is considered an interfering substance in the BCA assay?

Cysteine residues. (B) Signup and view all the answers

What does the de-stain solution for gels typically consist of?

Glacial acetic acid and methanol (B) Signup and view all the answers

What is hyperchromicity in the context of DNA?

An increase in absorption of single-stranded DNA compared to double-stranded DNA (D) Signup and view all the answers

How is the purity of a protein sample assessed in SDS-PAGE?

By counting the number of bands present (B) Signup and view all the answers

Which of the following factors does NOT affect light absorption by biological molecules?

Temperature fluctuations (C) Signup and view all the answers

What are monosaccharides also known as?

Simple sugars (D) Signup and view all the answers

What happens to reducing sugars when they are covalently linked in polysaccharides?

They cannot reduce oxidizing agents (A) Signup and view all the answers

How does the protonation state of NADH affect its absorption characteristics?

It exhibits different absorption spectra at wavelengths above 300nm compared to NAD (C) Signup and view all the answers

What is the primary limitation of intrinsic protein assays?

Each protein has a different absorption coefficient (C) Signup and view all the answers

Which method is commonly used to analyze the degree of triglyceride saturation?

Iodine number determination (B) Signup and view all the answers

What role do carbohydrates primarily serve in living organisms?

Energy storage and transport (A) Signup and view all the answers

Which biological molecules have significant UV absorption peaks centered around 260nm?

Nucleotides and nucleic acids (C) Signup and view all the answers

What does the presence of heme groups in cytochromes primarily facilitate?

Redox reactions in electron transport chains (B) Signup and view all the answers

What type of sugar does DNS reduce to indicate the presence of simple sugars?

Reducing sugars (A) Signup and view all the answers

What primarily composes triacylglycerols?

Fatty acids (D) Signup and view all the answers

What absorption characteristic is prominent in NADH compared to NAD?

A significant peak at 340nm (C) Signup and view all the answers

What is the primary benefit of using micro-volume spectrophotometers?

They allow for absorbance measurements using very low volumes of sample. (D) Signup and view all the answers

What is the purpose of a blank in spectrophotometry?

To remove constant background absorbance from measurements. (A) Signup and view all the answers

What does a higher iodine number indicate about triglycerides?

Lower saturation (D) Signup and view all the answers

What is the purpose of protein quantitation in biochemistry?

To assess protein concentration for various applications (B) Signup and view all the answers

Why are dual beam spectrophotometers typically more expensive than single beam spectrophotometers?

They allow for simultaneous measurement of the sample and blank. (B) Signup and view all the answers

What do colorimetric protein assays depend on?

Dyes that change their properties based on protein presence (D) Signup and view all the answers

What is a major structural component of plant tissues polysaccharides provide?

Cellulose (C) Signup and view all the answers

Why is analyzing triglyceride saturation important in the food industry?

For compliance with health regulations (C) Signup and view all the answers

What occurs during the denaturing of double-stranded DNA?

It interconverts to a single-stranded form that has higher absorption (A) Signup and view all the answers

What does Beer-Lambert Law state about absorbance?

Absorbance correlates directly with thickness, wavelength, and concentration of absorbing material. (A) Signup and view all the answers

How is absorbance calculated according to the information?

By calculating the log of the ratio of incident light to transmitted light. (A) Signup and view all the answers

What characterizes the fatty acids in simple triacylglycerols?

They contain only one type of fatty acid (C) Signup and view all the answers

Which of the following statements about the absorption of plant pigments is true?

They absorb light in the visible range (B) Signup and view all the answers

What is not a function of lipids?

Protein synthesis (D) Signup and view all the answers

What structures within a molecule are responsible for absorbing light?

Chromophores. (C) Signup and view all the answers

What is the role of redox state in the absorption characteristics of biological molecules?

It allows analysis by absorption spectroscopy (D) Signup and view all the answers

Why is calibration important in spectrophotometry?

It establishes a relationship between analyte concentration and instrument response. (A) Signup and view all the answers

What process describes the breakdown of starch into monosaccharides during fruit ripening?

Hydrolysis (A) Signup and view all the answers

Which method is often used for online protein detection?

Intrinsic absorbance measurements at 280nm (D) Signup and view all the answers

What is the relationship between photon energy and wavelength?

Photon energy is inversely correlated with wavelength. (A) Signup and view all the answers

Why do proteins absorb ultraviolet light at approximately 280nm?

Due to high content of tryptophan and tyrosine (C) Signup and view all the answers

What is the primary method used for protein analysis in a biochemistry lab?

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (C) Signup and view all the answers

Which amino acids are known to effectively absorb light in protein analysis?

Tryptophan and tyrosine. (B) Signup and view all the answers

What is NOT a typical application of protein quantitation?

Monitoring changes in protein structure (C) Signup and view all the answers

How does SDS-PAGE achieve separation of proteins?

Based on the length of their polypeptide chains (D) Signup and view all the answers

What happens to the three-dimensional structure of proteins when prepared for SDS-PAGE?

It is denatured (A) Signup and view all the answers

What is the main purpose of an absorption spectrum?

To display the amount of light absorbed at different wavelengths. (B) Signup and view all the answers

What are photomultiplier tubes, silicon photodiodes, and charge-coupled devices examples of?

Types of detectors. (C) Signup and view all the answers

What is the role of SDS in SDS-PAGE?

To coat proteins with negative charges (B) Signup and view all the answers

Which statement is true regarding the calibration curve?

It helps to establish a clear relationship needed for measuring unknown samples. (D) Signup and view all the answers

Why is it important to use a loading buffer with glycerol or sucrose?

To ensure the sample sinks to the bottom of the well (B) Signup and view all the answers

What kind of materials can impact the absorbance levels obtained in spectrophotometric measurements?

Both the spectrophotometer and the sample conditions. (A) Signup and view all the answers

What factors influence the migration velocity of proteins during electrophoresis?

The size, shape, and charge of the proteins (A) Signup and view all the answers

Which characteristic of a molecule affects its light absorption ability?

The arrangement of electrons in its chromophores. (C) Signup and view all the answers

What is the typical acrylamide concentration in the separating gel for SDS-PAGE?

6 to 15% (C) Signup and view all the answers

What is the purpose of the stacking gel in SDS-PAGE?

To concentrate the protein samples into a tight band (A) Signup and view all the answers

What occurs when the incident light intensity equals the transmitted light intensity?

The Absorbance equals zero. (B) Signup and view all the answers

Which staining method is commonly used to visualize proteins on an SDS-PAGE gel?

Coomassie blue staining (C) Signup and view all the answers

What is the purpose of heating protein samples before loading onto the SDS-PAGE?

To denature the proteins (B) Signup and view all the answers

What happens to the proteins after electrophoresis is completed?

They must be fixed to the gel (A) Signup and view all the answers

What temperature is typically used to heat protein samples for SDS-PAGE preparation?

95°C (D) Signup and view all the answers

What challenges do complex protein structures pose in electrophoresis?

Variable migration rates due to shape and size differences (B) Signup and view all the answers

Which component of the SDS loading buffer is responsible for breaking disulfide bonds?

B-mercaptoethanol (A) Signup and view all the answers

What does the iodine number of a lipid mixture indicate?

The degree of saturation of carbon-carbon bonds (B) Signup and view all the answers

What reagent is used to form molecular iodine from unreacted iodine chloride in the iodine number determination?

Potassium iodide (C) Signup and view all the answers

What happens during the titration of unreacted iodine with sodium thiosulfate?

The iodine is converted to iodide, lightening the solution (A) Signup and view all the answers

What is the role of starch in the iodine titration process?

As an indicator for the presence of iodine (A) Signup and view all the answers

Which chlorophyll pigment absorbs light most strongly in the violet/blue and orange/red regions?

Chlorophyll a (A) Signup and view all the answers

What characteristic structure do most plant pigments share that aids in light absorption?

Extensive conjugated double bonds (A) Signup and view all the answers

In paper chromatography, what phase is typically used as the stationary phase?

Solid cellulose phase (C) Signup and view all the answers

What is the main purpose of the mobile phase in paper chromatography?

To carry the solute molecules along the paper (C) Signup and view all the answers

How is the relative migration of solute molecules in chromatography compared?

Using RF values (B) Signup and view all the answers

What property of solute molecules determines their migration speed in paper chromatography?

Polarity of the molecules (C) Signup and view all the answers

Which compound is a provitamin derived from carotene that is converted in mammalian tissue to vitamin A?

Beta-carotene (C) Signup and view all the answers

What is the primary function of chlorophyll in plants?

Absorbing light energy for photosynthesis (A) Signup and view all the answers

What effect does the presence of double bonds in fatty acids have on the iodine number?

Increases the iodine number (A) Signup and view all the answers

What is the end appearance of the solution in the iodine titration when the reaction is complete?

Colorless (A) Signup and view all the answers

Study Notes

Monochromators and Slits

Spectrophotometers use monochromators to isolate specific wavelengths of light from a broadband source.
Monochromators typically use prisms or gratings to separate light into distinct wavelengths.
Gratings, with their precise line patterns, are more common for accuracy and uniformity.
Slits are used to select a narrow wavelength range of light from the dispersed wavelengths.
Mirrors and lenses focus the selected light onto the sample.
A lab spectrophotometer focuses light into a 1mm diameter beam, 8mm above the cuvette bottom, allowing handling without fingerprints affecting readings.

Cuvettes and Cuvette Holders

Cuvettes are specialized containers for holding samples in spectrophotometers.
Common cuvette volumes are 1mL and 3mL, with a standard path length of 1.00cm.
- Specialized cuvettes have longer path lengths.
Cuvettes must be transparent to the light used.
Materials and their light transmission ranges include:
- Polystyrene: 325-750nm
- PMMA: 245-750nm
- Quartz: 170-2500nm
Polystyrene cuvettes are inexpensive and disposable.
Quartz cuvettes are more expensive and transmit light at shorter wavelengths.
Cuvette holders ensure proper placement.
Micro-volume spectrophotometers use small sample volumes(1-2.5µL) placed between pedestals.
- Fiber optic cables handle light delivery and detection.

Detectors

Detectors gather and measure light intensity (some also wavelength).
Detectors convert light signals to electrical signals, amplifying them then sending the data to a recorder.
Different detectors include photomultiplier tubes (PMTs), silicon photodiodes, and charge-coupled devices (CCDs).

Absorbance and Beer-Lambert Law

Absorbance quantifies light absorption.
Absorbance (A) is calculated as: A = log(I₀/I), with I₀ as incident light intensity and I as transmitted intensity.
- Absorbance has no units.
- Absorbance cannot be negative (other than error).
Beer-Lambert Law describes factors affecting absorbance:
- Wavelength (probability of photon absorption).
- Path length of the absorbing material.
- Concentration of the absorbing material.

Blanks

Blanks are reference solutions identical to samples except for the analyte.
Blanks are used to eliminate background absorbance.
Dual-beam spectrophotometers measure blank and sample simultaneously, minimizing errors from fluctuating lamp intensity. Single-beam spectrophotometers do not split the beam.

Calibration and Calibration Curves

Spectrophotometer conditions affect absorbance readings.
Calibration curves are used to correct for potential errors by establishing relationships between analyte amount and instrument signal.
A best fit line plotted through the points is used to calculate analyte amount in samples of unknown concentration.

Absorption Spectra

Absorption spectra are graphs depicting absorbance across a range of wavelengths.
Absorption patterns have peaks where light is most strongly absorbed by the analyte or molecules.
- The wavelength at highest absorbance is λ_max.

Chromophores

Chromophores are light-absorbing structural features within molecules.
- Conjugated double bonds are common chromophores in the UV-visible spectrum.

Proteins

Most protein side chains do not absorb light strongly, except tryptophan, tyrosine, and phenylalanine.
- Intrinsic absorption of these can quantify proteins.
Prosthetic groups in some proteins, such as heme groups in myoglobin and hemoglobin, absorb strongly in the UV-Visible range.
- Coenzymes like flavins and nicotinamides absorb in the relevant range.

Nucleotides and Nucleic Acids

Aromatic rings in nucleotides and nucleic acids absorb strongly in the UV region around 260nm.

Other Light-Absorbing Biological Molecules

Plant pigments absorb light in the visible spectrum.

Factors Affecting Absorption

Solvent: Influences the stability of molecular states, thus affecting wavelengths absorbed.
Protonation: The protonation state of molecules impacts absorption spectrum.
- NAD and NADH show different absorption at specific wavelengths.
Redox state: Redox reactions change molecules' electron distribution, influencing their absorbance properties.
Interaction effects: Interactions with other molecules changes absorption, e.g., DNA denaturation.

Protein Quantitation and Enzyme Kinetics

Protein quantification is frequently essential in biochemistry.
Protein quantification methods include:
- Intrinsic protein assays exploit intrinsic absorption, rapid but limited to pure proteins.
- Colorimetric protein assays rely on interaction; typically destructive to the sample.
- Biuret assay: Older method, less sensitive.
- Lowry assay: Older method, moderately sensitive.
- Bradford assay: Commonly used due to speed, sensitivity, but limited by protein type and detergent compatibility.
- BCA assay: Highly sensitive and compatible with detergents, slightly longer than Bradford.

Introduction to Enzyme Assays

Enzymes are biological catalysts that accelerate reactions.
Enzyme assays measure enzyme activity, monitoring the change in substrate and product concentrations over time.
- Spectrophotometry may be used if the products and reactants exhibit different absorption properties.
- Chromogenic substrates enable easy spectrophotometric detection of reactions.
Reaction progress curves provide data for calculating reaction rates.

Enzyme Specificity

Enzymes demonstrate high specificity for their substrates and chemical reactions.
Active site structure limits substrate compatibility.
Enzymes like trypsin and chymotrypsin, differ fundamentally in their substrate selection based on amino acid location in the protein chain.

Michaelis-Menten Kinetics

Michaelis–Menten kinetics describe the relationship between reaction rate and substrate concentration for enzymes that saturate.
- V_max is the maximum reaction rate at saturation.
- K_m, the Michaelis–Menten constant, denotes the substrate concentration producing half the maximum reaction rate.
Lineweaver–Burk plot transforms the Michaelis–Menten equation to produce a linear relationship for determining K_m and V_max.

DNA Purification and Analysis

DNA must be isolated and purified for molecular biology applications.
Modern kits simplify and standardize the isolation procedure.
Quality control involves evaluating DNA purity and integrity.
- The 260/280nm absorbance ratio assesses purity.
- Agarose gel electrophoresis assesses DNA fragment size and integrity.

Restriction Enzymes and Molecular Cloning

Molecular cloning techniques allow the transfer or DNA fragments into new molecules.

Electrophoresis

Electrophoresis separates charged molecules in a solution using an electric field based on varying migration velocities.

Agarose Gel Electrophoresis

Agarose gels are porous materials used to separate DNA fragments by size in electrophoresis.
DNA ladders providing known DNA size markers are critical for size determination.

Protein Purification from Tissue/Cell Extracts

Protein isolation typically involves multiple steps for purification.
The initial steps may involve cell lysis—to release intracellular proteins.
Various techniques isolate proteins based on size, charge, affinity, or other properties.
- Techniques include ammonium sulfate precipitation, chromatography (ion exchange, affinity, size exclusion), and others.

Determining Protein Purity

Evaluation of protein purity involves methods to quantify the target protein relative to other molecules.
Specific activity of the target protein (enzyme) can be measured to assess purification progress.
SDS-PAGE is commonly performed to assess protein size and purity.

Protein Column Chromatography

Chromatography is a technique that separates molecules based on their interactions with stationary and mobile phases.
Protein purification relies on column chromatography with stationary phases packed in columns.
Common methods are ion-exchange, affinity, and size-exclusion chromatography.
- Ion-exchange: Separates proteins based on charge differences, utilizing a charged column.
- Affinity: Relies on specific binding interactions between protein and ligand.
- Size-exclusion: Separates proteins by size.

SDS-PAGE Analysis of Egg White Proteins

SDS-PAGE separates proteins based on size following denaturation.
Loading and visualization steps are essential for proper analysis.

Effect of Storage Conditions on Banana Ripening

The ripening of fruit involves the conversion of storage carbohydrates to accessible monomeric forms.
DNS reducing sugar assays measure the presence of simple sugars formed during ripening.

Iodine Number Determination of Triacylglycerols

Iodine number quantifies the degree of unsaturation in triacylglycerols.
- The degree of unsaturation is measured by the amount of iodine consumed.
- Iodine is consumed/reacted in the saturation of the double bonds.
- The calculation involving the amount of sodium thiosulfate is used to determine the amount of unreacted iodine.

Isolation and Analysis of Spinach Leaf Pigments

Plant pigments, such as chlorophyll a and b, absorb light to perform photosynthesis.
Paper chromatography separates pigments based on their polarity relative to a solvent and compared using R_f values.

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Description

Test your knowledge on the fundamental components and principles of spectrophotometry. This quiz covers essential topics such as monochromators, cuvettes, and the materials used in spectrophotometric analysis. Perfect for students and professionals looking to reinforce their understanding of this important analytical technique.