Biochemistry Chapter on Rate-Zonal Centrifugation

Questions and Answers

What is the primary technique used to determine the amino acid sequence of a protein before 1985?

Edman degradation (correct)

Nuclear magnetic resonance

X-ray crystallography

Mass spectrometry

What does Edman degradation rely on for amino acid identification?

Physical separation of proteins

Gene cloning techniques

Ionization of proteins

High-pressure liquid chromatography (correct)

What is the advantage of using recombinant DNA techniques over Edman degradation for sequencing proteins?

They directly analyze protein structure.

They are generally faster than chemical sequencing. (correct)

They can identify more amino acids at once.

They are more precise than chemical methods.

Which of the following steps is NOT involved in the Edman degradation process?

Ionizing the molecular sample Signup and view all the answers

What is required to initiate the Edman degradation process?

Phenylisothiocyanate (PITC) Signup and view all the answers

In the context of mass spectrometry, what role does the organic acid play?

It helps in the drying process of the sample. Signup and view all the answers

What has contributed to the rapid expansion of genome sequence databases?

Development of DNA sequencing methods Signup and view all the answers

How does the Edman degradation affect the polypeptide after each cycle?

It removes one amino acid from the N-terminus. Signup and view all the answers

What is the primary purpose of isoelectric focusing (IEF)?

To create a pH gradient for protein separation Signup and view all the answers

How do proteins migrate during isoelectric focusing?

According to their pI, or isoelectric point Signup and view all the answers

In two-dimensional gel electrophoresis, what is the second dimension based on?

The molecular weight of the proteins Signup and view all the answers

What advantage does two-dimensional gel electrophoresis have in studying cellular proteins?

It allows for the resolution of many proteins simultaneously Signup and view all the answers

What does liquid chromatography separate molecules based on?

Interactions with a solid surface Signup and view all the answers

Which of the following statements is true regarding the proteins during the SDS PAGE step?

They separate according to their mass. Signup and view all the answers

What is the role of the isoelectric point (pI) in protein separation techniques?

It is the pH at which a protein has no net charge. Signup and view all the answers

Why is isoelectric focusing effective for resolving proteins that differ by only one charge unit?

It establishes a precise pH gradient. Signup and view all the answers

What is the primary function of rate-zonal centrifugation?

To separate molecules of interest into distinct bands Signup and view all the answers

What can influence the sedimentation rate in rate-zonal centrifugation?

Both mass and shape of particles Signup and view all the answers

In rate-zonal centrifugation, what happens if the centrifuge is operated for too long?

All molecules will form a pellet at the bottom Signup and view all the answers

Which technique is mostly used for separating DNA or organelles?

Equilibrium density-gradient centrifugation Signup and view all the answers

What is the main principle behind electrophoresis?

Molecules separate according to their charge:mass ratio Signup and view all the answers

How does the charge of a molecule affect its speed during electrophoresis?

Greater net charge results in faster movement towards an electrode Signup and view all the answers

What is one common use of electrophoresis?

To resolve small molecules like nucleotides Signup and view all the answers

What factor is not relevant in determining how molecules migrate in electrophoresis?

Color of the molecule Signup and view all the answers

What is the major site of DNA synthesis as identified by autoradiographic studies using [3H]thymidine?

Nucleus Signup and view all the answers

After prolonged incubation with [3H]thymidine, where does most of the radioactivity remain?

In the nucleus Signup and view all the answers

What does the localization of autoradiographic grains over the cytoplasm indicate about RNA?

RNA is transported from the nucleus Signup and view all the answers

How long are the tracks created by the β particles emitted by tritium in autoradiographic studies?

0.47 μm Signup and view all the answers

What is the approximate accuracy of locating 3H-labeled structures within cells?

0.5–1.0 mm Signup and view all the answers

What factor significantly affects the localization ability of autoradiography?

The energy of particles emitted during radioactive disintegrations Signup and view all the answers

Which of the following statements about the emission of β particles by phosphorus-32 is true?

They generate very long streaks on photographic emulsion. Signup and view all the answers

In autoradiographic studies using [3H]uridine, where are the grains predominantly located after a long incubation period?

In both the nucleus and cytoplasm Signup and view all the answers

What is the primary use of x-ray diffraction in protein analysis?

To establish the detailed three-dimensional structure of proteins Signup and view all the answers

What challenge is associated with crystallizing large multisubunit proteins?

They often require precise conditions that are difficult to replicate Signup and view all the answers

How are the x-rays used in diffraction experiments to analyze protein structures?

They form a scattering pattern when passing through a crystal Signup and view all the answers

What is a key advantage of cryoelectron microscopy compared to x-ray crystallography?

It does not require crystallization of proteins Signup and view all the answers

Which wavelengths are used in x-ray diffraction studies?

0.1–0.2 nanometers Signup and view all the answers

What role do heavy metal modifications play in x-ray crystallography?

They aid in interpreting the diffraction pattern Signup and view all the answers

What is the outcome of scattering x-rays in x-ray diffraction experiments?

A diffraction pattern containing discrete spots Signup and view all the answers

What is a critical factor to consider when using electron microscopy for protein analysis?

Radiation should be minimized to prevent structural damage Signup and view all the answers

Study Notes

Rate-Zonal Centrifugation

Proteins are separated into bands or disks in a tube based on mass using rate-zonal centrifugation.
Samples must be centrifuged for optimal time; too short leads to insufficient separation, while too long results in pellet formation.
Sedimentation rates are influenced by both mass and shape, complicating precise molecular weight determination.
Rate-zonal centrifugation remains practical for separating varied polymers and particles.

Equilibrium Density-Gradient Centrifugation

Primarily used for separating DNA or organelles.
Employs a density gradient to allow different components to settle at their respective densities.

Electrophoresis Techniques

Electrophoresis separates molecules in mixtures using an electric field, influenced by their charge:mass ratio.
Molecules with the same mass and shape migrate at different speeds based on net charge.
Isoelectric focusing (IEF) resolves proteins by migrating them through a pH gradient until they reach their isoelectric point (pI).

Two-Dimensional Gel Electrophoresis

Combines IEF with SDS electrophoresis for dual separation based on charge and mass.
Allows up to 1000 proteins to be resolved simultaneously, aiding in gene expression studies.

Liquid Chromatography

Separates proteins, nucleic acids, and other molecules by their interaction with a solid surface in a liquid medium.
Critical for autoradiographic studies, identifying synthesis sites of molecules within cells, like DNA in the nucleus and RNA movement to the cytoplasm.

Autoradiography

Involves using a radiation-sensitive photographic emulsion over labeled cells to visualize radioactive incorporation sites.
Emission energy of isotopes impacts localization accuracy; e.g., tritium offers better resolution than phosphorus-32.

Edman Degradation

Classic method for amino acid sequencing; targets the N-terminus of polypeptides for sequential identification.
Involves labeling, cleavage, and identification, allowing for gradual reduction of the polypeptide chain.

Recombinant DNA Techniques

Offer faster methods for deducing protein sequences from mRNA or gene sequences than Edman degradation.
Genome sequencing advancements enhance protein research and understanding of large proteins.

Mass Spectrometry

Measures mass of proteins and peptides through ionization, acceleration, and detection of molecular ions.
Essential for establishing three-dimensional structures of proteins via x-ray diffraction patterns.

Cryoelectron Microscopy

Utilized for proteins that do not crystallize easily; captures low-resolution images of proteins in a frozen state.
The technique involves rapid freezing and electron microscopy, with computer analysis for three-dimensional structure reconstruction.

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Description

Explore the principles behind rate-zonal centrifugation, a key technique in biochemistry for separating proteins based on their masses. Understanding the balance of centrifugation time is crucial for achieving effective separation. Test your knowledge about this density-gradient method and its applications in protein analysis.