Proteins and Enzyme Cleavage Quiz

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Questions and Answers

What specific cleavage does cyanogen bromide (CNBr) perform on proteins?

C-terminal side of methionine residues (correct)

N-terminal side of arginine residues

C-terminal side of proline residues

N-terminal side of lysine residues

Which of the following accurately describes the function of endopeptidases?

Cleavage at the C-terminus of dipeptides

Cleavage at specific internal sites of proteins (correct)

Cleavage of all peptide bonds indiscriminately

Cleavage at the N-terminus of polypeptides

How many peptides would a protein with 9 lysine and 7 arginine residues typically yield when digested with trypsin?

17 (correct)

Which enzyme cleaves at the carboxyl side of aromatic residues such as phenylalanine, tyrosine, and tryptophan?

Chymotrypsin Signup and view all the answers

Which type of enzyme removes amino acids from the N-terminus of peptides?

Aminopeptidase Signup and view all the answers

What is the primary focus of purification techniques for proteins?

Size and charge Signup and view all the answers

Which step comes first in the process of extracting pure proteins from cells?

Homogenization Signup and view all the answers

What is the purpose of salting in & out during protein purification?

To enhance protein solubility Signup and view all the answers

Which type of chromatography separates proteins based on size?

Size-exclusion chromatography Signup and view all the answers

What is the principle behind dialysis in protein purification?

Diffusion and molecular weight cut-off Signup and view all the answers

In ion-exchange chromatography, what type of resin would you use for positively charged particles?

Anion exchanger Signup and view all the answers

At a starting pH of 6.5, which protein would likely be washed out first due to being unbound if it has a pI of 4.7?

Protein #4 Signup and view all the answers

Which of the following is NOT true about ammonium sulfate in protein purification?

It can lead to pure results without any crude elements. Signup and view all the answers

Under non-reducing conditions, which bands are observed for a particular protein molecule weighing 50 KDa?

40 KDa and 20 KDa Signup and view all the answers

Which of the following statements accurately describes the Edman Degradation method?

It cleaves the N-terminal residue step-by-step for identification. Signup and view all the answers

In an ELISA assay, what is a typical sensitivity limit for detecting substances?

10-9 g Signup and view all the answers

Which of the following applications is NOT typically associated with ELISA?

Sequencing of long peptides Signup and view all the answers

What happens to a protein under reducing conditions that differ from non-reducing conditions?

It exists as one band. Signup and view all the answers

What is a consequence of using the Edman Degradation method for sequencing?

It requires high-performance liquid chromatography for each residue. Signup and view all the answers

Which molecular weights are typically represented as bands under reducing conditions for a protein that exhibits a 30 KDa band under non-reducing conditions?

20 KDa and 10 KDa Signup and view all the answers

What is a significant characteristic of the ELISA method in terms of its format?

It is typically conducted in 96-well plates. Signup and view all the answers

What is the primary mechanism of affinity chromatography for protein purification?

Specific binding to a ligand covalently linked to the stationary phase Signup and view all the answers

What does SDS-PAGE primarily provide information about when using reducing conditions?

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

Under which condition would a protein with a 40-KDa band under non-reducing conditions show two bands at 20 KDa under reducing conditions?

The protein is a homodimer Signup and view all the answers

Which medium is typically used for the electrophoresis of proteins?

Polyacrylamide (PAGE) Signup and view all the answers

What is a significant property of proteins that is utilized in isoelectric focusing?

Different isoelectric points Signup and view all the answers

How can protein-ligand interactions be disrupted in affinity chromatography?

By changing the pH or ionic strength Signup and view all the answers

What is the distinguishing factor for using agarose versus PAGE in gel electrophoresis?

Agarose is used for nucleic acids, while PAGE is used for proteins Signup and view all the answers

In the native gel of acrylamide without SDS, how is protein mobility determined?

It is based on overall charge and shape Signup and view all the answers

Study Notes

Protein Purification and Characterization Techniques

Techniques primarily focus on protein size and charge.
Initial step is homogenization (e.g., grinding, homogenizer, sonication, freezing/thawing, detergents).
Differential centrifugation separates components based on density:
- 600 g: unbroken cells and nuclei
- 15,000 g: mitochondria
- 100,000 g: ribosomes and membrane fragments

Salting In & Out

Proteins solubility varies.
Salt stabilizes protein charges, increasing water polarity and solubility.
Ammonium sulfate is a frequent reagent in this step.
This technique is useful but the results can be crude.

Dialysis

Based on diffusion.
Separates molecules based on their molecular weight cut-off (MWCO).
Distinguishes between pure and crude samples. (diagram of dialysis process shown)

Column Chromatography

Based on stationary and mobile phases.
Separates based on interactions with phases.
Used for purification or analysis.
Applications include washing or elution, and various types of column chromatography are available.

Size-Exclusion Chromatography/Gel-Filtration Chromatography

Separates molecules based on size (molecular weight).
Uses cross-linked gel particles (e.g., dextran, agarose, polyacrylamide).
Separates molecules linearly within a range of molecular sizes (dependent on gel).
This method is useful for estimating molecular weights, size range extent, and cross-linking and pore size (exclusion limit) characteristics.

Molecular-Sieve Chromatography

Separates molecules based on size. (diagram showcasing the process.)
Larger molecules elute quickly, smaller molecules later.
Used for separating and purifying molecules.

Ion-Exchange Chromatography

Separation based on net protein charge.
Resin either positively charged (cation exchanger) or negatively charged (anion exchanger).
Uses a buffer to equilibrate the resin, which is bound to counterions.
Elution of proteins occurs with changing salt concentration, based on protein binding strength.
Common examples of resins include CM cellulose and DEAE cellulose

Problem (Protein Elution Order)

5 different proteins have different isoelectric points (pI).
Proteins are loaded onto a column set at a pH of 6.5, and unbound molecules are washed away.
Elution order in cationic and anionic exchange chromatography depends on the pI values, and the pH.

Affinity Chromatography

Purifies specific proteins using specific ligand bindings.
The ligand binds with the desired protein covalently linked to the stationary phase.
Protein is eluted by adding high-concentrated ligand or pH/ionic strength changes.
Purified proteins are usually very pure in this procedure. (diagrams showcased)

Electrophoresis

Separates charged macromolecules in an electric field.
Factors influencing molecule mobility are charge, size, and shape.
Common medium for separation is agarose or acrylamide.

Agarose or PAGE

Agarose is commonly used with nucleic acids, while PAGE (polyacrylamide gel electrophoresis) is used for proteins.
SDS (sodium dodecyl sulfate) is a detergent that denatures proteins, and is used in PAGE, so that their shapes are similar.
Shape/charge are approximately the same (size dictates separation).
Native gels separate proteins without denaturing them, therefore, the mobility of the proteins can illustrate their conformation but not necessarily their size.

Isoelectric Focusing

Technique to separate molecules based on their isoelectric points.
Uses a pH gradient in a gel medium.
Separated proteins are found at their respective pIs.

Questions (SDS-PAGE Results)

Provided questions regarding protein structures based on SDS-PAGE results (non-reducing and reducing conditions). (graphs relating to different scenarios are showcased). This section displays the variety of structures and the influence of reducing conditions on the size/structure of the proteins.

Enzyme-Linked Immunosorbent Assay (ELISA)

Detects and quantifies substances like proteins, antibodies, peptides, and hormones.
Commonly used in 96-well plates, which are a key feature.
Rapid, convenient and sensitive assays with a detection limit of 10⁻⁹ g are frequently performed using these techniques.

Protein Sequencing

Determining the amino acid sequence of a protein.
Edman degradation is a common method to sequence a protein.
This section is related to the sequencing procedure of a chosen protein. This procedure cleaves the N-terminal amino acid.

Protein Sequencing – Edman Method

Procedure for determining amino acid sequences in proteins or peptides.
Section explains the method using hydrolysis, heating, HCl, and separating amino acids with ion-exchange or HPLC. Shows reaction process using diagrams.

Procedure (Edman Degradation)

Procedure for determining the amino acid sequence in a protein or peptide.
Uses phenylisothiocyanate to label the N-terminal amino acid and then identifying the amino acid using chromatography. (Shows the reaction process with diagrams).

Advantages (Edman Degradation)

The remainder of the peptide stays intact, allowing the sequence to be repeated till all amino acids are identified.
Typically successful with less than 50 amino acid chains.

Cleavage Methods

Sequencing whole proteins can be done by cleaving them into small peptides.
Methods, like chemical digestion, endopeptidases, and exopeptidases are used frequently.

Chemical Digestion

Cleaves peptide bonds using a specific amino acid recognition.
Cyanogen bromide (CNBr) is a commonly used chemical reagent used to cleave peptide bonds.
It specifically cleaves at the carboxyl side of methionine residues.

Endopeptidases

Enzymes that cleave peptide bonds within the primary sequence of proteins.

Example (Endopeptidases e.g. Trypsin)

Trypsin cleaves at the carboxyl side of lysine and arginine residues yielding peptides.
An example is provided using the structure of the protein, displaying peptides after reaction.

Other examples (Enzyme Specificities)

Example of various other available cleavage enzymes. This section gives a broad view of various types.

Exopeptidases

Cleaves amino acids from the beginning or end of a protein.
Aminopeptidases cleave from the N-terminus, and carboxypeptidases cleave from the C-terminus.

Protein Sequencing – Prediction from DNA & RNA

If the gene sequence is known, this process is straightforward.
If unknown, techniques like sequencing of mRNA, complementary RNA, or PCR are used for gene sequencing.

Determination of 3° Structure

X-ray crystallography is a high-resolution method, where x-rays interact with protein samples in a crystal. Structural information about molecules is calculated based on the diffraction patterns.
2-D Nuclear magnetic resonance is a solution method.

X-Ray and NMR Data

These data methods are used to determine the protein's 3 dimensional structure of proteins. (Showcases graphs and diagrams explaining the methods and data characteristics)

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Description

Test your knowledge on protein cleavage and the functions of various endopeptidases. This quiz covers specific enzymes like cyanogen bromide and trypsin, as well as the characteristics of peptides formed during digestion. Challenge yourself to see how well you understand these important biochemical processes.