Biochemistry Quiz: Peptide Bond Reagents

Questions and Answers

Match the following reagents with their specific cleaving actions:

Chymotrypsin = Hydrolysis of peptide bonds on the carboxyl side of Lys and Arg Trypsin = Cleavage of peptide bonds on the carboxyl side of Met Dithiothreitol = Breakage of disulfide (—S—S—) bonds Edman reagent (phenylisothiocyanate) = Determination of the amino acid sequence of a peptide FDNB = Determining the amino-terminal amino acid in a polypeptide

What is a major step in determining the sequence of amino acid residues in a protein?

Use Edman degradation to determine the sequence of each peptide.

What is the likely explanation for the identification of Ala and Leu as amino-terminal residues in equal amounts?

The protein has some multiple of two subunits, with Ala and Leu as the amino-terminal residues.

What conclusion can be drawn about the protein that has undergone treatment with performic acid?

The protein has two subunits (Mr 35,000 and 45,000), joined by one or more disulfide bonds. Signup and view all the answers

Describe a key difference between chemical synthesis of polypeptides and synthesis in living cells.

Chemical synthesis proceeds from carboxyl terminus to amino terminus; in living cells, it starts at the amino terminus. Signup and view all the answers

What is the distinction between homologs, paralogs, and orthologs?

Homologs are any members of a protein family; paralogs are two homologs in the same species; orthologs are homologs in different species. Signup and view all the answers

How are 'signature sequences' useful in analyzing proteins?

They help construct evolutionary trees by indicating relationships among taxonomic groups. Signup and view all the answers

Which two amino acids differ from each other by only one atom?

Ser and Cys Signup and view all the answers

Identify the pair of peptides below that cannot be distinguished by tandem mass spectrometry.

VTSPLYANEGK and VTSPIYANEGK Signup and view all the answers

List three classes of conjugated proteins and their associated prosthetic groups.

Lipoproteins with lipid groups, glycoproteins with carbohydrate groups, hemoproteins with heme groups. Signup and view all the answers

What is a polymorphic protein?

A polymorphic protein is one whose amino acid sequence varies among individuals in the population. Signup and view all the answers

What is the native sequence determined from the tetrapeptide composed of (Lys) 2, Phe, and Gly?

His-Leu-Phe-Gly-Lys-Lys-Phe-Met-Gly Signup and view all the answers

When a gene is available and its sequence of nucleotides is known, what chemical studies are still necessary?

The location of disulfide bonds Signup and view all the answers

What does the term 'proteome' refer to?

The complement of proteins encoded by an organism's DNA Signup and view all the answers

What major problem did advances in mass spectrometry overcome?

Mass spectrometric analysis involved molecules in the gas phase Signup and view all the answers

Which tripeptides from the following options have the largest number of nonpolar R groups?

Leu-Val-Phe Signup and view all the answers

The amino acid ______ has no free alpha-amino group.

L-proline Signup and view all the answers

What are the structural characteristics common to all amino acids found in naturally occurring proteins?

All amino acids have an alpha carbon, a carboxylic acid, an amine, a hydrogen, and a variable side chain. Signup and view all the answers

Only one of the common amino acids has no free alpha-amino group.

True Signup and view all the answers

Which tripeptide will yield DNP-tyrosine when reacted with l-fluoro-2,4-dinitrobenzene and hydrolyzed in acid?

Tyr-Lys-Met Signup and view all the answers

What is the pI, and how is it determined for amino acids that have nonionizable R groups?

The pI is the isoelectric point determined by the average of a molecule's two pKa values: pI = 1/2 (pK1 + pK2). Signup and view all the answers

What factors affect the elution order of molecules during size-exclusion chromatography?

Smaller molecules elute after larger molecules. Signup and view all the answers

Define the primary structure of a protein.

The primary structure is its unique sequence of amino acids and any disulfide bridges present in the covalent bond structure. Signup and view all the answers

Which reagents are used in protein chemistry?

All of the above Signup and view all the answers

The chirality of an amino acid results from the fact that its alpha carbon:

is bonded to four different chemical groups. Signup and view all the answers

Of the 20 standard amino acids, only _ is not optically active. The reason is that its side chain _.

glycine; is a hydrogen atom Signup and view all the answers

Two amino acids of the standard 20 contain sulfur atoms. They are:

methionine and cysteine. Signup and view all the answers

All of the amino acids that are found in proteins, except for proline, contain a(n):

amino group. Signup and view all the answers

Which of the following statements about aromatic amino acids is correct?

On a molar basis, tryptophan absorbs more ultraviolet light than tyrosine. Signup and view all the answers

Which of the following statements about cystine is correct?

Cystine forms when the —CH2—SH R group is oxidized to form a —CH2—S—S—CH2— disulfide bridge between two cysteines. Signup and view all the answers

The uncommon amino acid selenocysteine has an R group with the structure —CH2—SeH (pKa ~5). In an aqueous solution, pH = 7.0, selenocysteine would:

be a fully ionized zwitterion with no net charge. Signup and view all the answers

Amino acids are ampholytes because they can function as either a(n):

acid or a base. Signup and view all the answers

Titration of valine by a strong base, for example NaOH, reveals two pK's. The titration reaction occurring at pK2 (pK2 = 9.62) is:

—NH3+ + OH- --> —NH2 + H2O. Signup and view all the answers

In a highly basic solution, pH = 13, the dominant form of glycine is:

NH2—CH3+—COO-. Signup and view all the answers

For amino acids with neutral R groups, at any pH below the pI of the amino acid, the population of amino acids in solution will have:

a net positive charge. Signup and view all the answers

At pH 7.0, converting a glutamic acid to gamma-carboxyglutamate, will have what effect on the overall charge of the protein containing it?

it will become more negative Signup and view all the answers

At pH 7.0, converting a proline to hydroxyproline, will have what effect on the overall charge of the protein containing it?

it will stay the same. Signup and view all the answers

What is the approximate charge difference between glutamic acid and alpha-ketoglutarate at pH 9.5?

½ Signup and view all the answers

The formation of a peptide bond between two amino acids is an example of a(n) ______________ reaction.

condensation Signup and view all the answers

The peptide alanylglutamylglycylalanylleucine has:

four peptide bonds. Signup and view all the answers

An octapeptide composed of four repeating glycylalanyl units has:

one free amino group on a glycyl residue and one free carboxyl group on an alanyl residue. Signup and view all the answers

At the isoelectric pH of a tetrapeptide:

the total net charge is zero. Signup and view all the answers

Which of the following is correct with respect to the amino acid composition of proteins?

Proteins with different functions usually differ significantly in their amino acid composition. Signup and view all the answers

The average molecular weight of the 20 standard amino acids is 138, but biochemists use 110 when estimating the number of amino acids in a protein of known molecular weight. Why?

The number 110 reflects the higher proportion of small amino acids in proteins, as well as the loss of water when the peptide bond forms. Signup and view all the answers

In a conjugated protein, a prosthetic group is:

a part of the protein that is not composed of amino acids. Signup and view all the answers

Prosthetic groups in the class of proteins known as glycoproteins are composed of:

carbohydrates. Signup and view all the answers

For the study of a protein in detail, an effort is usually made to first:

purify the protein. Signup and view all the answers

In a mixture of the five proteins listed below, which should elute second in size-exclusion (gel-filtration) chromatography?

immunoglobulin G Mr = 145,000 Signup and view all the answers

By adding SDS (sodium dodecyl sulfate) during the electrophoresis of proteins, it is possible to:

separate proteins exclusively on the basis of molecular weight. Signup and view all the answers

To determine the isoelectric point of a protein, first establish that a gel:

exhibits a stable pH gradient when ampholytes become distributed in an electric field. Signup and view all the answers

The first step in two-dimensional gel electrophoresis generates a series of protein bands by isoelectric focusing. In a second step, a strip of this gel is turned 90 degrees, placed on another gel containing SDS, and electric current is again applied. In this second step:

proteins with similar isoelectric points become further separated according to their molecular weights. Signup and view all the answers

The term specific activity differs from the term activity in that specific activity:

is the activity (enzyme units) in a milligram of protein. Signup and view all the answers

Which of the following refers to particularly stable arrangements of amino acid residues in a protein that give rise to recurring patterns?

Secondary structure Signup and view all the answers

Which of the following describes the overall three-dimensional folding of a polypeptide?

Tertiary structure Signup and view all the answers

The functional differences, as well as differences in three-dimensional structures, between two different enzymes from E.coli result directly from their different:

amino acid sequences. Signup and view all the answers

One method used to prevent disulfide bond interference with protein sequencing procedures is:

reducing disulfide bridges and preventing their re-formation by further modifying the —SH groups. Signup and view all the answers

A nonapeptide was determined to have the following amino acid composition: (Lys)2, (Gly) 2, (Phe) 2, His, Leu, Met. What are the results of the peptide's analysis with 1-fluoro-2,4-dinitrobenzene?

2,4-dinitrophenylhistidine Signup and view all the answers

Study Notes

Chirality and Amino Acids

Chirality in amino acids arises from the alpha carbon being bonded to four different groups.
Glycine is the only standard amino acid that is not optically active due to its hydrogen side chain.

Sulfur-Containing Amino Acids

Methionine and cysteine are the only standard amino acids that contain sulfur.

Amino Acids in Proteins

All standard amino acids, apart from proline, contain an amino group.
Aromatic amino acids absorb ultraviolet light, with tryptophan being more efficient than tyrosine.

Cystine and Disulfide Bonds

Cystine is formed when the thiol group of cysteine is oxidized, resulting in a disulfide bridge.

Zwitterion Form in Solutions

At pH 7.0, selenocysteine exists as a fully ionized zwitterion.
Amino acids function as ampholytes, acting as both acids and bases.

Titration and pKa Values

The titration curve for valine reveals two pKa values; at pK2, the reaction involves the deprotonation of the amino group.
In a basic solution, glycine predominantly exists as a protonated species.

Charge Properties of Amino Acids

Below the isoelectric point (pI), amino acids with neutral side chains carry a net positive charge.
Gamma-carboxyglutamate conversion at pH 7.0 makes proteins more negatively charged.

Peptide Bonds and Structure

Peptide bond formation is a condensation reaction resulting in the release of water.
The number of peptide bonds in a peptide is one less than the number of amino acids.

Protein Purification and Analysis

Protein purification is a crucial first step in detailed protein study.
The isoelectric point of a protein can be determined by creating a stable pH gradient in the gel.

Protein Structural Levels

Secondary structure refers to stable arrangements of amino acid residues, such as alpha-helices and beta-sheets.
Tertiary structure encompasses the overall three-dimensional folding of a polypeptide.

Mass Spectrometry and Protein Studies

Advances in mass spectrometry help address challenges related to analyzing macromolecules.

Identifying Protein Sequences

Techniques like trypsin digestion and cyanogen bromide help elucidate amino acid sequences in peptides.
The native sequence of peptides can be determined using HPLC and other biochemical methods.

Proteomics

"Proteome" refers to the full complement of proteins encoded by an organism's DNA, highlighting its complexity and functional diversity.### Mass Spectrometry and Proteins
Mass spectrometry advancements enable the analysis of macromolecules but face challenges related to gas-phase molecules.

Protein Sequence Comparison

Protein sequences from four different proteins analyzed for evolutionary relationships.
Evolutionary divergence is highlighted between proteins based on amino acid sequences.
Specific relationships include:
- Protein 4 exhibiting the greatest evolutionary divergence in column B.
- Proteins 1 and 2 have notably diverged evolutionarily.
- Protein 4 shows significant homology to protein 1.

Amino Acid Structure

All naturally occurring amino acids share a structure of an alpha carbon connected to:
- A carboxylic acid
- An amine group
- A hydrogen atom
- A variable side chain.
Amino acids exist predominantly in the L configuration.

Uncommon Amino Acids

Examples include 4-hydroxyproline, 5-hydroxylysine, and selenocysteine.
These amino acids typically arise from post-translational modifications of standard amino acids.

Zwitterion Formation

Amino acids become zwitterions in water because:
- Carboxylic acid groups ionize to form COO⁻.
- Amino groups become NH₃⁺ by protonation.

Titration and Buffering

At pH 9.5, the NH₃⁺ group in amino acids is titrated to NH₂.
The positively charged –NH₃⁺ stabilizes the negatively charged COO⁻, lowering its pKa.
The pH region with the greatest buffering capacity is around the amino acid's pKa, indicated by a leveling off in titration curves.

Protein Purification and Analysis

Specific activity of an enzyme is calculated as activity per protein concentration.
Peptide bonds are stable due to high activation energy for hydrolysis.
Techniques like ion-exchange and size-exclusion chromatography separate proteins based on charge and size, respectively.

Isoelectric Point (pI)

The isoelectric point is reached at a pH where a molecule exhibits no net charge.
Calculated as the average of two pKa values for amino acids without ionizable side chains.

Protein Structure Determination

Steps for deducing the sequence of an amino acid chain post-disulfide bond reduction:
- Edman degradation for each peptide.
- Use proteases like trypsin for additional peptide generation.
- Overlap peptides to reconstruct the full sequence.

Protein Relationships

Distinction between homologs (related proteins within a family), paralogs (duplicate genes within a species), and orthologs (genes in different species).
Signature sequences are instrumental in linking functionally related proteins, enhancing evolutionary and functional analysis.

Chemical vs. Biological Synthesis

Chemical synthesis is from carboxyl to amino terminus, while biological synthesis is the reverse.
Cells can synthesize larger and more complex proteins compared to current chemical methods, which are limited to shorter polypeptides.### Amino Acid Differences
Serine (Ser) and Cysteine (Cys) differ by only one atom.
Importance of identifying variations at the atomic level in amino acid sequences.

Tandem Mass Spectrometry

Certain peptides cannot be distinguished using tandem mass spectrometry due to similarities in their sequences.
Example pair: VTSPLYANEGK and VTSPIYANEGK are indistinguishable.

Conjugated Proteins

Conjugated proteins contain additional chemical substituents beyond amino acids.
Three classes of conjugated proteins include:
- Lipoproteins, associated with lipid groups
- Glycoproteins, associated with carbohydrate groups
- Phosphoproteins, associated with phosphoryl groups
- Hemoproteins, associated with heme groups
- Flavoproteins, associated with flavin nucleotide groups
- Metalloproteins, associated with metal ions (e.g., zinc, iron, calcium)

Polymorphic Proteins

Polymorphic proteins are characterized by varying amino acid sequences among individuals in the human population.
Variants of polymorphic proteins typically maintain similar function and activity.

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Description

Test your knowledge on various reagents used in the hydrolysis and cleavage of peptide bonds. This quiz covers important reagents like CNBr, Edman reagent, and enzymes such as chymotrypsin and trypsin, as well as their specific applications. Assess your understanding of protein chemistry and amino acid sequence determination.

Biochemistry Quiz: Peptide Bond Reagents

Questions and Answers

Match the following reagents with their specific cleaving actions:

What is a major step in determining the sequence of amino acid residues in a protein?

What is the likely explanation for the identification of Ala and Leu as amino-terminal residues in equal amounts?

What conclusion can be drawn about the protein that has undergone treatment with performic acid?

Describe a key difference between chemical synthesis of polypeptides and synthesis in living cells.

What is the distinction between homologs, paralogs, and orthologs?

How are 'signature sequences' useful in analyzing proteins?

Which two amino acids differ from each other by only one atom?

Identify the pair of peptides below that cannot be distinguished by tandem mass spectrometry.

List three classes of conjugated proteins and their associated prosthetic groups.

What is a polymorphic protein?

What is the native sequence determined from the tetrapeptide composed of (Lys) 2, Phe, and Gly?

When a gene is available and its sequence of nucleotides is known, what chemical studies are still necessary?

What does the term 'proteome' refer to?

What major problem did advances in mass spectrometry overcome?

Which tripeptides from the following options have the largest number of nonpolar R groups?

The amino acid ______ has no free alpha-amino group.

What are the structural characteristics common to all amino acids found in naturally occurring proteins?

Only one of the common amino acids has no free alpha-amino group.

Which tripeptide will yield DNP-tyrosine when reacted with l-fluoro-2,4-dinitrobenzene and hydrolyzed in acid?

What is the pI, and how is it determined for amino acids that have nonionizable R groups?

What factors affect the elution order of molecules during size-exclusion chromatography?

Define the primary structure of a protein.

Which reagents are used in protein chemistry?

The chirality of an amino acid results from the fact that its alpha carbon:

Of the 20 standard amino acids, only ___________ is not optically active. The reason is that its side chain ___________.

Two amino acids of the standard 20 contain sulfur atoms. They are:

All of the amino acids that are found in proteins, except for proline, contain a(n):

Which of the following statements about aromatic amino acids is correct?

Which of the following statements about cystine is correct?

The uncommon amino acid selenocysteine has an R group with the structure —CH2—SeH (pKa ~5). In an aqueous solution, pH = 7.0, selenocysteine would:

Amino acids are ampholytes because they can function as either a(n):

Titration of valine by a strong base, for example NaOH, reveals two pK's. The titration reaction occurring at pK2 (pK2 = 9.62) is:

In a highly basic solution, pH = 13, the dominant form of glycine is:

For amino acids with neutral R groups, at any pH below the pI of the amino acid, the population of amino acids in solution will have:

At pH 7.0, converting a glutamic acid to gamma-carboxyglutamate, will have what effect on the overall charge of the protein containing it?

At pH 7.0, converting a proline to hydroxyproline, will have what effect on the overall charge of the protein containing it?

What is the approximate charge difference between glutamic acid and alpha-ketoglutarate at pH 9.5?

The formation of a peptide bond between two amino acids is an example of a(n) ______________ reaction.

The peptide alanylglutamylglycylalanylleucine has:

An octapeptide composed of four repeating glycylalanyl units has:

At the isoelectric pH of a tetrapeptide:

Which of the following is correct with respect to the amino acid composition of proteins?

The average molecular weight of the 20 standard amino acids is 138, but biochemists use 110 when estimating the number of amino acids in a protein of known molecular weight. Why?

In a conjugated protein, a prosthetic group is:

Prosthetic groups in the class of proteins known as glycoproteins are composed of:

For the study of a protein in detail, an effort is usually made to first:

In a mixture of the five proteins listed below, which should elute second in size-exclusion (gel-filtration) chromatography?

By adding SDS (sodium dodecyl sulfate) during the electrophoresis of proteins, it is possible to:

To determine the isoelectric point of a protein, first establish that a gel:

The first step in two-dimensional gel electrophoresis generates a series of protein bands by isoelectric focusing. In a second step, a strip of this gel is turned 90 degrees, placed on another gel containing SDS, and electric current is again applied. In this second step:

The term specific activity differs from the term activity in that specific activity:

Which of the following refers to particularly stable arrangements of amino acid residues in a protein that give rise to recurring patterns?

Which of the following describes the overall three-dimensional folding of a polypeptide?

The functional differences, as well as differences in three-dimensional structures, between two different enzymes from E.coli result directly from their different:

One method used to prevent disulfide bond interference with protein sequencing procedures is:

A nonapeptide was determined to have the following amino acid composition: (Lys)2, (Gly) 2, (Phe) 2, His, Leu, Met. What are the results of the peptide's analysis with 1-fluoro-2,4-dinitrobenzene?

Study Notes

Chirality and Amino Acids

Sulfur-Containing Amino Acids

Amino Acids in Proteins

Cystine and Disulfide Bonds

Zwitterion Form in Solutions

Titration and pKa Values

Charge Properties of Amino Acids

Peptide Bonds and Structure

Protein Purification and Analysis

Protein Structural Levels

Mass Spectrometry and Protein Studies

Identifying Protein Sequences

Proteomics

Protein Sequence Comparison

Amino Acid Structure

Uncommon Amino Acids

Zwitterion Formation

Titration and Buffering

Protein Purification and Analysis

Isoelectric Point (pI)

Of the 20 standard amino acids, only _ is not optically active. The reason is that its side chain _.