Clinical Biochemistry: Proteins

Practice flashcards covering protein structure, classification, denaturation, hydrolysis, purification techniques, colorimetric assays, and misfolding pathology based on lecture notes.

What is the Greek origin and meaning of the word protein?

It comes from the Greek word proteios, which means "of first importance."

What is the average nitrogen content of proteins by mass?

The average nitrogen content of proteins is $15.4\%$ by mass.

How many amino acid residues must be present in a peptide chain for it to be classified as a protein?

A protein must contain at least $40$ amino acid residues.

What distinguishes a monomeric protein from a multimeric protein?

A monomeric protein consists of only one peptide chain, whereas a multimeric protein contains two or more peptide chains (subunits).

Who determined the primary structure and sequence of the $51$ amino acids of human insulin in $1953$?

Frederick Sanger.

What structural linkages hold together the two polypeptide chains (A chain and B chain) of human insulin?

Disulfide bonds (or linkages).

Which six specific atoms lie in the same plane along a peptide bond backbone?

The $\alpha\text{-carbon}$ atom and $C=O$ group of the first amino acid, and the $N-H$ group and $\alpha\text{-carbon}$ atom of the second amino acid.

What chemical property prevents free rotation around the $C-N$ bond in a peptide linkage?

The partial double-bond character of the $C-N$ bond.

What are the standard structural parameters (residues per turn, pitch, and rise per residue) of a right-handed $\alpha\text{-helix}$?

An $\alpha\text{-helix}$ contains $3.6$ amino acid residues per turn, has a pitch of $0.54\,\text{nm}$, and a rise of $0.15\,\text{nm}$ per residue.

Which two amino acids act as helix breakers that disrupt an $\alpha\text{-helix}$ structure?

Proline (due to its rigid secondary amino group inserting a kink) and Glycine (due to its high conformational flexibility from a small R group).

What structural motif connects adjacent antiparallel $\beta\text{-strands}$ in a monomeric protein by making a tight $180^\circ$ turn?

A $\beta\text{-bend}$ (or $\beta\text{-turn}$), which involves four amino acid residues with a hydrogen bond between the first and fourth residues.

What four types of chemical interactions stabilize the tertiary structure of a globular protein?

Disulfide bonds (covalent), electrostatic interactions (salt bridges), hydrogen bonds, and hydrophobic interactions.

In size-exclusion chromatography, which proteins elute from the column first?

Larger proteins elute first because they are too large to enter the pores of the cross-linked polymer beads and take a shorter path around them.

How does sodium dodecyl sulfate (SDS) prepare proteins for molecular weight separation in SDS-PAGE?

SDS denatures and unfolds proteins into rod-like shapes while imparting a large, uniform negative charge that overwhelms intrinsic charges, giving all proteins a similar charge-to-mass ratio.

At what point does a protein stop migrating during Isoelectric Focusing (IEF)?

When it reaches the position in the pH gradient where the local $pH$ equals its isoelectric point ($pI$), because its net charge becomes zero.

What two techniques are combined sequentially in two-dimensional (2D) gel electrophoresis?

Isoelectric Focusing (IEF) in the first dimension (separating proteins by $pI$) and SDS-PAGE in the second dimension (separating proteins by molecular weight, $M_r$).

What chemical principle causes the positive result in a Biuret test?

Cupric ions ($Cu^{2+}$) in alkaline solution coordinate with unshared electron pairs on peptide nitrogens in compounds containing two or more peptide bonds, forming a violet-colored complex.

Which two amino acid residues reduce the Folin-Ciocalteu reagent to produce an intense blue color in the Lowry protein assay?

Tyrosine and tryptophan.

What absorbance shift occurs when Coomassie Brilliant Blue G-250 dye binds to protein basic side chains in the Bradford assay?

The maximum absorbance wavelength of the dye shifts from $465\,\text{nm}$ to $595\,\text{nm}$.

What empirical equation corrects for up to $20\%$ nucleic acid contamination in spectrophotometric protein assays at $280\,\text{nm}$?

$$\text{Protein concentration (mg/mL)} = 1.55 A_{280} - 0.76 A_{260}$$

How does protein denaturation differ fundamental from protein hydrolysis?

Denaturation disrupts secondary, tertiary, and quaternary structures without breaking peptide bonds (primary structure remains intact), whereas hydrolysis breaks peptide bonds to yield free amino acids.

Why is a $70\%$ alcohol solution a more effective disinfectant than pure ($100\%$) alcohol?

Pure alcohol rapidly denatures and coagulates bacterial surface proteins, creating a barrier that prevents deeper penetration; $70\%$ alcohol denatures more slowly, permitting complete cellular penetration before surface coagulation occurs.

What misfolded protein components form extracellular plaques and intracellular tangles in Alzheimer's disease?

Extracellular amyloid plaques are formed by aggregated amyloid $\beta$ ($A\beta$) peptides in a $\beta\text{-pleated}$ sheet conformation; intracellular neurofibrillary tangles are formed by hyperphosphorylated, insoluble $\tau$ (tau) protein.

What structural transformation converts normal cellular prion protein ($PrP^C$) into the infectious scrapie form ($PrP^{Sc}$)?

A conformational change where several $\alpha\text{-helices}$ in $PrP^C$ are replaced by $\beta\text{-sheets}$ in $PrP^{Sc}$, making it resistant to proteolytic degradation.

Which enzyme family catalyzes the conversion of $X\text{-Pro}$ peptide bonds from the trans configuration to the cis configuration during protein folding?

Proline-cis, trans-isomerases (also known as cyclophilins).

What essential amino acid is limiting in gelatin?

Tryptophan.

By what mechanism is casein isolated from milk during laboratory procedure A?

Isoelectric precipitation at $pH = 4.6$, where net charge is zero and water solubility is lowest, causing casein to aggregate.

How does ammonium sulfate achieve the salting out of proteins like myoglobin?

High salt concentrations compete for water molecules in the protein's solvation layer, reducing hydration until protein-protein hydrophobic interactions cause aggregation and precipitation.