Biochemistry: Protein Structure, Function, and Purification

A collection of 50 vocabulary flashcards covering amino acid properties, chemical modifications, thermodynamics, and experimental protein purification and electrophoresis techniques.

Average molecular weight of an amino acid residue

Approximately $110\,\text{Da}$, determined by subtracting the molecular weight of water ($18\,\text{Da}$) lost during peptide bond formation from the average amino acid weight of $128\,\text{Da}$.

Trypsin

A protease from bovine pancreas that specifically cleaves polypeptide chains on the carboxyl-terminal (C) side of lysine (Lys) and arginine (Arg) residues.

Chymotrypsin

A protease from bovine pancreas that selectively cleaves peptide bonds on the carboxyl-terminal (C) side of aromatic amino acids: phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr).

Staphylococcus aureus V8 protease

A bacterial protease from S. aureus that specifically cleaves polypeptide chains on the carboxyl-terminal (C) side of aspartate (Asp) and glutamate (Glu) residues.

Asp-N-protease

A bacterial protease isolated from Pseudomonas fragi that cleaves peptide bonds on the amino-terminal (N) side of aspartate (Asp) and glutamate (Glu) residues.

Pepsin

A protease from porcine stomach that hydrolyzes peptide bonds on the amino-terminal (N) side of leucine (Leu), phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr) residues.

Endoproteinase Lys C

An enzyme isolated from the bacterium Lysobacter enzymogenes that specifically cleaves peptide bonds on the carboxyl-terminal (C) side of lysine (Lys) residues.

Cyanogen bromide

A chemical reagent used for protein fragmentation that specifically cleaves peptide bonds on the carboxyl-terminal (C) side of methionine (Met) residues.

280 nm detection wavelength

The standard ultraviolet wavelength used in spectrophotometry to quantify protein concentrations due to the light absorption by conjugated double bonds in aromatic amino acid side chains.

Tryptophan UV absorbance

The aromatic amino acid that exhibits the strongest absorbance peak in the range of $270\text{--}280\,\text{nm}$, significantly higher than tyrosine and phenylalanine.

Conservative substitution

An evolutionary mutation where an amino acid residue is replaced by another residue of similar size and chemical properties, such as substituting serine (Ser) with threonine (Thr).

Molar concentration of pure water

Approximately $55.5\,\text{M}$.

Hydrophobic effect

The thermodynamic drive wherein nonpolar regions cluster together in water, minimizing ordered clathrate water cages and increasing the overall entropy of the system.

Micelles

Thermodynamically stable structures of amphipathic compounds formed in water where hydrophobic nonpolar groups are sequestered within the interior and polar heads face the solvent.

Urushiol

The allergen present in poison ivy possessing an aromatic ring with long alkyl chains ($\text{p}K_a \approx 8$), most effectively neutralized and washed away using soap, water, and baking soda (sodium bicarbonate).

Protein kinase

An enzyme responsible for post-translational modification by transferring a phosphate group to hydroxyl-bearing amino acid residues (serine, threonine, or tyrosine).

4-Hydroxyproline

An uncommon amino acid formed by post-translational modification of proline residues after protein synthesis, found predominantly in collagen.

Pyrrolysine

An uncommon amino acid incorporated directly during protein synthesis that contributes functionally to methane biosynthesis.

Ornithine

A non-proteinogenic amino acid metabolite that functions as an intermediate in the arginine biosynthetic pathway.

Disulfide bond

A covalent cross-link connecting polypeptide chains formed by the oxidation of two cysteine thiol (\text{SH}) groups.

2-Mercaptoethanol (\beta-ME)

A chemical reducing agent used in biochemistry to cleave covalent disulfide bonds within or between polypeptide chains.

Buffering region

The relatively flat plateau of a titration curve spanning $\text{pH} = \text{p}K_a \pm 1$, where a weak acid and its conjugate base exist in equilibrium to effectively resist changes in pH.

Crude extract

The complex aqueous solution containing all released intracellular proteins and soluble components immediately following cell or tissue disruption.

Fractionation

The process of separating a protein mixture into distinct fractions based on physical or chemical properties such as size, charge, or solubility.

Salting in

The effect observed at low ionic strength where the addition of dissolved ions increases protein solubility by weakening electrostatic interactions that cause protein aggregation.

Salting out

A purification technique where high concentrations of dissolved ions outcompete proteins for water molecules, leading to protein aggregation and precipitation.

Ammonium sulfate ((NH_4)_2SO_4)

A highly soluble salt widely selected for salting-out fractionation because it precipitates target proteins effectively without denaturing or damaging them.

Isoelectric point (pI)

The specific pH at which a protein carries a net electric charge of zero, corresponding to the point where the protein is least soluble.

Dialysis

A separation method that uses a semipermeable membrane bag to separate large macromolecules, such as proteins, from small solutes and excess salt.

Stationary phase

The solid porous matrix packed inside a chromatography column that retards the migration of sample components based on their physical or chemical properties.

Mobile phase

The buffered solution that flows through the stationary phase in a chromatography column, percolating and carrying the protein mixture.

Ion-exchange chromatography

A chromatographic method that separates proteins according to the sign and magnitude of their net electric charge at the specific buffer pH used.

Cation exchanger

An ion-exchange resin functionalized with negatively charged groups (such as carboxymethyl) that binds positively charged proteins while allowing negatively charged proteins to elute first.

Anion exchanger

An ion-exchange resin carrying bound positively charged functional groups (such as DEAE) that attracts and retains negatively charged proteins.

Carboxymethyl (CM)

A commonly used cation-exchange group with the formula $\text{Matrix-CH}_2\text{-COO}^-$ that carries a negative charge at physiological pH.

Diethylaminoethyl (DEAE)

A frequently used anion-exchange functional group with the chemical formula $\text{Matrix-CH}_2\text{-CH}_2\text{-NH(CH}_2\text{CH}_3)_2^+$ that bears a positive charge.

Size-exclusion chromatography

Also called gel filtration; a chromatography technique that separates proteins by size where large molecules are excluded from the porous beads and elute first, whereas small molecules enter pores and elute last.

Included volume

The accessible internal porous volume within size-exclusion beads through which smaller molecules take labyrinthine paths, delaying their emergence.

Affinity chromatography

A highly specific purification technique based on the biological binding of a protein to a ligand covalently linked to a solid matrix, followed by elution with free ligand or salt.

His-tag affinity capture

An affinity method where a recombinant protein engineered with six histidine residues ($\text{His}_6$) binds specifically to an immobilized $\text{Ni}^{2+}$ matrix (such as Ni-NTA or Ni-TED).

Imidazole

A chemical analogue of the histidine side chain used to elute His-tagged proteins from $\text{Ni}^{2+}$ affinity resins by competitive binding.

Specific activity

The ratio of total enzyme catalytic activity (in units) to total protein mass (in mg), serving as a standard index of sample purity during purification.

Purification factor

The ratio of the final specific activity of a protein sample to its initial specific activity in the starting crude extract.

Percent yield (purification)

The percentage of original total enzyme activity that remains in the recovered sample following a specific purification step or sequence.

Polyacrylamide gel electrophoresis (PAGE)

An analytical method using a cross-linked polyacrylamide gel matrix to separate proteins in an electric field based on charge-to-mass ratio and hydrodynamic size.

Coomassie blue

An organic dye that binds to proteins in polyacrylamide gels, allowing visual detection and band identification following electrophoresis.

Sodium dodecyl sulfate (SDS)

An anionic detergent that unfolds proteins into linear conformations and coats them with a uniform negative charge, allowing electrophoretic separation strictly by molecular weight.

Electrophoretic mobility (\mu)

The migration rate of a molecule per unit electric field, defined by the relationship $\mu = \frac{V}{E} = \frac{Z}{f}$, where $Z$ is net charge and $f$ is the frictional coefficient.

Isoelectric focusing (IEF)

An electrophoretic technique that separates proteins along a stable pH gradient gel; each protein migrates until reaching the pH corresponding to its isoelectric point (pI).

Two-dimensional electrophoresis (2D PAGE)

A high-resolution protein separation method that couples isoelectric focusing (separating by pI in the first dimension) with perpendicular SDS-PAGE (separating by molecular weight in the second dimension).