Lecture 3 Flashcards
what is a method we can measure total protein concentration?
Absorbance Spectroscopy
Solubility increases with ionic strength - well-hydrated shell at the protein surface, preventing aggregation of the molecules.
salting in
At high ionic strengths, the salt competes for water and this leads to aggregation and precipitation of the proteins.
salting out
Size exclusion is a type of
chromatography
a method to separate protein due to porous beads within a column
Size exclusion
in ion exchange chromatography, separation is based on
charge
what are the two Stationary phases in ion exchange chromatography
cation exchange and anion exchange
in anion exchange, the matrix has
+ charged beads
in cation exchange, the matrix has
- charged beads
what are two ways to detach our proteins for the matrix in neither cation or anion exchange?
changing the pH or increasing salt levels
Any molecule that reversibly binds proteins
ligand
in Affinity chromatography, what is unique about its stationary phase?
Stationary phase in the column
has covalently attached ligand
specific for the protein of interest
how do we elude our protein of interest in Affinity chromatography?
excess free ligand or high salt
of enzyme units per milligram of total protein
specific activity
proteins that help speed up metabolism, or the chemical reactions in our bodies
enzyme
composition of a protein includes
which amino acids and how many are present
sequence of a protein includes
arrange of amino acids
a process that uses enzymes to cleave, or break, specific bonds in a substrate
Enzymatic cleavage
what are the two commonly used cleavage enzymes (talked about in class)?
trypsin, chymotrypsin
A polypeptide with a net positive charge at pH 7.4
most likely contains R groups that are:
basic R groups
In an a-helix, the R groups on the AA residues:
Are found on the outside of the helix spiral
Which amino acid might be expected to have the LEAST effect on the function of an enzyme if it replaces a Glu residue in the enzyme?
Aspartic acid
Which of the following contributes the MOST to the energetics of protein stability during folding of a new polypeptide?
the hydrophobic effect
the amount of myoglobin that has bound O2
fractional saturation