Chem 108

Why should proteins be kept cold during purification?

To prevent thermal denaturation and microbial growth

What can cause protein denaturation?

Heat, pH shifts, detergents, organic solvents, heavy metals, oxidation, and foaming

What does A280 measure?

protein concentration through absorbance of aromatic amino acids

What is the unit of absorbance A?

unitless

What absorbs at 280 nm?

aromatic amino acids in proteins (Trp,Tyr,Phe)

What absorbs at 340 nm?

NADH

What absorbs at 595 nm?

Coomassie dye in the Bradford assay

What is the structure of LDH?

Tetramer made of H and M subunits

What are LDH isozymes?

different combinations of M and H subunits

What are isozymes?

enzymes with the same function but different amino acid sequences and kinetics

What properties differ among isozymes?

Tissue expression, electrophoretic mobility, and KM/Vmax values

What is KM?

Substrate concentration at half Vmax, measure affinity

What is Vmax?

maximum reaction rate velocity, when all enzyme is bound to substrate

What is Kcat?

Turnover number, substrate molecules converted per enzyme per second

What changes in competitive inhibition?

Km increases, Vmax stays the same

What changes in noncompetitive inhibition?

Vmax decreases, KM unchanged

What changes in uncompetitive inhibition?

Vmax and Km decrease

How do you determine competitive inhibitor on a lineweaver-burk plot?

The lines intersect at the Y-axis

How do you determine noncompetitive inhibitor on a lineweaver-burk plot?

The lines intersect at the X-axis

How do you determine uncompetitive inhibitor on a lineweaver-burk plot?

They are parallel lines

What does the y-intercept represent on a lineweaver-burk plot?

1/Vmax

What does the x-intercept represent on a lineweaver-burk plot?

-1/KM

What is salting out?

Protein precipitation using ammonium sulfate?

How are proteins separated by ion exchange?

By charge, eluted using increasing salt (NaCl) concentration

What type of chromatography uses cibacron blue for LDH

Affinity chromatography

How does gel filtration work?

Separates by size, the larger proteins elute first

What is the principle of the Bradford assay?

Dye binds basic amino acids; absorbance shifts from 465 to 595

What is a drawback of Bradford assay?

It requires a standard curve, destroys the sample, not very fast

What are the advantages of A280

Fast, no standard curve, nondestructive

What are the disadvantages of A280?

many other materials absorb at A280, some proteins do not absorb significantly

What are the advantages of affinity chromatography?

very selective, fast, size/structure doesn’t need to be known

What are the disadvantages of affinity chromatography?

Need antibody, tag, or ligand attached to resin, expensive

How are bound proteins eluted in affinity chromatography?

Competition, by adding a solution that contains a species that binds tighter to the resin than the protein

What is the stationary phase for column chromatography?

The resin

What is the mobile phase for column chromatography?

Buffer solution

type of buffer is used to elute for ion exchange chromatography?

increasing concentration of salt buffers

How are proteins eluted for ion exchange chromatography?

ion exchange resins bind to the complimentary ions present in salts more tightly that the protein.

What does LDH do?

catalyzes the reduction of pyruvate to lactate through hydride anion transfer

Where is M form most prominent

anaerobic tissue

Where is H form most prominent

aerobic tissue

What does 1 represent

vmax

What does 2 represent

½ vmax

What does 3 represent

km

What is the michaelis-menten equation

v= vmax[S]o / Km+[S]o