Dental Biochemistry Protein Characterization/Purification

______ are covalently linked a-amino acids

polypeptides

something that can be tightly associated or covalently bound to polypeptides that contains functional non-amino acids or metal ions

cofactors

something that can be tightly associated or covalently bound to polypeptides that contains organic cofactors or NAD+ in lactate dehydrogenase

coenzymes

something that can be tightly associated or covalently bound to polypeptides that contains covalently attached cofactors

prosthetic groups

To study the structure-function of a protein, we must first _____ it

purify

Differential and sucrose-density centrifugation separate sample by ____/_____

size; weight

most proteins are ____ soluble at high salt concentrations, an effect called ____ ____

less; salting out

Increasing the salt concentration will result in ________ protein being present in the aqueous solution after pellet formation

less

After salting out, ______ can be used to remove excess salt

dialysis

Salts have a two-fold effect on protein solubility. At low concentrations, the ______ the solubility, called _____ _____

increase; salting-in

The isoelectric point (pI) is the pH at which protein has a ___ charge and at which it is _____ soluble

neutral; least

The _____ phase of column chromatography has an _____ for the ____ phase

stationary; affinity; mobile

Column chromatography separates proteins by ______-

charge

At acidic pH (_____ than pI) , the _____ group is protonated and the aa is in the _____ form

lower; carboxyl; cationic

At neutral pH-pI, the carboxyl group is ____ and the amino group is _____. the resulting ion is called a _______

deprotonated; protonated; zwitterion

At alkaline pH (_____ than pI), the amino group is _____ and the aa is in the _____ form

higher; neutral; anionic

In size-exclusion chromatography, the larger sample will be retained ______ in the column

lower

We use ______ with known molecular weights to determine the weight of the unknown.

standards

What separation technique requires you to know a great deal about the protein of interest?

affinity chromatography

Hydrophobic Interaction Chromatography is unique in that proteins bind at _____ salt concentration and elute at ____ salt concentration

high;low

HPLC =

high pressure liquid chromatography

Reversed-phase chromatography results from the adsorption of ________ molecules into a _____ solid support in a _____ mobile phase

hydrophobic; hydrophobic; polar

Separation in electrophoresis is based on ____, ____, and _____ of proteins

charge; size; shape

In electrophoresis, the gel separates proteins on the basis of what?

size and shape

In electrophoresis, the electric field separates proteins on the basis of what?

charge density

SDS is a ______, and gives all proteins a ______. Therefore all proteins will separate based on _____

detergent; negative charge; size

A higher concentration polyacrylamide gel will result in ______ proteins being retained

smaller

SDS-PAGE can be used to calculate the ______ of a protein

molecular weight

_______ _______ can be used to determine the pI of a protein

isoelectric focusing

_________ and _______ are combined in 2D electrophoresis

isoelectric focusing; SDS-PAGE

______ _____ refers to the units of activity per milligram of protein

specific activity

The aromatic amino acids absorb light in the ____ _____

UV Region

Proteins typically have UV absorbance maxima around _____ - ____ nm

275-280 nm

______ and ______ are the strongest chromophores

Tryptophan; tyrosine

Cyanogen bromide cleaves at the ___________

carboxyl side of methionine residues

Trypsin cleaves at the __________

Carboxyl side of lysine and arginine residues

Chymotrypsin cleaves primarily at the ____

carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, methionine

The classical method of protein sequencing

edman degradation

modern method of protein sequencing

mass spec