P1 Spring Pceutics 3/07 (Lecture 3)

Front 1

What substance denatures protein by breaking disulfide bonds?

Back 1

surfactants

Front 2

Dehydration versus hydrolysis?

Back 2

dehydration: forms peptide bond by removing an H2O

hydrolysis: breaks a peptide bond by adding an H2O

Front 3

Whats the temp called that a protein denatures at?

Back 3

Tm ("melting temp")

Front 4

List the non covalent factors that stabilize proteins

Back 4

1.H-bonding
2.Van der waals (permanent or induced dipoles)
3.Hydrophobic interactions
4.Electrostatic interactions
5.Protein hydration (H-bonds to protein in soln)

Front 5

List things that cause physical instability of proteins

Back 5

1.Surface adsorption
2.Precipitation
3.Aggregation
4.Denaturation

Front 6

Aggregation vs precipitation?

Back 6

Precipitation is large scale aggregation (visible aggregation)

Front 7

List things that cause chemical instability of proteins

Back 7

1.Hydrolysis
2.Oxidation
3.Racemization
4.Disulfide exchange
5.Deamidation
6.Deglycosylation

Front 8

Whats deamidation?

Back 8

•A form of hydrolysis
•Hydrolysis of the amine groups off of glutamine or asparagine
•Replaced by COO--, giving those amines a negative charge
•Typically alters the tertiary structure

Front 9

List parts of a protein susceptible to oxidation

Back 9

•Aromatic side chains
•Methionine (has an S)
•Cysteine (has SH)

Front 10

List things that influence oxidation

Back 10

•Temperature
•Oxygen tension
•pH
•Buffers
•Amino acid neighbors
•Catalysts

Front 11

Cause and result of racemization?

Back 11

cause: carbanion intermediate

result: some L-aminos become D-aminos, giving you a mix of the two

Front 12

Cause of disulfide exchange?

Back 12

Oxidaton of disulfide bonds, then exchange of bonds catalyzed by thiols

Front 13

Prevention of disulfide exchange?

Back 13

Thiol scavengers can prevent oxidative cleavage of bonds

Front 14

Whats shearing?

Back 14

Protein unfolds due to mechanical force, then refolds incorrectly

Front 15

Whats shaking?

Back 15

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Front 16

Whats SDS-PAGE (define acronym, purpose, steps)?

Back 16

•sodium dodecylsulfate-polyacrylamide gel
•purpose: determine MW of protein
steps:

1.add dye (B-ME or DTT), breaks SS bonds
2.SDS denatures and separates protein, gives pieces a neg charge
3.proteins migrate across gel (smaller ones go faster/farther)

Front 17

Purpose of Immunoblotting?

Back 17

Determines the relative amount of protein in a sample

Front 18

Another name for Immunoblotting?

Back 18

Western blotting

Front 19

Steps of Immunoblotting?

Back 19

1.Add protease inhibitor
2.Perform SDS-PAGE
3.Transfer separated proteins to a membrane
4.Coat with generic protein (eg milk)
5.Add primary Ab+dye
6.Add secondary Ab+chromogenic enzyme

Front 20

Steps for ELISA?

Back 20

1.Coat microtiter with antigen/protein
2.Add generic protein (eg milk)
3.Add primary Ab
4.Add secondary Ab+chromogenic enzyme (Ab is anti mouse IgG)
5.Add chromogenic substrate

Front 21

In what type of cells does glycosylation occur?

Back 21

In mammalian (not bacterial)