Dye Binding of Serum Albumin

Study of importance of interactions between biomolecules

Molecules with complementary surfaces interact

Dye Binding of Serum Albumin

Study of importance of interactions between biomolecules

Molecules with complementary surfaces make a “good fit” (“induced fit” “lock and key”)

ab

c

Substrate

+

Enzyme

ab c

ES complex

ab c

Dye Binding of Serum Albumin

Molecules with complementary surfaces make a “good fit”Antibody-antigen

Dye Binding of Serum Albumin

Molecules with complementary surfaces make a “good fit”Antibody-antigen

Dye Binding of Serum Albumin

Serum albumin - protein used to transport nonsoluble molecules through the bloodstreamCarries: bilirubin, fatty acids, hormones, dyes

bilirubin

Bromphenol blue(phenol red w/o Br)

Fatty acids

Dye Binding of Serum Albumin

Investigate specificity of protein for small dyes, bromphenol blue and phenol redDetermine whether NATIVE STRUCTURE of protein is required to bind dyes

Dye Binding of Serum Albumin

NATIVE STRUCTURE of protein vs. denatured

Denaturation affects weak interactions, such as H-bonds Denature proteins by:

Heat, extreme pH, add organics (alcohol, acetone)

Add urea, guanidine hydrochloride, detergent (SDS)

Add reducing agent to break S-S (-ME, DTT)Renaturation = regaining native structure and biological activityRenature proteins by: refolding protein (remake H-bonds, S-S, etc)

Dye Binding of Serum Albumin

NATIVE STRUCTURE of protein vs. denatured

Relatively strong,covalent disulfide bond

Relatively weak (e.g, H ) bonds

S SS S

Dye Binding of Serum Albumin

NATIVE STRUCTURE of protein vs. denatured

Increased thermal energy “disrupts” relatively weak bonds (ionic, H bonds)

S-S

S S

Heated @ 95oC for 4 min

Dye Binding of Serum Albumin

NATIVE STRUCTURE of protein vs. denatured

mercaptoethanol (DTT): Disrupts any covalent, disulfide bonds between cysteine amino acids

S-S

ME

Dye Binding of Serum Albumin

NATIVE STRUCTURE of protein vs. denatured

SDS is an amphipathic molecule with a strong negative charge

+ polypeptide

Dye Binding of Serum Albumin

Determine molecular weight of serum albuminStudy binding specificity of protein for dyes using columnDetermine effects of SDS on protein-dye interactions

Two samples:+SDS -SDSSerum albumin Serum albuminBromphenol blue Bromphenol bluePhenol red Phenol redSDS

1. Load -SDS sample onto your column and separate measuring Ve for colored dyes (BPB and PR)

2. Load +SDS sample onto your column and separate measuring Ve for colored dyes (BPB and PR)