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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”)
Substrate
a
c
b
a
+
b
ES complex
a
b
Enzyme
c
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 bloodstream
Carries: 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 red
Determine 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 activity
Renature proteins by: refolding protein (remake H-bonds, S-S, etc)
Dye Binding of Serum Albumin
NATIVE STRUCTURE of protein vs. denatured
Relatively weak
(e.g, H ) bonds
S S
S S
Relatively strong,
covalent disulfide bond
Dye Binding of Serum Albumin
NATIVE STRUCTURE of protein vs. denatured
Increased thermal energy “disrupts” relatively weak bonds
(ionic, H bonds)
Heated @ 95oC
for 4 min
S S
S-S
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 albumin
Study binding specificity of protein for dyes using column
Determine effects of SDS on protein-dye interactions
Two samples:
+SDS
Serum albumin
Bromphenol blue
Phenol red
SDS
-SDS
Serum albumin
Bromphenol blue
Phenol red
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)