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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 - PowerPoint PPT Presentation
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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”)
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)