Dye Binding of Serum Albumin

1. Dye Binding of Serum Albumin Study of importance of interactions between biomolecules Molecules with complementary surfaces interact

2. 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 + c b ES complex a c b Enzyme

3. Dye Binding of Serum Albumin Molecules with complementary surfaces make a “good fit” Antibody-antigen

4. Dye Binding of Serum Albumin Molecules with complementary surfaces make a “good fit” Antibody-antigen

5. 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

6. 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

7. 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)

8. Relatively weak (e.g, H ) bonds S S S S Dye Binding of Serum Albumin NATIVE STRUCTURE of protein vs. denatured Relatively strong, covalent disulfide bond

9. S-S S S 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

10. S-S Dye Binding of Serum Albumin NATIVE STRUCTURE of protein vs. denatured  mercaptoethanol (DTT): Disrupts any covalent, disulfide bonds between cysteine amino acids  ME

11. Dye Binding of Serum Albumin NATIVE STRUCTURE of protein vs. denatured SDS is an amphipathic molecule with a strong negative charge + polypeptide

12. 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 -SDS Serum albumin Serum albumin Bromphenol blue Bromphenol blue Phenol red Phenol red SDS 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)