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Lecture

bch10-12.docx

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Department
Biochemistry
Course Code
BCH210H1
Professor
Charles Deber

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Description
Lectures 10 to 12 Hemoglobin A Vs. S  Hemoglobin is a oxygen carrier with 4 subunits. HBA=wild type HBS=mutant  Hemoglobin S differs from HBA in one amino acids in the Beta chain Glu-6 to Val-6  This is a non-conservative mutation: Glu is a polar charged amino acid, Val is a highly non polar amino acid  HBS is more positive then HBA and will be closer to the negative charge in the SDS-PAGE  HBS tends to aggregate and form aggregated fibers which precipitates into clumps  Apparently there is a hydrophobic patch on deoxyhemoglobin S which has a complementary site on another deoxyhemoglobin S molecule (aggregation happens in the deoxy form) Why does HBS aggregate?  CF p-values show that Glu is a higher helix former and Val is a higher Beta sheet former  In HBA can be either in a helix or beta sheet but in HBS there is increased beta-sheet potential  Since beta sheets are inter- rather than intra molecular structures CF analysis predicts the change in protein properties. (aggregating is using inter molecular bonds)  Suggestion that genetic disease arising from mutant proteins result from biophysical interactions Glucagon  Glucagon is a small protein secreted when blood sugar is low  Spectroscopic measurements by CD show that glucagon changes structure as a function of concentration  At low concentration glucagon is Ahelical  At high concentrations glucagon is B sheet  Concentrated solutions of glucagon from a gel like gelatin  The CF values for the residues 19-27 are interchangeable between Palpha and P beta. There cannot be a distinction between the two possible secondary structures  Biological implication: glucagon segment may change conformation during its biological action- receptor binding hormone  since hormone /receptor interactions are inter molecular, possibly glucagon converts to Bsheet to binds its receptor Types of chromatography -chromatography used to separate proteins from a protein mixture and acquire a pure sample Gel Chromatography  Gel chromatography separates by size  The column has pores with varying size  The smaller proteins get stuck in the pores and are slowed down while the larger proteins get eluded first. (use if your protein has a significantly larger size than the others) Affinity chromatography  affinity chromatography column separates proteins b selectively binding specific proteins (can only use if your protein has something that is different for all other proteins like His tags or your protein specifically binds to something like Glucose)  column contains covalently bound substrate or ligand that recognises one protein in the mixture and binds it exclusively  selected protein molecules remain bound to the column when all unbound proteins are eluted through the column  once separated a substrate is added in column in excess: this completes the bound protein and the protein will come off in pure form HPLC  separates proteins by hydrophobicity  the more hydrophobic the longer it will attach to the hydrophobic columns and will have longer retentions times and be eluted last Ion Exchange Chromatography  separates by positive or negative charges  the column will contain the oppositely charge of your protein and your protein will stick to the column  used in amino acid analysis to separate amino acids to determine protein composition Composition of proteins  determine the percentage of each of the 20 amino acids in the overall sequence  boil protein in concentrated hydrochloric acid then analyze monomeric amino acid content and ratios Protein sequence  to determine the sequence of the unknown protein  determining sequence from the N-terminal end use the edman degradation reagent combines with the N-terminal amino acid, cleaves i
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