Study guide CHEM 4510
Popular in Biochemistry I
Popular in Chemistry and Biochemistry
This 3 page Study Guide was uploaded by Juliana Herran on Tuesday September 20, 2016. The Study Guide belongs to CHEM 4510 at University of Northern Iowa taught by Dr. Ira Simet in Fall 2016. Since its upload, it has received 4 views. For similar materials see Biochemistry I in Chemistry and Biochemistry at University of Northern Iowa.
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Date Created: 09/20/16
Last minute checks… - Acids, bases, buffers [Base] pH=pka+log? [Acid] Smaller pka = stronger acids Larger ka = stronger acids (Within weak acids, strong acids completely dissociate) Mix weak acid + its conjugate base = buffer o Add Acid → More HA, less A- o Add Base → Less HA, more A- Buffering regions are 2 pH units wide H O2+ CO ↔(2orry for the arrows, there are no equilibrium arrows in word) H 2O ↔3CO ↔CO3 3- o Acidosis: Blood pH LESS THAN 7.35 (Breath faster) o Alkalosis: Blood p H MORE THAN 7.35 (Breath slower, need more CO2) If p H is a unit or more above the pka then you have the base form. - Amino acids 19 at alpha amino acids Know your titration curves Rxn of COOH group: Deprotonation, esterofocation, amidation Rxn of NH2 group: Protonation, Amidation, Nucleophilic rxns Amidation is the important one There are 9 Hydrophobic chains, 5 hydrophilic (charged), 4 hydrophilic (uncharged) pI= pka1+pka2 2 Choose isoelectric form of the amino acid for paper electrophoresis Beta branched: val, ile, thr Aromatic: phe, trp, tyr, his Acid labile: met, trp, asn (asp), gln (glu), ser, thr Peptide = less than 50 amino acids (MW less than 5000) Polypeptide = more than 50 amino acids (MW more than 5000-typically 10000-200000) Simple proteins have aa’s only Conjugated proteins have aa’s and prosthetic groups - Protein purification Start with large volumes, non-specific (batch methods) Finish with small volumes, specific (Chromatographic methods) You need a good source, an assay (specific-color based, non specific- buret assay for peptide bonds, dye assay) –Can be used for detection and quantification. Cell disruption (sonication, homogenation) Solubility methods (batch): ACIDIFICATION/ALKALINIZATION SALTING OUT + Easy + Easy +Good for large volumes + Does not ruin shape of protein -Non specific + Fairly mild -Harsh -Non specific -Protein loses its shape and function Size base methods (batch): DIALYSIS ULTRAFILTRATION + Mild + Mild + Good for large volumes + Fast -Non specific +Can be used to concentrate solutions -Slow -Non specific -Useful if you know size -More of a clean-up step than actual purification Size based (Chromatographic methods): GEL FILTRATION PAGE SEDIMENTATION VELOCITY + Mild + Increased resolution Proteins move + Used to estimate MW + Overcame shape/size through -Slow problem (SDS) homogeneous -Partially specific + Specific medium under -VERY Shape dependent +Very good for influence of -Size dependent characterization in centrifugal force. Large stuff is excluded addition to purification +Mild (Volume eluted) -Harsh + Used for Medium and Small (Total -Protein recovery almost characterization impossible Volume) are included + Preparative/Analytic al/ Recoverable -Precision less than other methods -Slow -Shape dependence -Rate of movement depends on molecular size Charge based methods (Chromatographic methods): ION EXCHANGE CHROMATHOGRAPHY ISOELECTRIC FOCUSING Least charged is eluted first + Very mild + Mild + Very specific + Fairly specific -Proteins sometimes precipitate + If modified can separate proteins with (Protein solubility is at its lowest when different amounts of negative charge the protein is neutral) -Retrieval sometimes difficult (can -Slow happen with concentrated salt solutions) -Small scale Other methods… EQUILIBRIUM SEDIMENTATION AFFINITY CHROMATOGRAPHY Heterogeneous medium under Column/Ligands centrifugal force. (Protein density – + Extremely specific Medium density) + Mild + Mild if sucrose gradient is used + Easy -Retrieval can be difficult + Good for separating proteins from other substances -Limited to proteins with recognition -BAD for protein-protein separations functions (ligands) -Slow - Limited to ligands that can be coupled Total amino acid composition does not help with the sequence, just tells what is in there
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