7 - Wednesday, October 7th, 2009 Biochemistry 405 Lecture...

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1 Wednesday, October 7 th , 2009 Biochemistry 405 – Lecture #4 Kane Hall 130 10:30- 11:20 am Lecturer: Wim Hol Slide Set #1
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2 Outline 1. Primary Structure. - The universe of proteins. 2. Protein purification techniques. - Assay for the protein of interest - Separation techniques. - Electrophoresis - Mass spectrometry. 3. Evolutionary studies using protein sequences. Proteins: Primary Structure, Purification, and Evolution
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3 Gly Ser ASP His Ala Val Ile Leu Met Phe Tyr Pro Gln Cys Thr Asn Glu Lys Arg Trp The Twenty Amino Acids – Ultra-schematic The Building Blocks of All Proteins
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Linking amino acids by forming peptide units. The order of the amino acids is called the “Primary Structure” of a protein A Polypeptide Chain
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5 How many proteins are possible? The average protein chain is ~ 400 residues in length. At each position any of the 20 amino acids could occur, so that the number of possibilities is: 20 400 = 2.6 x 10 520 . The number of atoms in the universe is estimated as 9 x 10 78 . So the number of possible proteins of length 400 residues would exceed the universe in size by many orders of magnitude. Protein sizes range from ~ 40 to ~ 3,000 residues. VVP 2/e Fig on p. 94 VVP 3/e Fig on p. 91
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6 Proteins chains differ considerably in length From the third column you see that proteins often contain multiple chains. The last column gives only the Molecular Mass of a single chain! You have to know that the average Molecular Mass of an amino acid residue is about 110. (You do not need to know the names and numbers in the Table – unless they come back explicitly in later lectures)
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7 Concentration determination of a specific protein It is important to find a rapid & reliable assay for the specific protein you wish to purify. Popular methods are: - Enzymatic activity (But your protein needs to be an enzyme). - Antigenic specificity such as ELISA (Enzyme-Linked Immunosorbent Assay) (But you need to generate specific anti-bodies).
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8 Protein concentration determination and A 280 Proteins usually contain several Trp or Tyr or Phe residues. The side chains of Trp and Tyr absorb quite strongly UV light at 280 nm Hence, ideal for concentration determination by “absorption spectroscopy” Hence this method is often used. But not all proteins contain Trp or Tyr or Phe and then other methods are needed And the UV method is not very sensitive, rarely lower than 50 to 100 μ g per mL (Note that the vertical scale is logarithmic)
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9 Protein concentration determination Coomassie brilliant blue binds to proteins. In acidic solutions, the absorbance shifts from 465 to 595 nm upon binding to proteins. Hence the 595 nm absorbance provides a way to measure protein concentration In the so-called “Bradford” assay, which uses this absorption shift of Coomassie, protein concentrations as low as 1 μ g per mL can be detected
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10 Protein Production Very often large amounts of proteins are required for 3D structure determination or drug screening: these usually require multi-milligram
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This note was uploaded on 06/03/2010 for the course BIOL BIO 101 taught by Professor Drumheller during the Spring '10 term at University of Washington.

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7 - Wednesday, October 7th, 2009 Biochemistry 405 Lecture...

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