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Proteins.pptAmino Acids and Proteins
Structure of -amino acids
The 20 Amino Acids Found in Proteins
Formation of a Peptide
Polypeptide backbone
9.bin
10.bin
- Proteins are made of 20 amino acids linked by peptide bonds
- Polypeptide backbone is the repeating sequence of the N-C-C-N-C-C… in the peptide bond
- The side chain or R group is not part of the backbone or the peptide bond
Proteins
- Make up about 15% of the cell
- Have many functions in the cell
- Enzymes
- Structural
- Transport
- Motor
- Storage
- Signaling
- Receptors
- Gene regulation
- Special functions
Motor- myosin
Storage- ferritin, transport- hemoglobyn
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Importance of Proteins
- Main catalysts in biochemistry: enzymes (involved in virtually every biochemical reaction)
- Structural components of cells (both inside and outside of cells in tissues)
- Regulatory functions (if/when a cell divides, which genes are expressed, etc.)
- Carrier and transport functions (ions, small molecules)
Levels of Protein Structure
- Primary Structure - amino acid sequence in a polypeptide
- Secondary Structure - local spatial arrangement of a polypeptide’s backbone atoms (without regard to
side chain conformation)
- Tertiary Structure - three-dimensional structure of entire polypeptide
- Quaternary Structure - spatial arrangement of subunits of proteins composed of multiple polypeptides (protein complexes)
3-D Structure of Myoglobin
- People with proteinuria have urine containing an abnormal amount of protein. The condition is often a sign of kidney disease.
- Healthy kidneys do not allow a significant amount of protein to pass through their filters. Kidney disease often has no early symptoms. One of its first signs may be proteinuria that's discovered by a urine test done during a routine physical exam. Blood tests will then be done to see how well the kidneys are working.
- Both diabetes and high blood pressure can cause damage to the kidneys, which leads to proteinuria.
Proteinuria (Protein in Urine)
Proteinuria (Protein in Urine)
Methods of Protein Estimation
Quantitative
- Biruet method
- Bradford method
- Folin-Lowry method
- Kjeldahl method
- Bicinchoninic acid method (BCA method)
- UV method
- Flourimetric method
- Mass spectrometry
Protein Determination assay
Bicinchoninic Acid (BCA) method
Principle
- The BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantification of total protein.
- This method combines the well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium (the biuret reaction)
- Highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a unique reagent containing bicinchoninic acid.
- The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion.
- This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20-2,000 μg/ml).
The reaction of BCA with cuprous ion. Two molecules of BCA bind to each molecule of copper that had been reduced by a peptide-mediated biuret reaction.
Spectrophotometry
- Determines concentration of a substance in solution
- Measures light absorbed by solution at a specific wavelength
- The amount of light radiation absorbed by a sample is measured
- The light absorbed is directly related to the concentration of the specific compounds in a sample
- As concentration increases light absorbed increases linearly
- The data obtained from the transmittance of light is converted by the spectrophotometer into absorbance
Lamp
Io I
A = 0.012
l
Monochromator
Cuvette
Detector
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0 25 250 500 1000 2000
μg/mL
Abs
2
1
0.5
0.25
0.15
0.1
0
Unknown Sample
ABS
0.4
650μg/mL
Standard curve
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Assignment
- Please finish the quiz within the class hour. This quiz will be counted as your attendance.
- Please write the lab report based on the video and results. Please calculate the protein concentration of unknown samples based on the standard curve(which already provided in the pdf documents) The example of how to make the standard curve has been put under the module.
- In the discussion part, please discuss the harms of proteinuria, how to prevent it by our diets.
