Lab 3

Question 1

general definition of protein?

Answer 1

large biological molecules consisting of one or more long chains of amino acid residues

Question 2

protein structure

Answer 2

• primary structure:linear conformation
• secondary structure: alpha helix and beta sheets
• tertiary structure: three dimensional
• quaternary structure: three dimensional structure of multiple subunit

Question 3

four spectroscopic methods:

Answer 3

• Measurements of the protein’s intrinsic UV absorbance
• The Lowry
• Smith Assay
• The Bradford dye assay

Question 4

UV absorbance

Answer 4

-proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm

PROBLEMATIC BECAUSE:
-because different protiens have widely varying absorption characteristics there may be considerable error, especially for unknown or protein mixture

• requires solution that is free of other UV absorbing agent
• Quartz cuvette required for most accurate readings (expensive)

Question 5

lowry and smith test

Answer 5

-based on reduction of Cu+2 and Cu+1 by amides

PROBLEMATIC:
-careful preparation of several reagents

• length, precisely timed incubations
• immediate absorbance measurements required
• sensitive to other chemicals used in preparation especially SDS which is usually used to dissolve samples

Question 6

Bradford assay

Answer 6

• based on the ability of the coomassie brilliant blue dye to bind to proteins in acidic solution
• Measure the concentration of protein in solution

under acidic conditions the red form of dye is converted into its bluer form to bind to the protein being assayed

ADVANTAGES

• faster with fewer mixing steps
• no heating required
• not expensive

DISADVANTAGES:

• affected by reagents involved in preparation such as detergent
• better or more sensitive for lower concentrations of proteins 20-2000 microg/ml

Question 7

Coomassie brilliant blue

Answer 7

• two variants: R-250, or G-250
• at different pH, the dye has different colors
• max absorbance at 595 nm

Question 8

spectrophotometery

Answer 8

• measures the intensity of light beam before and after it passes through a sample
• transmittance(T) of a sample is the ratio f intensity of the light that has pass through the sample (I) to the intensity of the light before it passes the sample I/I0

absorbance- is a measure of the capacity of a substance to absorbs light of a specified wavelength. It is equal to logarithm of reciprocal of the transmittance.

absorbance=log10(I0/I)