Lab 1: Spectrophotometrc Estimation of Protein Concentration Flashcards
Proteins are polymers of ____
amino acids
What kind of bonds link amino acids together to form a chain?
peptide bonds
a chain of amino acids is called a
polypeptide chain
a fibrous protein is a _____ structure that is held together via ______ type of bonds. What’re the two structure shapes of a fibrous protein?
a fibrous protein is a SECONDARY structure that is held together via HBOND type of bonds.
2 structures: alpha helix and beta pleated sheet
in the alpha helix, H bonding occurs where?
within the polypeptide chain
in the beta sheet, H bonding occurs where?
between sheets of polypeptide
How are globular proteins held together?
by various interactions between the R groups of the amino acids.
A protein that has more than one polypeptide chain is known to have a ____ level of structure
a QUATERNARY level of structure.
2 reasons as to why knowing the concentration of protein in a biological system is important.
1) to measure the activity of an enzyme. You must standardize the activity to the amount of protein present.
2) the measure the nutritional state of a tissue.
before knowing the true amount of enzymatic activity in a sample, you must:
You must standardize the activity to the amount of protein present. One sample may have a lot more activity than the other, but if the amount of protein present is higher, the activity may actually just be equivalent to a sample with a slightly smaller amount of activity but a lesser amount of overall protein
3 main tests to determine protein concentration
1) colorimetric
2) UV spec
3) NanoDrop Spec
Two main colorimetric tests that are good for quantifying proteins
1) biuret
2) lowry
General mechanism of a colorimetric test
Copper ions and a base will form a purple complex in the presence of two or more peptide bonds, allowing for quantification via spectrophotometry.
- a standard curve (absorbance vs concentration) by reacting copper ions+base with samples of KNOWN protein concentration allows you to determine the concnetration of an unknown protein by using the SLOPE.
T/F the copper ions used in colorimetric tests will stain free Amino Acids as well as proteins
False. Free AAs do not react with copper ions. Only protein can be measured
T/F: Cu ions are specific to the type of protein in a substance and can indicate purity
false. All types of proteins will be stained by copper. It will not detect purity.
How does the biuret test work?
uses copper SULFATE to create a violet color when it comes in contact with peptide bonds.
T/F: A biuret test is specific to the type of protein
false. It is a type of colorimetric test. It will stain to all proteins
Which colorimetric test is more sensitive?
Lowry test is more sensitive and can detect lower concentrations of protein than the biuret. the biuret will only give accurate readings if the concentration of protein is high
How does the lowry test work?
uses copper IONS and FOLIN REAGENT to create a blue color when it comes in contact with peptide bonds. Is still not specific to a type of protein, but it is more SENSITIVE than the biuret and can detect protein at a lower concentration.
How does the lowry test have increased sensitivity?
because of the addition of the folin reagent. The folin reagent reacts with tyrosine and phenol rings, in addition to the copper ions reacting with the peptide bonds, showing a more drastic color change, allowing for detection of smaller protein concentrations.
How does UV spectrophotometry quantify protein? Which specific amino acids are involved?
tryptophan, tyrosine and phenylalanine absorb in the UV range, allowing for quantification
How does NanoDrop quantify protein?
it is like UV spec and relies on aromatic AA’s to absorb at the UV range, however, it does not use cuvettes, they use surface tensions of the sample to form a micro-column between the pedestals where light can shine through.
T/F a standard curve is needed for both UV spec and Nanodrop
false. You need a standard curve for UV but there is already a preprogrammed standard curve for nanodrop
why did we make multiple dilutions of the protein sample prior to conducting any type of spectrophotometry
because some samples owuld not fit on the standard curve- they may be too dilute or too concentration. we needed to find one whose absorbances fall within the appropriate linear range.
pros and cons of the lowry colorimetric test
pros;
- more sensitive
- easy to conduct
- sensitive and good for small protein concentration
cons;
- more reagents needed because you need to folin reagent.
- long incubation time for the folin reagent.
- will give inaccurate readings if concentration of protein is too hgh
- folin reagent is toxic
- sensitivity hinges on tyrosine being present, what if the sample doesn’t contain tyrosine?
pros and cons of the biuret test?
pros:
- less reagent needed than the lowry
- fast
- results were seemingly closer to the nanodrop
- shorter incubation time because no folin reagent
cons;
- not as sensitive
- will give inacurrate readings if the protein concentration is too low.
nanodrop test pros and cons
pros
- only small amount of sample needed
- no reagents needed
- very fast, no standard curve needed
cons
- expensive
- uses aromaticity of amino acids to abaorb at UV range, what if the protein doesn’t have much AA’s?
UV spec pros and cons
pros
-less time consuming, no reagents needed
cons
- uses aromativity of AA’s to absorb at UV range, what if the protein doesn’t have much AA’s
