Lab 1: Spectrophotometrc Estimation of Protein Concentration

Question 1

Proteins are polymers of ____

Answer 1

amino acids

Question 2

What kind of bonds link amino acids together to form a chain?

Answer 2

peptide bonds

Question 3

a chain of amino acids is called a

Answer 3

polypeptide chain

Question 4

a fibrous protein is a _____ structure that is held together via ______ type of bonds. What’re the two structure shapes of a fibrous protein?

Answer 4

a fibrous protein is a SECONDARY structure that is held together via HBOND type of bonds.

2 structures: alpha helix and beta pleated sheet

Question 5

in the alpha helix, H bonding occurs where?

Answer 5

within the polypeptide chain

Question 6

in the beta sheet, H bonding occurs where?

Answer 6

between sheets of polypeptide

Question 7

How are globular proteins held together?

Answer 7

by various interactions between the R groups of the amino acids.

Question 8

A protein that has more than one polypeptide chain is known to have a ____ level of structure

Answer 8

a QUATERNARY level of structure.

Question 9

2 reasons as to why knowing the concentration of protein in a biological system is important.

Answer 9

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.

Question 10

before knowing the true amount of enzymatic activity in a sample, you must:

Answer 10

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

Question 11

3 main tests to determine protein concentration

Answer 11

1) colorimetric
2) UV spec
3) NanoDrop Spec

Question 12

Two main colorimetric tests that are good for quantifying proteins

Answer 12

1) biuret

2) lowry

Question 13

General mechanism of a colorimetric test

Answer 13

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.

Question 14

T/F the copper ions used in colorimetric tests will stain free Amino Acids as well as proteins

Answer 14

False. Free AAs do not react with copper ions. Only protein can be measured

Question 15

T/F: Cu ions are specific to the type of protein in a substance and can indicate purity

Answer 15

false. All types of proteins will be stained by copper. It will not detect purity.

Question 16

How does the biuret test work?

Answer 16

uses copper SULFATE to create a violet color when it comes in contact with peptide bonds.

Question 17

T/F: A biuret test is specific to the type of protein

Answer 17

false. It is a type of colorimetric test. It will stain to all proteins

Question 18

Which colorimetric test is more sensitive?

Answer 18

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

Question 19

How does the lowry test work?

Answer 19

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.

Question 20

How does the lowry test have increased sensitivity?

Answer 20

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.

Question 21

How does UV spectrophotometry quantify protein? Which specific amino acids are involved?

Answer 21

tryptophan, tyrosine and phenylalanine absorb in the UV range, allowing for quantification

Question 22

How does NanoDrop quantify protein?

Answer 22

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.

Question 23

T/F a standard curve is needed for both UV spec and Nanodrop

Answer 23

false. You need a standard curve for UV but there is already a preprogrammed standard curve for nanodrop

Question 24

why did we make multiple dilutions of the protein sample prior to conducting any type of spectrophotometry

Answer 24

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.

Question 25

pros and cons of the lowry colorimetric test

Answer 25

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?

Question 26

pros and cons of the biuret test?

Answer 26

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.

Question 27

nanodrop test pros and cons

Answer 27

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?

Question 28

UV spec pros and cons

Answer 28

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