Lecture 9 - Protein (part 2)

Question 1

what is the dumas method based on?

Answer 1

the conversion of organic N in the food sample to N2

Question 2

what are the 2 steps in the dumas method?

Answer 2

1. combustion

2. measurement of elemental N2

Question 3

what color does the biuret method form when ____ ions form a complex with ____ under ____ conditions?

Answer 3

1. violet to purple
2. Cu2+
3. peptide bonds
4. alkaline

Question 4

what is the biuret reagent?

Answer 4

CuSO4 + NaOH + potassium sodium tartrate

Question 5

procedure in biuret reagent?

Answer 5

1. mix sample with biuret reagent
2. allow it to stand for 15-30 mins
3. measure absorbance at 540nm

Question 6

what are the most common conversion factors that are used?

Answer 6

6.25 and 6.38

Question 7

in the biuret reagent, what is the role of NaOH or KOH?

role of copper sulfate?

role of potassium sodium tartrate?

Answer 7

1. NaOH or KOh: provides alkaline conditions
2. CuSO4: provides Cu2+ ions
3. potassium sodium tartrate: stabilizes the complex

Question 8

what type of complex is formed in the biuret method?

what ion is in the middle?

Answer 8

a chelate complex

contains a Cu2+ ion

Question 9

what does a positive or negative biuret test look like?

Answer 9

positive:

• blue to purple = proteins present
• blue to pink = peptides present

negative: no change in color

Question 10

the biuret method is ____ to peptide bonds but not ____

Answer 10

this method is (selective) to Peptide bonds. But not (sensitive)

only proteins and peptides are measured

Question 11

does biuret reagent contain biuret?

Answer 11

no

it is named biuret reagent because it gives a positive test when it reacts with biuret

Question 12

what is the lowry method?

Answer 12

combines the biuret reagent with folin-ciocalteau phenol reagent

Question 13

what is the folin-coicalteau phenol reagent?

Answer 13

phosphomolybdic acid and phosphotungstic acid

Question 14

what color is formed in this method?? how is it formed?

Answer 14

1. Cu1+ is produced after reduction of Cu2+ by peptide bonds
2. complexed with folin-ciocalteau phenol reagent
3. forms a BLUISH color (measured at 500nm or 750nm)

Question 15

in the lowry method, what is phosphomolybdotungstate reduced to?

Answer 15

heteropolymolybdenum

Question 16

the lowry method reaction is _____ sensitive

Answer 16

pH

pH should be 10-10.5

Question 17

in the lowry method, reading at 750nm enhances specificity because…?

Answer 17

noise from the matrix is less because most of the other organic molecules don’t absorb at 750nm

Question 18

The lowry method _____ sensitive to low concentrations of protein?

Answer 18

more sensitive

Question 19

the lowry method is selective to ____ and the amino acids ____ and ____

Answer 19

1. peptide bonds

2. tyrptophan and tyrosine

Question 20

what is the lowry method normally used for?

Answer 20

quantifying purified or extracted protein

interference from buffers, drugs, nucleic acids and sugars reduce specificity

Question 21

what reaction happens in the BCA method?

what does BCA stand for?

Answer 21

BCA = bicinchonic acid method

reaction:
1. proteins reduce cupric ions (Cu+2) into cuprous ions (Cu1+) under alkaline conditions
2. Cu1+ reacts with BCA reagent to for a purple complex, measured at 562nm

Question 22

peptide bonds and what 4 AA contribute to the color formation in BCA?

Answer 22

cysteine
cystine
typtophan
tyrosine

Question 23

describe the anionic dye binding method

Answer 23

the protein containing sample is mixed with negatively charged anionic dye in a buffered solution

proteins form an insoluble complex with the dye b/c of the electrostatic attraction and unbound dye remains soluble

the remaining amount of unbound dye is separated and determined by measuring absorbance

Question 24

in the anionic dye binding method, the anionic dye binds with what?

Answer 24

binds with the cationic groups of the basic AA residues (imidazole of histidine, guanidine or arginine and e-amino group of lysine) and the free amino terminal group of the protein

Question 25

in the anionic dye binding method, how is the amount of unbound dye related to the protein content of the sample?

Answer 25

inversely related

because more binding to amino groups means less dye

Question 26

describe the bradford method

what color change occurs?

what absorbance is it measured at?

how is the change in absorbance related to the protein concentration of the sample?

Answer 26

coomassie brilliant blue G-260 binds electrostatically to proteins.

color change: red becomes blue

absorbance is measured at 595nm

change in absorbance is proportional to protein conc of the sample

Question 27

describe the ultraviolet 280nm absorption method

what amino acids causes absorption?

what is the major disadvantage of this method?

Answer 27

proteins (solubilized in buffer or alkali) absorb in the region of UV radiation at 280nm due to tryptophan and tyrosine residues in the proteins

disadvantage: nucleic acids also absorb UV strongly at 280nm. This could interfere with protein measurement