Lecture 9 - Protein (part 2) Flashcards
what is the dumas method based on?
the conversion of organic N in the food sample to N2
what are the 2 steps in the dumas method?
- combustion
2. measurement of elemental N2
what color does the biuret method form when ____ ions form a complex with ____ under ____ conditions?
- violet to purple
- Cu2+
- peptide bonds
- alkaline
what is the biuret reagent?
CuSO4 + NaOH + potassium sodium tartrate
procedure in biuret reagent?
- mix sample with biuret reagent
- allow it to stand for 15-30 mins
- measure absorbance at 540nm
what are the most common conversion factors that are used?
6.25 and 6.38
in the biuret reagent, what is the role of NaOH or KOH?
role of copper sulfate?
role of potassium sodium tartrate?
- NaOH or KOh: provides alkaline conditions
- CuSO4: provides Cu2+ ions
- potassium sodium tartrate: stabilizes the complex
what type of complex is formed in the biuret method?
what ion is in the middle?
a chelate complex
contains a Cu2+ ion
what does a positive or negative biuret test look like?
positive:
- blue to purple = proteins present
- blue to pink = peptides present
negative: no change in color
the biuret method is ____ to peptide bonds but not ____
this method is (selective) to Peptide bonds. But not (sensitive)
only proteins and peptides are measured
does biuret reagent contain biuret?
no
it is named biuret reagent because it gives a positive test when it reacts with biuret
what is the lowry method?
combines the biuret reagent with folin-ciocalteau phenol reagent
what is the folin-coicalteau phenol reagent?
phosphomolybdic acid and phosphotungstic acid
what color is formed in this method?? how is it formed?
- Cu1+ is produced after reduction of Cu2+ by peptide bonds
- complexed with folin-ciocalteau phenol reagent
- forms a BLUISH color (measured at 500nm or 750nm)
in the lowry method, what is phosphomolybdotungstate reduced to?
heteropolymolybdenum
the lowry method reaction is _____ sensitive
pH
pH should be 10-10.5
in the lowry method, reading at 750nm enhances specificity because…?
noise from the matrix is less because most of the other organic molecules don’t absorb at 750nm
The lowry method _____ sensitive to low concentrations of protein?
more sensitive
the lowry method is selective to ____ and the amino acids ____ and ____
- peptide bonds
2. tyrptophan and tyrosine
what is the lowry method normally used for?
quantifying purified or extracted protein
interference from buffers, drugs, nucleic acids and sugars reduce specificity
what reaction happens in the BCA method?
what does BCA stand for?
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
peptide bonds and what 4 AA contribute to the color formation in BCA?
cysteine
cystine
typtophan
tyrosine
describe the anionic dye binding method
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
in the anionic dye binding method, the anionic dye binds with what?
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
in the anionic dye binding method, how is the amount of unbound dye related to the protein content of the sample?
inversely related
because more binding to amino groups means less dye
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?
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
describe the ultraviolet 280nm absorption method
what amino acids causes absorption?
what is the major disadvantage of this method?
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
