Lecture 8 - Protein analysis Flashcards
why do we analyze for protein content in food?
- nutritional quality
- functional properties
- economic consideration
defne a protein
complex organic compound that consists of amino acids linked by polypeptide bonds
what characteristics of proteins are useful for their detection?
- determination of N
- peptide bonds
- aromatic amino acids
- dye-bind capacity
- UV absorption of proteins
write the equation for % crude protein
% Crude protein = total (N) x conversion factor
what is total N comprised of?
non-protein nitrogen + protein nitrogen
what are examples of non-protein nitrogens?
- free amino acids
- peptides
- some phospholipids
- amino sugar
- nucleic acids
- urea
- nitrates
- nitrites
true or false: proximate analysis measures the crude protein content and not the true protein content
true
again, give the equations for crude protein and total nitrogen
% crude protein = total (N) x conversion factor
total nitrogen = true protein nitrogen + non-protein nitrogen
what’s a conversion factor (crude protein)?
nitrogen-to-protein conversion factor that assumes that 1 Kg of plant or animal proteins contain a specific amount of N
what methods can be used to determine crude protein?
1) Kjeldahl (N measured as ammonia NH3)
2) Dumas (N measured as molecular N2)
describe the 2 steps of the Kjeldahl method
1) digestion (conversion of all N to NH3)
2) measurement of NH3 (distillation & titration, colorimetric, ammonium electrode)
what reagents are required during the digestion, neutralization, distillation, and titration steps of the Kjeldahl method?
Digestion - sulfuric acid - K2SO4 - catalyst Neutralization & Distillation - NaOH - Sodium thiosulfate (Na2S2O3) - distillation in boric acid solution Titration - HCl
what’s the purpose of H2SO4 in Kjeldahl method?
- oxidizes organic matter and combines with NH4+ to form nonvolatile ammonium sulfate (NH4)2SO4, which is stable in acid solution
- C and H elements converted to CO2 and H2O
at what temperature is the digestion step of the Kjeldahl method done? how long?
350-400C; 25 min- 2h
true or false: the digestion step of the Kjeldahl method releases toxic gases
true
sample (N) + conc. H2SO4 –>?
CO2, NH4, SO4, H2O
then NH4 reacts with H2SO4 to create a stable ammonium salt
what does the catalyst do in the Kjeldahl method? examples?
serves as a O carrier in oxidation process to speed up oxidation
- Mercury oxide (HgO)
- Selenium
- Copper
why is K2SO4 added during the digestion step of the Kjeldahl method?
increases boiling point of H2SO4 from 290 to where it can function
describe the distillation step of the Kjeldahl method
- ammonium salt (NH4)2SO4 is distilled with NaOH to increased the pH of solution to 9.2. this forms NH4OH + Na2SO4
- NH4OH breaks down into NH3 and H2O in the presence of heat
- ammonia is distilled into boric acid solution (H3BO3) to produce NH4 and H2BO3- (borate anion)
- sodium thiosulfate (Na2S2O3) is added to break NH4+ metal complexes
describe the titration step of the Kjeldahl method
borate anions are titrated with dilute HCL to form H3BO3
what is the molar ratio of HCl and NH3? what method am I talking about?
1:1. this is the Kjeldahl method.
list some colorimetric methods of determining ammonium
NH4 can form colored compounds with different reagents:
Nessler reaction: forms red-orange. measured at 440 nm
Berthelot Reaction: forms blue. measured at 630 nm
Ammonium ions can be measured by ISE. what’s ISE stand for?
Ion Selective Electrode.
what do ISEs measure?
ion activity, which depends on ionic strength.
why aren’t ISEs used that much in determining NH3?
low reliability due to interfering ion activity and it’s expensive
list advantages of the Kjeldahl method
- internationally accepted by AOAC
- used as standard method for comparison against all other methods
- high precision and good reproducibility
list disadvantages of the Kjeldahl method
- does NOT measure TRUE protein content
- different proteins need different conversion factors because they have different amino acid sequences
- hazardous reagents used
- time consuming
on what principle does the Dumas method operate?
conversion of organic nitrogen in the food sample to N2
what are the 2 general steps of the Dumas method?
1) combustion
2) measurement of elemental N2
how do you combust your sample for the dumas method?
put the sample at 875-900C in the presence of a catalyst (CuO/V2O5)
what compounds of interest are produced after the combustion step in the dumas method?
molecular nitrogen, nitrogen oxides
CO2 and H2O is also produced
what’s the purpose of the catalyst in combustion during Dumas?
- reduces nitrogen oxides to molecular nitrogen
- excess oxygen bound to Cu or tungsten in reduction column at high temp (600C)
how is water removed from the sample in the dumas method?
condenser filled with magnesium perchlorate, diphosphorus pentoxide or other drying agents
how do you remove interfering compounds in the dumas method?
silver wool (absorbent material)
what are considered interfering compounds?
volatile halogen, sulphur compounds
there’s 3 tubes used for the dumas method. what are they?
- combustion tube
- post combustion tube
- reduction tube
what happens in the combustion tube?
- temp = 900C
- CuO oxidizes sample to NOx, CO2, H2O, and other gases
what happens in the post combustion tube
- temp = 900C
- CuO/Pt oxidizes CH4 and CO from incomplete combustion
- silver wool removes interfering compounds (halogens, sulphur compounds)
what happens in the reduction tube?
- temp = 830C
- tungsten turns NOx to N2; removes O2 and sulfur compounds
- CuO/Cu: traces of CO and NOx turned into CO2 and N2
- zinc removes halogens and sulfur compounds
what are adulterants can be added to increase N content?
- urea
- ammonia
- melamine
can the Kjeldahl and Dumas methods detect adulterants
nope
what can the TCA method measure?
non-protein nitrogen (adulterants)
describe the TCA method
- stick sample (solution or dry sample) into TCA solution. let precipitate for 5 mins
- filter mixture and centrifuge at 30,000g to retain proteins while other components are washed away
- determine nitrogen content by Kjeldahl method
- convert nonprotein N to protein equivalent with conversion factor
expand TCA
trichloro acetic acid
what kind of chemistry is the Biuret method based on?
complexing of Cu with peptide bonds
describe the Biuret method
- mix sample with biuret reagent
- biuret reagent = CuSO4 + NaOH + Potassium sodium tartrate
- if the solution goes from blue to purple or pink, proteins or peptides are present
- measure absorbance at 540 nm
what do each of the reagents in the Biuret reagent do?
NaOH (or KOH) = alkaline conditions
CuSO4 = provider of Cu2+ ions
potassium sodium tartrate = complex stabilizer
true or false: Biuret method is not selective but sensitive
false
- selective
- not sensitive
only proteins and peptides are measured
true or false: biuret reagent contains biuret
false. it only gives a positive test when proteins/peptides react with it
what’s the lowry method?
biuret: who are you?
lowry: i’m you but better. i’m blue dabadee dabadai
the lowry method uses Folin-Ciocalteau phenol reagent which stabilizes the complexes formed
measured at 500 or 750 nm*(we’ll go over this in another card)
why does reading at 750 nm for the lowry method enhance specificity?
more sensitive to low concentrations of proteins
for what purpose is the lowry method used?
quantify purified/extracted proteins
not good if buffers, drugs, nucleic acids, and sugars are present (reduces specificity)
expand BCA
bicinchonic acid method
comes in the form of a sodium salt
what kind of chemistry is the BCA method based on?
copper chemistry: the reduction of cupric (Cu2+) to cuprous ions (Cu1+)
describe the BCA method
- proteins reduce copper (Cu2+ to Cu1+)
- cuprous ions react with BCA reagent –> purple complex
- measure at 562 nm
what is the BCA method selective for?
- peptide bonds
- cysteine
- cystine
- tryptophan
- tyrosine
what’s the anionic dye-binding method????????
- based on insoluble complexes formed between proteins and dyes because of electrostatic attraction
- known excess of negatively charged dye is used to assess sample. the amount of unbound (soluble) dye is measured using its absorbance
bound dye = ?
excess dye - unbound dye
list examples of anionic dyes
- sulfonic acid
- acid orange 12
- orange G
- amido black 10B
what do anionic dyes bind to?
basic amino acid residues - imidazole of histidine - guanidine of arginine - e-amino group of lysine free amino terminal group of protein
the amount of unbound dye is ______ related to protein content of the sample
inversely
what’s the bradford method?
we did this in lab
- uses coomassie brilliant blue reagent
- changes from red to BLUE
- measure at 595 nm
(referring to bradford method)
the change in absorbance at 595 nm is ______ to the protein concentration of the sample
proportional
what proteins show strong absorption in the region of UV radiation (280 nm)
tryptophan, tyrosine
what’s the major drawback of the UV absorbance method??
nucleic acids also absorb UV light strongly at 280 nm and can act as interferences
