8-protein Flashcards
why is protein analysis important in food?
- nutritional quality
- functional properties
- economic consideration
ex. of functional properties
gliadin and glutenins in wheat flour for bread making,
casein in
milk for producing cheese products, egg albumin for foaming
ex. of economic consideration
the cost of certain commodities is based on the protein
content e.g. cereal grains and milk
what makes important to determine the protein content of food product?
The nutritional and functional implications of proteins in food system
what is protein?
Protein is a complex organic compound that consists of amino acids linked by polypeptide bond with characteristic structure.
how many structures does protein have? descibe
4
primary AAs
secondary a-helix
tertiary -polypeptide, b-globin polypeptide
quaternary- b-globin polypeptide, a-globin polypeptide
Characteristics of protein that is useful for their____
detection
20 AA can classify to ___ groups, based on ___, what are they
5, R group
- -nonpolar, aliphatic R groups
- -polar, uncharge R groups
- -aromatic R groups
- -positively charged R groups
- -negatively charged R groups
which AA is in nonpolar, aliphatic R groups?
glycine alanine valine leucine methionine isoleucine
which AA is in polar uncharged R groups?
serine threonine cysteine proline asparagine glutamine
which AA is in aromatic R groups?
phenylalamine
tyrosine
tryptophan
which AA is in + charged R groups?
lysine
arginine
histidine
which AA is in - charged R groups?
asparate
glutamate
how to form peptide bond?
COO- + H3N form H20
oc-NH
Proteins contain _____ in their structure.
C, H, O, N and S
______ is the most distinguishing element present in proteins.
Nitrogen (N)
Nitrogen content in various food proteins ranges from ___% to ___%.
13-19
The basic principles of these methods to measure protein content include ___? 5 point
1- the determination of N, 2- peptide bonds, 3- aromatic amino acids, 4- dye-binding capacity, 5- ultraviolet absorptivity of proteins,
% crude protein= ?
total N x conversion factor
total N include?
non protein nitrogen NPN
true protein nitrogen TPN
ex of NPN
- Free amino acids -Peptides
- Some phospholipids -Amino sugar -nucleic acids
- Urea
- Nitrates
- Nitrites
Proximate analysis measures the ____ content and not _____content.
crude protein content
true protein content
In proximate analysis, the _____ present in a food sample is multiplied by a ___to give the ____.
total nitrogen
factor
crude protein content
Total Nitrogen=?
“true-protein nitrogen” + “non-protein nitrogen”.
%Crude protein=?
Total (N) x Conversion factor.
what is conversion factor?
It is a Nitrogen-to-protein (N: P) conversion factor which assumes that 1Kg of plant or animal proteins contain a specific amount of Nitrogen.
Nitrogen content in various food proteins ranges from __% to __%. what does it mean?
13-19
This means that Nitrogen content ranges from 130g to 190g/ Kg protein.
factor= ?
%N x factor=?
100/%N in protein
100
The factor is used to convert __ to ___; Most proteins contain __% N, so the universal conversion factor would be ___.
% N to % crude protein
16%
6.25
which 2 methods can measure crude protein?
kjeldahl method
dumas method
how can crude p measured?
%N x factor
N is measured as ___ in kjeldahl method?
ammonia NH3
N is measured as ___ in dumas method?
element N2
what is the basic principle of Kjeldahl method?
Kjeldahl Method is based on the conversion of organic nitrogen (Total N) in the food sample to ammonia (NH3).
the step of Kjeldahl method?
- digestion: convert all N to NH3
2. measure NH3 by distillation followed by titration –colorimetric methods –ammonium NH4 electrode technique
write down 3 step on measuring NH3
digestion: sample+sulfuric acid+catalyst
neutralization&distillation: NaOH
titration:HCl
describe the digestion step
Protein nitrogen is release to form ammonium ions (NH4).
+
H2SO4 oxidizes organic matter and combines with NH4 to form nonvolatile
ammonium sulfate (NH4)2SO4 (very stable in acid Sol.).
Carbon and hydrogen elements are converted to CO2 and H2O.
what are the final products of digestion step?
CO2 H20 (NH4)2SO4 NH4+ SO4
what is the properties of (NH4)2SO4?
clear solution
very stable salt in acid solution
nitrogen is trapped as ammonium salt
what condition does digestion step need?
350-400 oc
catalyst
45min-2h
Catalyst speeds up _____; it serves as a _____in oxidation process.
digestion/ oxidation
O carrier
Catalysts used : _____ was originally used but it is___, so it is replaced by ___ or____
Mercury oxide (HgO)
toxic,
Selenium (Se)
3-Copper (Cu).
Boiling point of H2SO4 is ___0C, and it is not enough to convert ____ to ____; so _____ is used to increase the ___ of H2SO4.
290
all protein N to NH3
potassium sulfate (K2SO4)
bp
distillation technique: (NH4)2SO4+ 2NaOH or (KOH)—>
2NH4OH+ Na2SO4
distillation technique include?
distillation + titration
2 NH4OH–>
2NH3 + H2O
how to form NH3 from (NH4)SO4
(NH4)2SO4+NaOH form NH4OH NaSO4
NH4OH form 2NH3 + H2O
how to form NH3 from N?
protein N is release to form NH4+
NH4+ and H2SO4 for (NH4)2SO4
(NH4)2SO4+NaOH form NH4OH NaSO4
NH4OH form 2NH3 + H2O
The ammonia formed is distilled into a ___ solution
boric acid
NH3+H3BO3–>
NH4 + H2BO3-
Addition of NaOH or KOH to increase pH of solution to >__
9.2
Some NH4+ forms complexes with _____ (e.g. Cu-NH4 and Hg-NH4
complexes). Addition of _____ breaks these complexes
metals Sodium thiosulfate (Na2S2O3)
NH2-Hg+Na2S2O3–> with H+
NH3+HgS2O3
what is sodium thiosulfate?
Na2S2O3
____ is titrated with diluted HCl
Borate anion
H2BO3-+H+ –>
H3BO3
Moles of HCl=
moles of NH3
= moles of N in the sample
%N=?
N HCl x corrected acid volume x 14 / g of sample x 1000
x100
%N x 6.25=
% protein
colormetric methods: NH4 can form ___compounds with different reagents:
colored
2 reaction in colorimetric methods, what are they?
Nessler reaction
Berthelot reaction
write down the equation of Nessler reaction
4NH4OH + 2HgI2 + 4KI + 3KOH–> NH2Hg2IO+ 7KI + 2H2O
what is NH2Hg2IO? what color?
Ammonium dimercuric iodide, red-orange, 440nm
write down the equation of Berthelot reaction
NH3+phenol+hydrochloride–> indophenol
what is the color of indophenol
blue 630nm
Ammonium ion electrode: Ammonium ions are measured by ?
Ion Selective Electrode (ISE).
what is ISE
ion selective electrode
what does ISE measure?
ion activity which depends on ionic strength
an ISE is designed to respond to _____ but often other ions will_____.
is designed to respond to a specific ion but often other ions will interfere.
disadv of ISE
not use frequently
low reliability
expensive
adv of Kjeldahl method?
Widely used and internationally accepted (AOAC)
Used as standard method for comparison against all other methods High precision and good reproducibility
disadv of Kjeldahl method
Is not a measurement of the true protein content (low accuracy)
Different proteins need different convertion factors because they have
different amino acid sequences.
Use of hazardous reagents
Time consuming to carry-out.
dumas is also known as
combustion method
dumas is based on?
the conversion of organic nitrogen in the food sample to N2.
dumas: which two essential steps does it involve?
combustion
measurement of N2
dumas burn in _oC, with __ gas
875-900
oxygen
the product after sampleN–> with O2 ,875-900
NO, NO2,N2O, H2O,SO2,N2,CO2
NO,NO2,N2O ____ of NOx BY ___ , get __
reduction
Cu
N2
measure N2 to get ___
%total N
what is needed for measurement? dumas
KOH
mercury seal
nitrometer
thermal conductivity dector
write down the steps of dumas method
- Samples (approximately 100–500 mg) are weighed into a tin capsule
- Introduced to a combustion reactor
- Combustion under high temperature (9000C) in presence of catalyst (CuO/V2O5)
- Volatile decomposition products (mainly molecular nitrogen, nitrogen oxides, carbon dioxide, and water vapour) are transported by the carrier gas
- Nitrogen oxides are reduced to molecular nitrogen and the excess of oxygen is bound to the copper or tungsten in the reduction column at a high temperature 6000C
- Water is removed by means of a condenser filled with magnesium perchlorate, diphosphorus pentoxide or other drying agents
- Interfering compounds (e.g. volatile halogen and sulphur compounds) are removed by absorbents materials (e.g. silver wool)
- The nitrogen in the residual gas mixture, consisting of nitrogen and carrier gas, is passed through a thermal conductivity detector.
describe CPR, what is C
C-combustion tube at 900oC
CuO: oxidize sample to CO2,NOX, H2O and other gases
describe CPR, what is P
P-post combustion tube at 900oC
CuO/Pt: oxidize CH4 and CO drom incompl comb.
Ag wool remove halogen
describe CPR, what is R
R-reduction tube at 830oC
tungsten: turn NOx toN2, remove O2 and sulfur comp
CuO/Cu: traces of CO and NOx turn into CO2 and N2
zinc: remove halogen and sulfur comp.
what is adulterant used to ?
increase the N content in food
how can food adulterated? ex.
with urea, ammonia, and melamine to increase the N content for economic benefits
can Kjeldahl and Dumas detect adulteration?
no
what does CuO DO?
Oxidize sample to CO2, H2O AND NOx
what does CuO/Pt DO?
oxidize CH4 and CO from imcompl. comb
what does Ag wool DO?
remove halogen
what does tungsten DO?
turn NOx to N2, remove O2 and sulfur comp
what does CuO/Cu DO?
trace CO and NOx turn into CO2 and N2
what does zinc DO?
remove halogen and sulfur comp.
how to determine non protein nitrogen? which reagent is used?
TCA trichloro acetic acid
how to determine non protein nitrogen? steps
- Precipitate out proteins from a solution or dry sample using 10% TCA
- Mix the reaction mixture thoroughly and let precipitate settle for 5 min.
- Filter the mixture through Whatman No. 1 filter paper or centrifuge at 30,000g to retain the proteins while other components are washed away.
- Determine nitrogen content of the filtrate by the Kjeldahl method.
- Convert nonprotein nitrogen to protein equivalent with conversion factor.
how to Precipitate out proteins from a solution or dry sample
10% TCA
Determine nitrogen content of the filtrate by the ___method.
Kjeldahl
what does Biuret method depends on?
on the formation of a violet to purple color when Cu+2 ions form a complex with peptide bonds under alkaline conditions.
Biuret method procedure
mixing of the sample with Biuret reagent and stand for 15-30 min, then the absorbance is measured at 540nm using spectro-photometer
what does biuret reagent contain?
(CuSO4 + NaOH + potassium sodium tartrate)
protein sample+ biret reagent form?
chelate complex
Biuret Reagent: what does NaOH/KOH do?
provide the alkaline conditions.
Biuret Reagent: what does CuSO4 do?
provides Cu+2 ions.
Biuret Reagent: what does potassium sodium tartrate do?
stabilizes the complex.
what does + test show?
From Blue to purple- proteins present.
From Blue to Pink- peptides present.
what does - test show?
no color change
This method is ____ to Peptide bonds. But not ____
This method is selective to Peptide bonds. But not sensitive
Only _____are measured
proteins and peptides
does Biuret reagent contain Biuret?
no
why It is named ‘Biuret reagent’
it gives a positive test when it reacts with biuret.
lowry method combines the ____ reagent with _____ reagent ((phosphomolybdic acid + phosphotungstic acid)
combines the Biuret reagent with Folin-Ciocalteau phenol reagent ((phosphomolybdic acid + phosphotungstic acid)
what does Folin-Ciocalteau phenol reagent contain?
((phosphomolybdic acid + phosphotungstic acid)
lowry method is sensitive to ?
ph
ph should be 10-10.5
Phosphomolybdotungstate is reduce to?
heteropolymolybdenum
how is CU+ produced?
after reduction of Cu2+ by peptide bonds is complexed with Folin-Ciocalteau phenol reagent
what does it form?
a stable a bluish color which can be measured at 500 nm or 750 nm.
why reading 750nm enhance specificity?
minimum the interfere
This method is selective to?
peptide bond and the amino acids
tryptophan and tyrosine.
Lowry Normally used for?
quantifying purified/extracted protein
what reduce specificity?
Interference from buffers, drugs, nucleic acids, and sugars
More sensitive to ___ concentrations (0.01 mg of protein/mL) of
proteins than the biuret method.
low
what is bca method?
Bicinchoninc Acid Method
BCA:Proteins reduce __into __under __conditions; then the Cu+1 reacts with BCA reagent to form a __complex which is measured colorimetically at __nm.
Proteins reduce cupric ions (Cu+2) into cuprous ions (Cu+1) under alkaline conditions; then the Cu+1 reacts with BCA reagent to form a purple complex which is measured colorimetically at 562nm.
what contribute the color formation with BCA
Peptide bonds and 4 amino acids (Cysteine, cystine, tryptophan, and tyrosine)
write 2 steps of BCA
- protein+Cu2+ –> with OH- to form Cu1+(temp dependent)
2. Cu1+ react with BCA reagent to form a purple complex
Anionic Dye-Binding Method: The protein-containing sample is mixed with a known _____ in a ____solution.
a known excess of a negatively charged (anionic) dye
buffer
Anionic Dye-Binding Method: The proteins form an __complex with the dye. why?
insoluble
because of the electrostatic attraction and the unbound (free) dye remains soluble.
Anionic Dye-Binding Method: The remaining amount of the unbound dye is ___(e.g. by ___) and then determined by measuring its ___.
separate
centrifugation
absorbance
Anionic Dye-Binding Method: write down the steps
- protein+excess dye–>protein-dye complex (insoluble) and unbound soluble dye
- separation them by filtration or centrifugation
- the supernatant contain unbound soluble dye, measure it
- bound dye=excess dye-unbound dye
Anionic Dye-Binding Method: give an ex of dye
anionic sulfonic acid dye
Anionic Dye-Binding Method:The amount of the unbound dye is ___ related to the ___ content of the sample
inversely
protein
Anionic Dye-Binding Method: Anionic dye binds _____ and the ___
cationic groups of the basic amino acid residues
free amino terminal group of the protein
Bradford method:Coomassie Brilliant blue G-250 binds ____ to proteins; this leads to changing the dye’s color from___to ___
electrostatically
reddish to bluish.
Bradford method: The change in the absorbance at __nm is ___to the protein concentration of the sample.
The change in the absorbance at 595nm is proportional to the protein concentration of the sample.
Bradford method steps
- protein -basic and aromatic side chain+ coomassie G-250
- form protein-dye complex, change color from red to blue
- measure at 595nm
Ultraviolet 280nm Absorption Method: Proteins (solubilized in buffer or alkali) show ____ in the region of ultraviolet radiation at 280 nm, primarily due to __ and ___ residues in the proteins.
Proteins (solubilized in buffer or alkali) show strong absorption in the region of ultraviolet radiation at 280 nm, primarily due to tryptophan and tyrosine residues in the proteins.
Ultraviolet 280nm Absorption Method: what kind of protein?
aromatic aa
what are + aa
his arg lys
what is the major adisadv of ultraviolet 280nm absorption method?
nucleic acids also absorb UV strongly at 280 nm and could therefore interfere with the measurement of the protein.
A=abc
absorbance=absorptivity* cell path length*concentration
