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FDSC 300 > Lecture 8 - Protein analysis > Flashcards

Lecture 8 - Protein analysis Flashcards

1
Q

why do we analyze for protein content in food?

A
  • nutritional quality
  • functional properties
  • economic consideration
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2
Q

defne a protein

A

complex organic compound that consists of amino acids linked by polypeptide bonds

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3
Q

what characteristics of proteins are useful for their detection?

A
  • determination of N
  • peptide bonds
  • aromatic amino acids
  • dye-bind capacity
  • UV absorption of proteins
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4
Q

write the equation for % crude protein

A

% Crude protein = total (N) x conversion factor

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5
Q

what is total N comprised of?

A

non-protein nitrogen + protein nitrogen

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6
Q

what are examples of non-protein nitrogens?

A
  • free amino acids
  • peptides
  • some phospholipids
  • amino sugar
  • nucleic acids
  • urea
  • nitrates
  • nitrites
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7
Q

true or false: proximate analysis measures the crude protein content and not the true protein content

A

true

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8
Q

again, give the equations for crude protein and total nitrogen

A

% crude protein = total (N) x conversion factor

total nitrogen = true protein nitrogen + non-protein nitrogen

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9
Q

what’s a conversion factor (crude protein)?

A

nitrogen-to-protein conversion factor that assumes that 1 Kg of plant or animal proteins contain a specific amount of N

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10
Q

what methods can be used to determine crude protein?

A

1) Kjeldahl (N measured as ammonia NH3)

2) Dumas (N measured as molecular N2)

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11
Q

describe the 2 steps of the Kjeldahl method

A

1) digestion (conversion of all N to NH3)

2) measurement of NH3 (distillation & titration, colorimetric, ammonium electrode)

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12
Q

what reagents are required during the digestion, neutralization, distillation, and titration steps of the Kjeldahl method?

A 
Digestion
- sulfuric acid
- K2SO4
- catalyst
Neutralization & Distillation
- NaOH
- Sodium thiosulfate (Na2S2O3)
- distillation in boric acid solution
Titration
- HCl
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13
Q

what’s the purpose of H2SO4 in Kjeldahl method?

A
  • 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
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14
Q

at what temperature is the digestion step of the Kjeldahl method done? how long?

A

350-400C; 25 min- 2h

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15
Q

true or false: the digestion step of the Kjeldahl method releases toxic gases

A

true

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16
Q

sample (N) + conc. H2SO4 –>?

A

CO2, NH4, SO4, H2O

then NH4 reacts with H2SO4 to create a stable ammonium salt

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17
Q

what does the catalyst do in the Kjeldahl method? examples?

A

serves as a O carrier in oxidation process to speed up oxidation

  • Mercury oxide (HgO)
  • Selenium
  • Copper
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18
Q

why is K2SO4 added during the digestion step of the Kjeldahl method?

A

increases boiling point of H2SO4 from 290 to where it can function

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19
Q

describe the distillation step of the Kjeldahl method

A
  • 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
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20
Q

describe the titration step of the Kjeldahl method

A

borate anions are titrated with dilute HCL to form H3BO3

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21
Q

what is the molar ratio of HCl and NH3? what method am I talking about?

A

1:1. this is the Kjeldahl method.

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22
Q

list some colorimetric methods of determining ammonium

A

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

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23
Q

Ammonium ions can be measured by ISE. what’s ISE stand for?

A

Ion Selective Electrode.

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24
Q

what do ISEs measure?

A

ion activity, which depends on ionic strength.

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25
Q

why aren’t ISEs used that much in determining NH3?

A

low reliability due to interfering ion activity and it’s expensive

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26
Q

list advantages of the Kjeldahl method

A
  • internationally accepted by AOAC
  • used as standard method for comparison against all other methods
  • high precision and good reproducibility
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27
Q

list disadvantages of the Kjeldahl method

A
  • does NOT measure TRUE protein content
  • different proteins need different conversion factors because they have different amino acid sequences
  • hazardous reagents used
  • time consuming
28
Q

on what principle does the Dumas method operate?

A

conversion of organic nitrogen in the food sample to N2

29
Q

what are the 2 general steps of the Dumas method?

A

1) combustion

2) measurement of elemental N2

30
Q

how do you combust your sample for the dumas method?

A

put the sample at 875-900C in the presence of a catalyst (CuO/V2O5)

31
Q

what compounds of interest are produced after the combustion step in the dumas method?

A

molecular nitrogen, nitrogen oxides

CO2 and H2O is also produced

32
Q

what’s the purpose of the catalyst in combustion during Dumas?

A
  • reduces nitrogen oxides to molecular nitrogen

- excess oxygen bound to Cu or tungsten in reduction column at high temp (600C)

33
Q

how is water removed from the sample in the dumas method?

A

condenser filled with magnesium perchlorate, diphosphorus pentoxide or other drying agents

34
Q

how do you remove interfering compounds in the dumas method?

A

silver wool (absorbent material)

35
Q

what are considered interfering compounds?

A

volatile halogen, sulphur compounds

36
Q

there’s 3 tubes used for the dumas method. what are they?

A
  • combustion tube
  • post combustion tube
  • reduction tube
37
Q

what happens in the combustion tube?

A
  • temp = 900C

- CuO oxidizes sample to NOx, CO2, H2O, and other gases

38
Q

what happens in the post combustion tube

A
  • temp = 900C
  • CuO/Pt oxidizes CH4 and CO from incomplete combustion
  • silver wool removes interfering compounds (halogens, sulphur compounds)
39
Q

what happens in the reduction tube?

A
  • 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
40
Q

what are adulterants can be added to increase N content?

A
  • urea
  • ammonia
  • melamine
41
Q

can the Kjeldahl and Dumas methods detect adulterants

A

nope

42
Q

what can the TCA method measure?

A

non-protein nitrogen (adulterants)

43
Q

describe the TCA method

A
  • 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
44
Q

expand TCA

A

trichloro acetic acid

45
Q

what kind of chemistry is the Biuret method based on?

A

complexing of Cu with peptide bonds

46
Q

describe the Biuret method

A
  • 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
47
Q

what do each of the reagents in the Biuret reagent do?

A

NaOH (or KOH) = alkaline conditions
CuSO4 = provider of Cu2+ ions
potassium sodium tartrate = complex stabilizer

48
Q

true or false: Biuret method is not selective but sensitive

A

false
- selective
- not sensitive
only proteins and peptides are measured

49
Q

true or false: biuret reagent contains biuret

A

false. it only gives a positive test when proteins/peptides react with it

50
Q

what’s the lowry method?

A

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)

51
Q

why does reading at 750 nm for the lowry method enhance specificity?

A

more sensitive to low concentrations of proteins

52
Q

for what purpose is the lowry method used?

A

quantify purified/extracted proteins

not good if buffers, drugs, nucleic acids, and sugars are present (reduces specificity)

53
Q

expand BCA

A

bicinchonic acid method

comes in the form of a sodium salt

54
Q

what kind of chemistry is the BCA method based on?

A

copper chemistry: the reduction of cupric (Cu2+) to cuprous ions (Cu1+)

55
Q

describe the BCA method

A
  • proteins reduce copper (Cu2+ to Cu1+)
  • cuprous ions react with BCA reagent –> purple complex
  • measure at 562 nm
56
Q

what is the BCA method selective for?

A
  • peptide bonds
  • cysteine
  • cystine
  • tryptophan
  • tyrosine
57
Q

what’s the anionic dye-binding method????????

A
  • 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
58
Q

bound dye = ?

A

excess dye - unbound dye

59
Q

list examples of anionic dyes

A
  • sulfonic acid
  • acid orange 12
  • orange G
  • amido black 10B
60
Q

what do anionic dyes bind to?

A 
basic amino acid residues
- imidazole of histidine
- guanidine of arginine
- e-amino group of lysine
free amino terminal group of protein
61
Q

the amount of unbound dye is ______ related to protein content of the sample

A

inversely

62
Q

what’s the bradford method?

A

we did this in lab

  • uses coomassie brilliant blue reagent
  • changes from red to BLUE
  • measure at 595 nm
63
Q

(referring to bradford method)

the change in absorbance at 595 nm is ______ to the protein concentration of the sample

A

proportional

64
Q

what proteins show strong absorption in the region of UV radiation (280 nm)

A

tryptophan, tyrosine

65
Q

what’s the major drawback of the UV absorbance method??

A

nucleic acids also absorb UV light strongly at 280 nm and can act as interferences

FDSC 300 (9 decks)

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