Chapter 10 Amino acid & Protein Analysis Qijun Wang 2005-4-12
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Transcript of Chapter 10 Amino acid & Protein Analysis Qijun Wang 2005-4-12
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Chapter 10 Amino acid & Protein
Analysis
Qijun Wang2005-4-12
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Chapter 10-1 Amino Acids Analysis
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What is amino acid?
What is amino acid? Amino Acid: aminated carboxylic acid (R-
COOH) R grou
p
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NH2CH2COOH-amino acetic acid
NH2CH2CH2COOH
-amino propanoic acid
NH2CH2CH2CH2CH2CHCOOHNH2
, -2 amino caproic acid
Examples
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Classification of Amino Acid
1. By the location of Amino-group : / / -AA2. By its acidity : neutral/ acidic/ basic AA ratio of Amino-group to carboxylic group3. By whether containing phenyl group aromatic / non aromatic AA 4. By its occurrence in protein Protein / non protein AA5. By polarity of R group : polar / apolar side chain AA 6. By its nutrient value to human: Essential AA and non-essential AA
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20 -AA commonly found in proteins
-aa structure Cn name En name甘氨酸丙氨酸
缬氨酸 *
亮氨酸 *
异亮氨酸 *
glycine
alanine
valine
leucine
isoleucine
NH2
CH2COOHNH2
CHCOOHCH3
NH2
CHCOOHCH3 CHCH3
NH2
CHCOOHCH3 CHCH3
CH2
NH2
CHCOOHCH3 CH2
CH3
CH
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Continued -aa structure Cn name En name
蛋氨酸 *
脯氨酸苯丙氨酸 *
色氨酸 *
丝氨酸
methionine
proline
phenylalanine
tryptophan
serine
NH2
CHCOOHCH3SCH2CH2
NHCHCOOH
CH2
CH2CH2
NH2
CHCOOHCH2
NH2
CHCOOHCH2N
HNH2
CHCOOHCH2HO
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continued-aa structure Cn name En name
苏氨酸 *
半胱氨酸
酪氨酸天门冬酰胺
谷酰胺
threonine
cystine
tyrosine
asparagine
glutamine
NH2
CHCOOHHO
CH3CHNH2CHCOOHCH2HS
NH2CHCOOHCH2HO
NH2CHCOOHCOCH2NH2
NH2CHCOOHH2NCOCH2CH2
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continued-aa structure Cn name En name
天门冬氨酸谷氨酸
赖氨酸 *
精氨酸组氨酸
Aspartic acid
Glutamic acid
lysine
arginine
histidine
CH2
NH2CHCOOHHOOC
CH2CH2
NH2CHCOOHHOOC
NH2CHCOOHH2NCH2CH2CH2CH2
NH2CHCOOHCH2CH2CH2H2N
NHC NH
=
H
NH2CHCOOHCH2
NN
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Physical characteristics
ALL the pure -AAs are Colorless crystal , with quite high melting point (>200 deg.C) and water solubility,
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AA are ampholyte
Anion, when at high pH
zwitterion, when at isoelectric point (pI)
Cation, when at low pH
NH2 CH COO-R
H+
OH- NH3+ CH COO-
RH+
OH-
RCHCO2HNH2
RCHCO2-
N+H3H
NaOH
HCL
NOTE: peptide or protein also have both acid and base properties. They share the same property of being positively charged at low pH and negatively charged at high pH.
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Isolectric Point (pI) of AAs
Anion in basic sol’n.
RCHCO2HNH2
RCHCO2-
N+H3
H+RCHCO2HN+H3
HO-RCHCO2-
NH2
+++++++++
----
-
-
-
-
--
Zwitterion in pI sol’n. No move to either of Electrode. And with Lowest solubility
Cation in acidic sol’n.
pH 溶液<pI
pH 溶液>pI
anode cathode
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Note on pI
The Acidity of neutral AA is stronger than its Basicity, which means the dissociation degree of reaction (i) is larger that of reaction(ii). Therefore the pH of neutral AA water solution is less than 7 。
H2N-CHR-COOH +H2O H2N-CHR-COO- +H3O+ (i)
H2N-CHR-COOH +H2O H3N+-CHR-COOH +OH-
(ii) * therefore, [anion] >[cation], to get pI, more acid is to
be added. pI of neutral AAs is around 5.6~6.3 , pI of acidic AAs is around 2.8~3.2 ; pI of basic AAs is around 7.6~10.8 。
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Ninhydrin Reaction
RCHCOOH
NH2
O
O
OH
OH+
O
O
N
O
O
+ RCHO + CO2 + 3H2O
Note:
1, Ninhydrin solution is made in basic solution of phosphate buffer (pH8.04).
2, The reaction products of all AAs except proline, are purpule-bule(540nm), and for proline is yellow(440nm)
3, protein and peptide also can occur this reaction due to their containing amino group.
4. This reaction can be used to quantitative and qualitative analysis, with the help of spectrophotometer, TLC.
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Automatic AA analyzerFrom pH2.2 to pH 6.4
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Separation principles
NH2CHCOOHH2NCH2CH2CH2CH2
CH2
NH2CHCOOHHOOC
Separate aspartic acid and lysine by cation exchange resin.
H+-type ion exchange Resin: consists of relatively chemically inert polymer, which has quite strong acidic side-chain constituents such as –SO3H,-CH2SO3H
*suitable for A/B/Neutral conditions-
+
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Automatic AA analyzer Absorbent: cation ion exchange resin , Eluting sol’n: citric acid buffer of
pH2.2,pH3.3,pH4.0 and pH6.4 Extracted and evaporated AAs is
required to be dissolved in pH2.2 citric acid.
Eluting order: acidic AAs,polar AAs, apolar AAs, and basic. For AAs in a same catalog, low mass AA is eluted out first.
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Aromatic AAs
Aromatic AAs absorb light in the near ultraviolet (230-300nm).
NH2CHCOOHCH2HO
NH2
CHCOOHCH2
NH2
CHCOOHCH2N
H
phenylalanine
tyrosine
tryptophan
Note: This UV absorption property of protein is solely determined by the content of these 3 aromatic AAs. However, far ultraviolet (190nm)absorption of protein stems from the peptide bond.
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AAs assay by GC
Principle:
C CR
H
NH2
O
OH + C H9OH4 O
O
NH3Cl
H
R CC 4H9C
-++ H2O
+ -C H94C CR
H
NH3Cl
O
O + (CF3C
O
) O2 C H94C CR
H O
O
N
H O
C CF3
HCl100
Non-volatile AA
Volatile AA-derivate
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Chapter 10-2 PROTEIN ANALYSIS
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Protein Analysis
What is protein: polymer of 20 - amino acids, with mol.wt from 5000
to1000,000 daltons. N is most distinguished element: among the
composing elements of C,H, N, O, S, for some proteins: P, Cu, Fe, I.
N content in different proteins ringing from 13.4% -19.1%, and averagely 16%.
Therefore protein coefficient is 6.25 for most proteins. 5.70 is only for wheat and its products proteins according to AOAC method.
Most abundant component in cells: 50% of dry cells by weight
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Cereals : ( % ) Brown Rice 7.9 Polished rice 7.1Wheat flour, whole-grain 13.7Corn flour, whole-grain 6.9Corn starch 0.3legumes :Soybean, raw 36.5 Beans, kidney, raw 23.6Tofu, raw, regular 8.1Fruits & vegetables :Apple, raw, with skin 0.2Strawberry, raw 0.6lettuce , raw 1.0
Meat, poultry, fish: Beef 18.5Dry beef 29.1Chicken, breast meat, raw 23.1Ham 17.6Egg, raw, whole 12.5Finfish, raw, 17.9Dairy products :Milk, whole, fluid 3.3Milk, skim, dry 36.2Cheese, cheddar 24.9Yogurt 5.3
* High quality protein ?
Protein content in food (%)
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Conversion factors for FoodsN to Protein conversion factorsFoods factorsEgg or meat 6.25Dairy products 6.38Wheat 5.70Other cereal grains and oilseeds 6.25Almonds 5.18Peanuts 5.46Other tree nuts and coconut 5.30
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Proteins functions
NO proteins no life! 1, Structural proteins: Such as keratin; myosin, actin; glycoprotein, lipoprotein, 2, biological active proteins : Such as: Enzymes, hemoglobin, myoglobin, ferritin, antibody, glycoprotein, lipoprotein
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1. According to whether containing non-proteins components :
Simple protein: only containing AAs upon hydrolysis, such as Egg Albumin; myosin, actin, insulin;
Conjugated protein: AAs + non-AAs upon hydrolysis; Such as lipoprotein; glycoprotein; hemoglobin,
ferritin; majority of enzymes
2. According to theirs solubility: Non-water soluble protein: filament protein: myosin,
actin, keratin Water soluble proteins: hydrophilic groups outsides
(apolar groups), and hydrophobic groups (-OH, -SH, -COOH,-NH2) insides, most global proteins, enzymes.
* Enzymes working conditions : mild conditions 。
Classification
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Main Properties of Protein
1. As polymer of AAs, proteins have both acid and base properties. zwitterion & pI& electrophoresis.
2. Most proteins are water soluble, and unable pass through dialysis membrane.
3. Denaturation: denaturants such as heat, acid, alkali, salt, detergents can altered solubility and functional properties of proteins.
reversible/non-reversible denaturation.4. Ultraviolet absorption at 280 nm, due to the
presence of 3 aromatic acids residues, i.e. tyrosine, tryptophan, phenylalanine.
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Principles: 1. Digest the organic compounds with strong sulfuric acid
in the presence of catalysts while heating. 2. The total organic N is converted to ammonium
sulphate. 3. Neutralize the digested sol’n with abundant alkali.
Here, the N is converted to ammonium hydroxide, and then being distilled into a boric acid solution and converted to ammonium borate.
4. Titrate ammonium borate with strong acid. (please notice that N: HCl = 1:1)5. N content in proteins is averagely 16%.
Kjeldahl’s method
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催化剂
煮沸
1. Digestion
NCOC + H2SO4 ( NH4 ) 2SO4 + CO2 + SO2 + H2O
2. Neutralization &distillation
2NaOH + ( NH4 ) 2SO4 2NH3↑+Na2SO4 + 2H2O
3. Absorption by boric acid :
2NH3 + 4H3BO3 ( NH4 ) 2B4O7 + 5H2O
4. Titration by strong acid
( NH4 ) 2B4O7 + 5H2O + 2HCl 2NH4Cl + 4H3BO3
( * NCOC, N containing organic compounds, N:HCl = 1:1)
Principles of Kjeldahl’s method
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Apparatus used in Kjeldahl
I. Digestion apparatusII. Distillation & absorption apparatus
(I) (II)
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1. Amount of protein sample and reagents used should be proportional.
2. All the working solution should be prepared with ammonia-free distilled water
3. Mildly heating When digestion, so that no sample to spatter onto flask wall.
4. Rotate the flask while digestion. 5. Add antifoam (silica oil) if necessary.6. 30% hydrogen peroxide can accelerate
the digestion. 7. At the end of fully digestion, the solution
should be clear light-blue or greenish.
Points that need your close attention
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Points that need your close attention
8. Digestion should be carried out in a ventilating cabinet.
9. Connect well the distillation apparatus before adding alkali into digested solution.
10. Add abundant alkali until there are red copper hydroxide formed.
11. Absorption solution should be less than 40 deg.C throughout the absorption. Cold water bath is a good choice to lower the temp.
12. Using indicating paper to help for the determination of distillation terminus.
13. Indicators of methylene blue and methyl red are added to absorption bottle before carrying on the distillation.
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Other methods for protein assay
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Micro Kjeldahl Method
Notes: 1. Applicable to all types of foods; 2. accurate, as an official method for crude protein content.3. poorer precision than the biuret method.
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Q : 1. Is this suitable for AAs? 2. what is Biuret Reaction ?
The Biuret Method
O=CNH2
NHO=C
NH2
2 CuSO4
NaOH
O=CNH2
NHO=C
NH2Na
OH
OH
HNC=O
C=O
NaH2N
OH
H2N
OH
Cu
Principle: This reaction is characterized by the development of a purple coloration from the complexing of cupric ions with peptide bonds in an alkaline medium. The wavelength , however, varies with the nature of the protein: from 540 to 650 nm, often at 550 nm.
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Application and advantages Applications: Suitable for cereals, meat, soybean, and isolated proteins.
But not for milk, as reducing sugars (lactose) can reduce copper ion. Advangtages: 1. less expensive than Kjeldahl method, rapid, simplest methods for protein
analysis. 2. color deviations are encountered less frequently than with Folin-Lowry,
UV absorption, or turbidimetric methods. 3. very few substances other than proteins in foods interfere with the biuret
reaction. 4. Detect N only from the peptide and protein sources. Disadvantages: 1. Not very sensitive as compared to the Folin-Lowry Method. Require at
least 2-4 mg protein for assay. 2. Bile pigments, high conc of ammonium salts interferes with the reaction.3. Color varies with different proteins. Gelatin gives a pinkish-purple color.4. Not an absolute method: color must be standardized against known
proteins(e.g., BSA) * Introduction of 30% isopropyl alcohol can reduce the reaction time from
35 to 10 mins. You also can try “Heating”.
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Dumas (N combustion) Principle: Samples are combusted at high temp (700-1000 deg.C). The N
released is quantitated by GC using a thermal conductivity detector (TCD).
Procedure:Samples (100-500 mg) are weighed into a tin capsule and
introduced to a combustion reactor in automated equipment. The N released is measured by a built-in GC.
Advantages: 1, Applicable for All proteins; 2, No hazardous chemicals; 3, Saving time: within 3 mins; 4, High performance:
recent automated instruments can analyze up to 150 samples without attention.
Disadvantages:Measures total organic nitrogen, not just Protein N.
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Principle:• Folin reagent ( phosphomolybdic and
phosphotungstic acid ) is reduced to a blue molybdenum complex, mainly by the phenolic groups of tryptophan and tyrosine.
2. Lowry greatly increased the sensitivity of the determination by preceding the reaction by pretreatment with a copper reagent in a basic medium. (Mistake in Sol’n B at p163)
The lowry’s Method (Folin-phenol method)
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Procedures of lowry’s Method1. Dilute protein sample to contain 20-100 ug.2. K Na tartrate-Na2CO3 solution is added after
cooling and incubated at RT for 10 min.3. CuSO4- K Na tartrate-NaOH solution is added
after cooling and incubated at RT for 10 min.4. Freshly prepared Folin Reagent is added, then
the reaction mixture is mixed and incubated at 50 deg.C for 10 min.
5. Absorbance is read at 650 nm.6. A standard curve of BSA is carefully constructed
for estimating protein conc of the unknown.
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Applications: Widely used in protein biochemstry, because of its simplicity and
sensitivity. But not widely used in Food proteins analysis without extracting proteins from the food mixtrue.
Advantages:1. 50-100 times more sensitive than biuret method, and 10-20 times than
280 nm UV absorption method.2. Less affected by turbidity of the sample.3. More specific than most other method. 4. Relatively simple, can be done in 1-1.5 hours. Disadvantages:1. Color varies with different proteins to a greater extent than the biruet
method. 2. Color is not strictly proportional to protein conc.3. The reaction is Interfered with to a varying degree by sucrose, lipids,
phosphate buffers, monosaccharides, and hexoamines.4. high conc., of reducing sugars, ammonium salfate, and sulfhydryl
compounds interfere with the reaction.
Properties of lowry’s Method
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UV 280 nm absorption MethodPrinciple: 1. Proteins show strong absorption at UV 280 nm, primarily due to
aromatic amino acids of tryptophan and tyrosine residues in proteins. 2. The content of Try and Tyr in proteins from each food sources is fairly
constant. Thereby, the extinction coefficient(E280) or molar absorptivity (Em) must be determined for individual proteins for protein content estimation.
Applications: 1. Used for protein content of meat and milk product. This technique is
better applied in a purified protein system or to proteins that have been extracted in alkali or denaturing agents such as 8 M urea.
Advantages: 1. Rapid; 2. Non interference from ammonium sulfate. 3. Non destructive. Disadvantages: 1. aromatic amino acids contents in proteins from various food sources
differs considerably!
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Stain with Coomassie blue
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1. ReagentsCoomassie Blue R-250Methanol Glacial acetic acid 2. Gel Stainning solution, 1 L 1.0 g Commassie Blue R-250 450 ml water 450 ml methanol 100 ml Glacial acetic acid3. Gel destaining solution, 1 L 100 ml Methanol 100 ml Glacial acetic acid 800 ml Water 4. Staining Procedure 1. Pick up the gel into (20 ml, usually enough) Staining solution in a container and agitate for 10 min for 0.75 mm Gel and 20 min and 1.5 mm gel. The staining solution can be reused several times.2. Take the gel out and rinse the gel with a few changes of water in a new container3. Add 50 ml destaining solution. Strong bands are visiable immediately on a light box, and 1 hour usually is enough.4. To destain completely, change destaining solution 2-3 times and agitate overnight.5. Scan or take photo to record the result.
Coomassie Blue staining procedures
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2-DE: the technique to separate proteins in the first dimension according to their isoelectric point, by Isoelectric Focusing(IEF), and in the second dimension according to their molecular weight, by SDS-PAGE. 2-DE combined with protein identification basing on microsequencing, amino acid composition and Mass spectrometry, provides an invaluable tool for proteomic studies.
Step 1 — Sample PrepStep 2 — First-Dimension (IEF) Separation Step 3 — Second-Dimension (SDS-PAGE) Separation Step 4 — Protein Detection by Staining /Destaining
Two dimensional Electrophoresis
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THANKS