10 amino acid and protein analysis

8/13/2019 10 Amino Acid and Protein Analysis

1/44

Chapter 10

Amino acid & Protein Analysis

Qijun Wang

2005-4-12


2/44

Chapter 10-1Amino Acids Analysis


3/44

What is amino acid?

What is amino acid? Amino Acid: aminated carboxylic acid (R-COOH)

R group


4/44

NH2CH2COOH

-aminoacetic acid

NH2CH2CH2COOH

-aminopropanoic acid

NH2CH2CH2CH2CH2CHCOOH

NH2

-2 amino caproic acid

Examples


5/44

Classification of Amino Acid

1. By the location of Amino-group / / -AA2. By its acidityneutral/acidic/ basic AA

ratio of Amino-group to carboxylic group

3. By whether containing phenyl group

aromatic / non aromaticAA

4. By its occurrence in protein

Protein/ non proteinAA

5. By polarity of R group :

polar/ apolarside chain AA

6. By its nutrient value to human:

EssentialAA and non-essentialAA


6/44

20 -AA commonly found in proteins

-aa structure Cn name En name

***

glycine

alanine

valine

leucine

isoleucine

NH2

CH2COOH

NH2

CHCOOHCH3

NH2

CHCOOHCH3 CH

CH3

NH2

CHCOOHCH3 CH

CH3

CH2

NH2

CHCOOHCH3 CH2

CH3

CH


7/44

Continued


*

*

*

methionine

proline

phenylalanine

tryptophan

serine

NH2

CHCOOHCH3SCH2CH2

NH

CHCOOH

CH2

CH2CH2

NH2

CHCOOHCH 2

NH2

CHCOOHCH2

N

H

NH2

CHCOOHCH2HO


8/44

continued


*

threonine

cystine

tyrosine

asparagine

glutamine

NH2

CHCOOH

HO

CH3CH

NH2

CHCOOHCH2HS

NH2

CHCOOHCH2HO

NH2

CHCOOHCOCH2NH2

NH2

CHCOOHH2NCOCH2CH2


9/44

continued


*

Asparticacid

Glutamic

acid

lysine

arginine

histidine

CH2

NH2

CHCOOHHOOC

CH2CH2

NH2

CHCOOHHOOC

NH2

CHCOOHH2NCH 2CH2CH2CH2

NH2

CHCOOHCH2CH2CH2H2N

NH

C NH

=

H

NH2

CHCOOHCH2

N

N


10/44

Physical characteristics

ALL the pure -AAs are Colorless

crystal, with quite high meltingpoint (>200 deg.C) and watersolubility,


11/44

AA are ampholyte

Anion,when athigh pH

zwitterion, whenat isoelectricpoint (pI)Cation,when atlow pH

NH2 CH COO-

RH+

OH- NH3

+CH COO

-

R

H+

OH-

RCHCO2H

NH2

RCHCO2-

N+H3

H

NaOH HCL

NOTE: peptide or protein also have both acid and base properties.They share the same property of being positively charged atlow pH and negatively charged at high pH.


12/44

Isolectric Point (pI) of AAs

Anion inbasic soln.

RCHCO2H

NH2

RCHCO2-

N+H3

H+

RCHCO2H

N+H3

HO-

RCHCO2-

NH2

++

+++++

++

--

--

-

-

-

-

--Zwitterion inpI soln Nomove to eitherof ElectrodeAnd with Lowestsolubility

Cation inacidic soln.

pH pI

anode cathode


13/44

Note on pI

TheAcidity of neutral AAis stronger than itsBasicity, which means the dissociation degreeof reaction (i) is larger that of reaction(ii).Therefore the pH of neutral AA water solutionis 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 beadded.

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


14/44

Ninhydrin Reaction

RCHCOOH

NH2

O

O

OH

OH

+

O

O

N

O

O

+ RCHO + CO2+ 3H2O

Note:1, Ninhydrin solution is made in basic solutionof 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 totheir containing amino group.

4. This reaction can be used to quantitative and qualitative

analysis, with the help of spectrophotometer, TLC.


15/44

Automatic AA analyzer

From pH2.2 to pH 6.4


16/44

Separation principles

NH2

CHCOOHH2NCH 2CH2CH2CH2

CH2

NH2

CHCOOHHOOC

Separate aspartic acid and lysine by cation exchangeresin.

H+-type ion exchange Resin:consists of relatively chemicallyinert polymer, which has quitestrong acidic side-chainconstituents such as SO3H,-CH2SO3H*suitable for A/B/Neutral conditions

-+


17/44

Automatic AA analyzer

Absorbent: cation ion exchange resin Eluting soln: citric acid buffer ofpH2.2,pH3.3,pH4.0 and pH6.4 Extracted and evaporated AAs is required tobe dissolved in pH2.2 citric acid. Eluting order: acidic AAs,polar AAs, apolarAAs, and basic. For AAs in a same catalog,low mass AA is eluted out first.


18/44

Aromatic AAs

Aromatic AAsabsorb light in the nearultraviolet (230-300nm).

NH2

CHCOOHCH2HO

NH2

CHCOOHCH 2

NH2

CHCOOHCH2

N

H

phenylalanine tyrosine

tryptophan

Note: This UV absorption property ofproteinis solely determined bythe content of these 3 aromaticAAs. However, far ultraviolet(190nm)absorption of protein stemsfrom the peptide bond.


19/44


20/44

Chapter 10-2PROTEIN ANALYSIS


21/44

Protein Analysis

What is protein:

polymerof 20 -amino acids, with mol.wt from 5000to1000,000 daltons.

N is most distinguishedelement: among the

composing elements of C,H, N, O, S, for someproteins: P, Cu, Fe, I.

N content in different proteins ringing from 13.4% -19.1%, and averagely 16%.

Therefore protein coefficientis 6.25 for mostproteins. 5.70 is only for wheat and its productsproteins according to AOAC method.

Most abundant component in cells: 50% of dry cellsby weight


22/44

Cereals %Brown Rice 7.9

Polished rice 7.1

Wheat flour, whole-grain 13.7

Corn flour, whole-grain 6.9

Corn starch 0.3

legumesSoybean, raw 36.5

Beans, kidney, raw 23.6

Tofu, raw, regular 8.1Fruits & vegetablesApple, raw, with skin 0.2

Strawberry, raw 0.6

lettuce , raw 1.0

Meat, poultry, fish:Beef 18.5

Dry beef 29.1

Chicken, breast meat, raw 23.1

Ham 17.6

Egg, raw, whole 12.5

Finfish, raw, 17.9

Dairy productsMilk, whole, fluid 3.3

Milk, skim, dry 36.2Cheese, cheddar 24.9

Yogurt 5.3

* High quality protein ?

Protein content in food (%)


23/44

Conversion factors for Foods

N to Protein conversion factorsFoods factors

Egg or meat 6.25

Dairy products 6.38Wheat 5.70

Other cereal grains and oilseeds 6.25

Almonds 5.18Peanuts 5.46

Other tree nuts and coconut 5.30


24/44

Proteins functions

NO proteins no life!

1, Structural proteins:

Such as keratin; myosin, actin;glycoprotein, lipoprotein,

2, biological active proteinsSuch as: Enzymes, hemoglobin,myoglobin, ferritin, antibody,

glycoprotein, lipoprotein


25/44

1. According to whether containing non-proteinscomponents :

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 conditionsmild conditions

Classification


26/44

Main Properties of Protein

1. As polymer of AAs, proteins have both acidand base properties.zwitterion & pI&electrophoresis.

2. Most proteins are water soluble, and unable

pass throughdialysis membrane.3. Denaturation: denaturantssuch as heat, acid,

alkali, salt, detergents can altered solubilityand 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.


27/44

Principles:

1. Digestthe organic compounds with strong sulfuric acidin the presence of catalysts while heating.

2. The total organic Nis converted to ammonium sulphate.

3. Neutralizethe digested soln with abundant alkali. Here,the N is converted to ammonium hydroxide, and thenbeing distilled into a boric acid solution and convertedto ammonium borate.

4. Titrate ammonium borate with strong acid.(please notice that N: HCl = 1:1)

5. N content in proteins is averagely 16%.

Kjeldahls method


28/44

1. Digestion

NCOC+ H2SO4 NH42SO4+ CO2+ SO2+ H2O2. Neutralization &distillation

2NaOH +NH42SO4 2NH3+Na2SO4+ 2H2O3. Absorption by boric acid :

2NH3+ 4H3BO3 NH42B4O7+ 5H2O4. Titration by strong acid

NH42B4O7+ 5H2O + 2HCl 2NH4Cl + 4H3BO3(* NCOC,N containing organic compounds, N:HCl = 1:1)

Principles of Kjeldahls method


29/44

Apparatus used in Kjeldahl

I. DigestionapparatusII. Distillation

absorptionapparatus

(I) (II)


30/44


31/44

Points that need your close attention

8. Digestion should be carried out in a ventilatingcabinet.

9. Connect well the distillation apparatus beforeadding alkali into digested solution.

10.Add abundant alkaliuntil there are redcopper hydroxide formed.11. Absorption solution should be less than 40

deg.Cthroughout the absorption. Cold waterbath is a good choice to lower the temp.

12. Using indicating paperto help for thedetermination of distillation terminus.13.Indicatorsof methylene blue and methyl red

are added to absorption bottle before carryingon the distillation.


32/44

Other methods for protein assay


33/44

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.

The Biuret Method


34/44

Q1. Is this suitable for AAs? 2. what is Biuret Reaction

The Biuret Method

O=C

NH2

NH

O=C

NH2

2 CuSO 4

NaOH

O=C

NH2

NH

O=C

NH2Na

OH

OH

HN

C=O

C=O

NaH2N

OH

H2N

OH

Cu

Principle: This reaction is characterized by the development ofa purple coloration from the complexingof 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.


35/44

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 expensivethan Kjeldahl method, rapid, simplest methods for proteinanalysis.

2. color deviations are encountered less frequentlythan with Folin-Lowry,UV absorption, or turbidimetric methods.

3. very few substancesother than proteins in foods interfere withthe biuretreaction.

4. Detect N only from the peptide and protein sources.

Disadvantages:

1. Not very sensitive as compared to the Folin-Lowry Method. Require atleast 2-4 mg protein for assay.

2. Bile pigments, high conc of ammonium saltsinterferes 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 knownproteins(e.g., BSA)

* Introduction of 30% isopropyl alcohol can reduce the reaction time

from 35 to 10 mins. You also can try Heating.


36/44

Dumas (N combustion)

Principle:

Samples are combusted at high temp (700-1000 deg.C). The Nreleased is quantitated by GCusing a thermal conductivitydetector (TCD).

Procedure:

Samples (100-500 mg) are weighed into a tin capsuleandintroduced to a combustion reactorin automatedequipment. 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 150samples without attention.

Disadvantages:

Measures total organic nitrogen, not just Protein N.


37/44

Principle:

Folin reagent phosphomolybdic and

phosphotungstic acid is reduced to a bluemolybdenum 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.

(Mistakein SolnB at p163)

The lowrys Method (Folin-phenol method)


38/44


39/44

UV 280 b i M h d


40/44

UV 280 nm absorption Method

Principle:

1. Proteins show strong absorption at UV 280 nm, primarily due toaromatic amino acidsof 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 proteinsfor

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!


41/44

Stain with Coomassie blue


42/44

1. ReagentsCoomassie Blue R-250MethanolGlacial acetic acid

2. Gel Stainning solution, 1 L1.0 g Commassie Blue R-250450 ml water

450 ml methanol100 ml Glacial acetic acid

3. Gel destaining solution, 1 L100 ml Methanol100 ml Glacial acetic acid800 ml Water

4. Staining Procedure

1. Pick up the gel into (20 ml, usually enough) Staining solution in a container andagitate for 10 min for 0.75 mm Gel and 20 min and 1.5 mm gel. The staining solutioncan 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 lightbox, 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


43/44


44/44

THANKS

10 amino acid and protein analysis

Documents