Chapter 10 Amino acid & Protein

Analysis

Qijun Wang2005-4-12

Chapter 10-1 Amino Acids Analysis

What is amino acid?

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

COOH) R grou

p

NH2CH2COOH-amino acetic acid

NH2CH2CH2COOH

-amino propanoic acid

NH2CH2CH2CH2CH2CHCOOHNH2

， -2 amino caproic acid

Examples

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

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

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

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

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

Physical characteristics

ALL the pure -AAs are Colorless crystal , with quite high melting point (>200 deg.C) and water solubility,

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.

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

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 。

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.

Automatic AA analyzerFrom pH2.2 to pH 6.4

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-

+

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.

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.

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

Chapter 10-2 PROTEIN ANALYSIS

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

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 (%)

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

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

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

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.

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

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

Apparatus used in Kjeldahl

I. Digestion apparatusII. Distillation & absorption apparatus

(I) (II)

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

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.

Other methods for protein assay

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.

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.

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”.

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.

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)

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.

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

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!

Stain with Coomassie blue

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

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

THANKS