Chapter 10
Amino acid & Protein Analysis
Qijun Wang
2005-4-12
Chapter 10-1
Amino Acids Analysis
What is amino acid?
What is amino acid?

Amino Acid: aminated carboxylic acid (R-COOH)
R group
Examples
NH2CH2COOH
NH2CH2CH2COOH
-amino
acetic acid
-amino
propanoic acid
NH2CH2CH2CH2CH2CHCOOH
NH2
，-2 amino caproic acid
Classification of Amino Acid
1. By the location of Amino-group ： / / -AA
2. By its acidity： neutral/ acidic/ basic AA
ratio of Amino-group to carboxylic group
3. By whether containing phenyl group
aromatic / non aromatic AA
4. By its occurrence in protein
Protein / non protein AA
5. 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
NH2
CH2COOH
NH2
CH3 CHCOOH
甘氨酸
glycine
丙氨酸
alanine
CH3 NH2
CH3 CH CHCOOH
缬氨酸*
valine
CH3
NH2
CH3 CH CH2 CHCOOH
亮氨酸*
CH3 NH2
CH3 CH2 CH CHCOOH
异亮氨酸*
leucine
isoleucine
Continued
-aa structure
NH2
Cn name
En name
蛋氨酸*
methionine
脯氨酸
proline
苯丙氨酸*
phenylalanine
色氨酸*
tryptophan
CH3SCH2CH2 CHCOOH
CH2 NH
CH2
CH2 CHCOOH
NH2
CH2 CHCOOH
NH2
CH2 CHCOOH
N
H
NH2
HO CH2 CHCOOH
丝氨酸
serine
continued
-aa structure
HO NH2
CH3CH CHCOOH
NH2
HS CH2 CHCOOH
HO
NH2
CH2 CHCOOH
NH2
NH2 COCH2 CHCOOH
NH2
H2NCOCH2CH2 CHCOOH
Cn name
En name
苏氨酸*
threonine
半胱氨酸
cystine
酪氨酸
tyrosine
天门冬酰胺
asparagine
谷酰胺
glutamine
continued
-aa structure
NH2
HOOC CH2 CHCOOH
NH2
HOOC CH2CH2 CHCOOH
Cn name
En name
天门冬氨酸
Aspartic
acid
Glutamic
acid
谷氨酸
赖氨酸*
lysine
NH2
NH
H2N C NH CH2CH2CH2 CHCOOH
精氨酸
arginine
组氨酸
histidine
=
NH2
H2NCH2CH2CH2CH2 CHCOOH
NH2
CH2 CHCOOH
N
N
H
Physical characteristics

ALL the pure -AAs are Colorless
crystal , with quite high melting
point (>200 deg.C) and water
solubility,
AA are ampholyte
RCHCO2H
R
NH2 CH COO
Anion,
when at
high pH
HCL
NH2
NaOH
+
H
-
OH
RCHCO2
-
+
N H3
zwitterion, when
at isoelectric
point (pI)
+
H
-
OH
R
+
NH3 CH COOH
Cation,
when at
low pH
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
anode
+
+
+
+
+
+
+
+
+
cathode
RCHCO2H
NH2
RCHCO2
NH2
Anion in
basic sol’n.
pH溶液>pI
-
HO-
RCHCO2
-
H+
N+ H3
Zwitterion in
pI sol’n. No
move to either
of Electrode.
And with Lowest
solubility
RCHCO2H
N+H3
Cation in
acidic sol’n.
pH溶液<pI
-
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
O
O
OH
OH
O

+ RCHCOOH
NH2
O
+ RCHO + CO 2 + 3H2O
N
O
O
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 analyzer
From pH2.2 to pH 6.4
Separation principles
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
+
NH2
HOOC CH2 CHCOOH
NH2
H2NCH2CH2CH2CH2 CHCOOH
Separate aspartic acid and lysine by cation exchange
resin.
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).
CH2
NH2
CHCOOH
phenylalanine
NH2
CH2 CHCOOH
N
H
tryptophan
HO
NH2
CH2 CHCOOH
tyrosine
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:
H O
H O
R C C OH + C4 H9OH HCl
100
NH 2
Non-volatile AA
H O
O
R C C OC4 H9 + ( CF 3C ) O
2
NH+
3Cl-
R C C OC4 H9
+ H 2O
NH+
3ClH O
R C C OC4 H9
N C CF 3
H O
Volatile AAderivate
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
Protein content in food (%)
Cereals：
（%）
Meat, poultry, fish:
Brown Rice
7.9
Beef
18.5
Polished rice
7.1
Dry beef
29.1
Wheat flour, whole-grain 13.7
Chicken, breast meat, raw 23.1
Corn flour, whole-grain
6.9
Ham
17.6
Corn starch
0.3
Egg, raw, whole
12.5
Finfish, raw,
17.9
legumes：
Soybean, raw
36.5
Dairy products：
Beans, kidney, raw
23.6
Milk, whole, fluid
3.3
Tofu, raw, regular
8.1
Milk, skim, dry
36.2
Cheese, cheddar
24.9
Yogurt
5.3
Fruits & vegetables：
Apple, raw, with skin
0.2
Strawberry, raw
0.6
lettuce , raw
1.0
* High quality protein ?
Conversion factors for Foods
N to Protein conversion factors
Foods
Egg or meat
Dairy products
Wheat
Other cereal grains and oilseeds
Almonds
Peanuts
Other tree nuts and coconut
factors
6.25
6.38
5.70
6.25
5.18
5.46
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
Classification
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。
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.
Kjeldahl’s method
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%.
Principles of Kjeldahl’s method
1. Digestion
催化剂
NCOC + H2SO4
煮沸
（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)
Apparatus used in Kjeldahl
I. Digestion
apparatus
II. Distillation
& absorption
apparatus
(I)
(II)
Points that need your close attention
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
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.
The Biuret Method
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.
OH
NH 2
O=C
NH
2
O=C
NH 2
OH
NH 2 Cu H2N
CuSO 4
O=C
NaOH
O=C
C=O
NH
HN
C=O
NH 2Na NaH 2N
OH
OH
Q：1. Is this suitable for AAs? 2. what is Biuret Reaction？
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.

The lowry’s Method (Folin-phenol method)
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)
Procedures of lowry’s Method
1. 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.
Properties of lowry’s Method
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.

UV 280 nm absorption Method
Principle:
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
Coomassie Blue staining procedures
1. Reagents
Coomassie Blue R-250
Methanol
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 acid
3. 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 container
3.
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.
Two dimensional Electrophoresis
2-DE: the technique to separate proteins in the first dimension according to t
heir isoelectric point, by Isoelectric Focusing(IEF), and in the second dimensi
on according to their molecular weight, by SDS-PAGE. 2-DE combined with
protein identification basing on microsequencing, amino acid composition an
d Mass spectrometry, provides an invaluable tool for proteomic studies.
Step 1 — Sample Prep
Step 2 — First-Dimension (IEF) Separation
Step 3 — Second-Dimension (SDS-PAGE) Separation
Step 4 — Protein Detection by Staining /Destaining
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