PROTEIN DETERMINATION METHODS

1. Kjeldahl Method.
2 Dye binding Method.
3. Biuret Method.
4. Lowry Method.
5. Ultraviolet Method.

1. Kjeldahl Method --- nitrogen determination principles.

(1) Digestion
+ conc. H2SO4
+ a catalyst
nitrogen converted into an ammonium ion.

(2) Neutralize to get NH3 with NaOH

(3) Steam distillation of NH3 and trap in boric acid.

(4) Titrate with hydrochloric acid.

Calculation:
Gram nitrogen/ gram of sample =

*(ml of sample - ml of blank) ∞ N standard acid × 0.014g/meq

weight of sample

* ml of hydrochloric acid required to titrate sample solution.

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Disadvantages: not all N is protein.
Purine
Pyrimidine DNA, RNA, etc.
Urea
Many plant tissues have > 50% non-protein N.
% N × 6.25 = % Protein

6.25 6.38 5.83 5.70 5.30

Corns Milk Whole wheat Wheat flour Nuts
Eggs Barley
Peas Oats
Meat Rye
Beans Millet

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2. Dye Binding Method:
Principle: At low pH, basic groups of protein are (+) charged. These
will quantitatively bind a (-) charged dye.

What are these basic groups?

+
NH 3
CH 2 H
+
CH 2 CH 2 N C NH 2
Lysine
CH 2 CH 2 NH 2
CH 2 O CH 2 Arginine
H
CH N C CH
N C C N
H H
O CH2
C NH +

HC CH Histidine
N
H

Acid Orange 12:

-
SO3
HO
N=N

Procedure:
1. Mix protein, dye, buffer pH = 2.
2. Filter or centrifuge.
3. Measure optical density (O.D.) of filterate.

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O.D. dye bound by protein = O.D. dye initial - O.D. filterate

O.D. at 470 nm

Skim milk

6 8 10 12 14 16

% Protein (Kjeldahl)

Factors Influencing Dye Binding determination:

1. Temperature
2. Non-proteins.
3. Buffers systems.
4. Protein quality.

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 ++
3. Biuret Method: Cu in alkaline solution form complexity with
peptide bonds - give pinkish-purple color.

Measure the intensity of color at 540 nm.

A at 540 nm

% Protein (Kjeldalh)

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4. Lowry Method: (one of most sensitive methods)
++
1. Cu in alkaline solution to form complexity with protein.
++
2. Cu catalyses oxidation of phenol group of tyrosine with
phosphomolybdic-phosphotungstic acid.

A at 750 nm

µ g 0f protein (Kjeldalh)

5. Ultra-violet Absorption (UV) at 280 nm

1. Chromophoric side chains of aromatic amino acids (Trosine,
Tryptophan).
2. Absorption at 280 nm. “Non-destructive means to
determine protein”.
3. Calculation protein conc. based upon absorption.

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6. Fluorescence Method:
Tyrosine is a fluorescent compound.
Tryptophane is a fluorescent compound.
Excite the amino acids at 280 nm.
Measure emission at 348 nm.

Advantage: more sensitive than UV absorption.

Intensity of Emitted Fluorescence

at 348 nm

mg of protein/ml of solution

What is fluorescence and how to measure it?

Excited State Emits radiation
(fluorescence)
Decay yields
fluorescence at
longer wavelength

Ground State

By using specific λ (wavelength) to excite and measure output at a specific λ. It is rather

specific.
Problems:
Turbidity/Quenching (self or others)/Expensive/Quantitation is difficult.

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Amino Acid Determination:

A. Hydrolysis
0
1. Overnight in 6 M HCl at 100 C.
2. Enzymes.

B. Separation by ion exchange.

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MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

+ pH 2
Na
+
H3 N
-
SO3
COOH
+
Na
OH
- +
So3 H3N
COOH
Exchange Resin

-
SO3 H3N+
pH3.5
COOH
OH
-
So 3 +
H3 N
+ - + -
Na COO H OH = H 2 O

+
Na

-
SO3 H3 N
+

- + -
COO H OH = H 2 O
-
So3 Na+ pH4.5

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Moles/Liter VAL

ALA

HIS LEU

ASP
LYS GLU

pH 2.25 pH 3.25 pH4.25

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Some Important Reactions of Proteins

Denaturation
o o o
Changes in 2 , 3 , 4 structure.
By heat.
Heavy metals (Hg is most common).
pH (trichloroacetic acid, phosphotungstic acid)
Salt (NaCl or ammonium sulfate [NH4]2 SO4)

Reasons for Precipitating Proteins

1. Purify, concentrate protein.
2. Remove protein which cause: turbidity/emulsion/troublesome.

Manifestation of Denaturation
1. Decreased solubility.
2. Alteration of size and shape
3. greater reactivity
4. Decreased biological activity (enzyme + immune proteins)
5. Increased sensitivity to electrolytes.
6. Nutritive value.

What is essential amino acids?

Amino acids which the body cannot make (or make enough of) for protein
synthesis due to lack of enzymes.

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Essential Amino Acids:
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine

Limiting amino acid is the essential amino acid which is lacking in the protein to have a
balanced protein.

Product Limiting Amino Acid

Corn Lysine
Oats Lysine
Rice Lysine
Wheat Lysine
Sesame Seed Lysine
Cow’s Milk Methionine
Potato Methionine
Chick Pea Methionine
Green Pea Methionine
Cotton Seed Isoleucine
Beef Valine
Chicken Tryptophan

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PROTEIN QUALITY DETERMINATION

1. Protein Efficiency Ratio.

2. Biological Value.
3. Net Protein Utilization.

What are the measurements of protein quality?

For labeling purposes, one needs to know the protein efficiency ratio.
1. If PER ε casein (2.5), the RDA = 45 g/day.
2. If 0.5 < PER < 2.5, then RDA = 65 g/day.
3. If PER < 0.5 (20% of casein), then “not a significant source of protein”.

How does one determine PER?

1. Male lab rats ε 21 days, ≤ 28 days of age, at least 10 rats/group.
2. Feed a standardized diet containing salt mix, vitamins, cotton seed oil, cellulose,
starch or sucrose + water for 28 days.
3. Measure weight gain and food intake at regular intervals, not > 7 days.
4. PER = Weight Gain/Gram of Protein in Diet.
5. Usually normalized for casein = 2.5.
6. Determine protein quality of sample as ratio of sample PER to reference casein
PER.

Protein Efficiency Ratio = Gain in weight per gram protein eaten.

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Protein efficiency ratio is a number that descries how well a given protein supplies the
building blocks for rapid growth.

Product PER

Rice 100% 2.30

Rice 70% Black Beans 30% 2.70
50% 50% 2.60
20% 80% 1.30
100% NIL
Corn
+ 0.4% Lysine
+ 0.07% Tryptophan 2.14
Corn (50%) + Black Beans (50%) 2.05

Percentages refer to the percent of the protein supplied by that source.

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Product PER

Soybean 2.32
Cotton Seed Meal 2.25
Egg 3.90
Sesame Seed 1.77
Chick Peas 1.68
Peanuts (ground nuts) 1.65
Kidney Beans 0.88

OTHER PROTEIN QUALITY DETERMINATION

Biological Value (BV)

Net Protein Utilization (NPU)

BV = Retained Nitrogen (nitrogen intake - fecal & urinary nitrogen)/Absorbed Nitrogen

(nitrogen intake - fecal nitrogen)

NPU = Retained Nitrogen/Intake Nitrogen = BV × Digestibility

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