Color Reactions Intact Protein (Gluten) Basic Hydrolysis

RESULTS AND DISCUSSIONS:
1. Acid hydrolysis of intact protein

- Myoglobin was chose as a test protein and 5.5 pg was hydrolysed in
CF3C02H/HCl (1 : 2) at various temperatures for 10 and 25 min. A 25-min
hydrolysis at 170°C resulted in a recovery of 94%. Myoglobin was
hydrolysed at 160 "C for 15 min with a mixture of propionic acid:HCl (1 :
I), according to the conditions of [lo] and for 30 min as an additional
hydrolysis time. Hydrolysis was incomplete after 15 min (73 %) and
reached 86 % even after 30 min. The data in Table 3 suggest that these
conditions can be improved. The hydrolysis temperature was studied
further between 150 "C and 170 "C to investigate the recovery of amino
acids and for reducing side reactions using glucagon and myoglobin. Our
conclusion is that the optimum conditions are between 165 "C and 170 "C.
The composition of myoglobin obtained at 166 "C. The degradation of
threonine and serine was slightly greater than that found in experiments
with free amino acids, but the zero-time extrapolation values were almost
exactly the figures expected. With the exception of isoleucine, nearly
quantitative recovery was obtained for all amino acids. The low recovery
of isoleucine can be explained by the presence of an Ile-Ile sequence in
myoglobin. Amino acid composition results for lysozyme and several other
proteins are listed in Table4. In general, the conditions were quite
acceptable. Yields of valine and isoleucine after 72-h hydrolysis by the
conventional method were about the same as those obtained after 25 min
by the new method.
2. Alkaline hydrolysis of intact protein

- Alkaline hydrolysis works by breaking down chemical bonds (peptide) of molecules down to its
building blocks. Using NaOH as a catalyst, the process requires extreme heat to speed up the
reaction. A polypeptide or proteein is a result of the condesation of the carboxyl groups of two
amino acids with the elimination of water. Hydrolysis reverses this reaction thus leaving behind
samples amino acid and smaller peptide. The results obtained for the qualitative color reactions
of gluten and the basic hydrolysis. The following test are used to detect presence of amino acids
and proteins and distinguish between them.
COLOR REACTIONS INTACT PROTEIN BASIC HYDROLYSIS

(GLUTEN)
BIURET TEST Purple solution Light blue solution
NINHYDRIN TEST Clear solution Violet solution
XANTHOPROTEIC TEST Yellow precipitate Yellow solution
MILLON’S TEST Salmon/flesh Turbid solution
HOPKINS-COLE TEST Formation of violet ring Clear solution
SAKAGUCHI TEST Turbid yellow solution Clear solution
NITROPRUSSIDE TEST Turbid white with sediment Salmon colored
FOHL’S TEST Formation of black sediment Brown solution
TEST FOR AMIDES Red to blue litmus paper Red to blue litmus paper
PAULY TEST Orange red solution Red orange solution
3. Biuret Test
- The normal color of biuret reagent is blue. The reagent turns violet in the presence of
peptide bonds -- the chemical bonds that hold amino acids together. The proteins detected
must have at least three amino acids, which means that the protein must have at least two
peptide bonds. The reagent’s copper ions, with a charge of +2, are reduced to a charge of +1
in the presence of peptide bonds, causing the color change. The techniques of absorption
spectroscopy, which identify the electromagnetic frequencies a sample will absorb, allow
testers to quantify the concentration of protein in a sample.
4. Ninhydrin Test
- This test is a general test and thus given by all amino acids. This test is due to a reaction
between a amino group of free amino acid and ninhydrin. Ninhydrin is a powerful oxidizing
agent and its presence, amino acid undergo oxidative deamination liberating ammonia, CO2,
a corresponding aldehyde and reduced form of ninhydrin ( hydrindantin). The NH3 formed
from a amino group reacts with another molecule of ninhydrin and is reduced product
( hydrindatin) to give a blue substance diketohydrin ( Ruhemanns complex). However, in
case of imino acid like proline and hydroxyproline, a different product having a bright yellow
color is formed. Asparagine, which has a free amide group, reacts to give a brown colored
product.
5. Xanthroproteic Test
- Xanthoproteic test is used to detect amino acids containing an aromatic nucleus (tyrosine,
tryptophan and phenylalanine) in a protein solution which gives yellow color nitro
derivatives on heating with conc. HNO3. The aromatic benzene ring undergoes nitration to
give yellow colored product. Phenylalanine gives negative or weakly positive reaction
though this amino acid contains aromatic nucleus because it is difficult to nitrate under
normal condition. On adding alkali to these nitro derivative salts, the color change fro
yellow to orange.
6. Millon’s test
- Positive Millon’s test: Brick red color (Tyrosine and phenol solution)Negative Millon’s test:
no red color ( arginine)
Compounds containing hydroxybenzene radical react with Millon’s reagent to form red
complexes. The only amino acid having hydroxybenzene ring is tyrosine. Thus, this test is
specific for the amino acid tyrosine and the protein containing this amino acid. Tyrosine
when reacted with acidified mercuric sulphate solution gives yellow precipitate of mercury-
amino acid complex. On addition of sodioum nitrate solution and heating, the yellow
complex of mercury-amino acid complex converts to mercury phenolate which is in red
color.
7. Hopkins – Cole test

- The Hopkins-Cole reaction, also known as the glyoxylic acid reaction, is a chemical test used
for detecting the presence of tryptophan in proteins. A protein solution is mixed with
Hopkins Cole reagent, which consists of glyoxylic acid. Concentrated sulfuric acid is slowly
added to form two layers. A purple ring appears between the two layers if the test is
positive for tryptophan. Nitrites, chlorates, nitrates and excess chlorides prevent the
reaction from occurring.
The indole group of tryptophan reacts with glyoxylic acid in the presence of conc. H2SO4 to
give a purple colored complex. Glyoxylic acid is prepared by reducing Oxalic acid with
magnesium powder or sodium amalgam. Glacial acetic acid which has been exposed to the
sunlight also contains glyoxylic acid and can thus be used for this test.
Positive Hopkin’s cole test: purple color at the interface. ( tryptophan and egg albumin)
Negative Hopkin’s cole test: glycine
8. Sakaguchi test
- Sakaguchi tests for the amino acid arginine. The reaction with a-naphthol and bleach gives a
deep red solution as a positive result. Arginine is an essential nutrient that animals can not
make on their own; they need to obtain it from their diet. Many functions of arginine in the
human body include healing wounds, eliminating waste products from livers, and
maintaining hormone and immune function.
Samples two, three, and five were red, meaning they contained amounts of arginine, and was tested
positive. Since two and three were both amino acids in past testing, number five is the sample
containing arginine.
9. Nitroprusside Test
- Based on the experiment conducted the result show that the change of color occurs from
clear solution to bright yellow solution.
10. Fohl’s test

- Diazotized sulphanilic acid couples with amines, phenols and imidazole to form highly
colored azo compounds. This coupling reaction must be done in cold condition since
diazonium compound is formed in cold.
11. Test for Amides

- an amide test by an experiment called The Sodium Hydroxide Hydrolysis of Amides. During
the reaction, if ammonia was formed, it would indicate that the compound was a primary
amide by turning pink litmus paper blue when the solution was heated.
- Colorless solution
12. Pauly’s Test

- Amino acids tyrosine or histidine coupled with diazonium salt in alkaline condition to form
red coloured azo dye.
Positive Pauly test: red colored azo dye ( tyrosine and histidine)
Negative Pauly test: no red color ( glycine)
13. Gluten from wheat flour

- Gluten is a storage protein found naturally in wheat, barley and rye
From the experiment, the characteristic of gluten is that it is white and a bit rubbery.
14. Myoglobin from muscle
- Myoglobin is a hemoprotein found in the skeletal muscle of mammals that functions in oxygen
storage and diffusion.1 A hemoprotein is a protein that contains a heme prosthetic group.
Myoglobin can reversibly bind a dioxygen molecule to regulate the transportation of oxygen
from red blood cells to mitochondria when skeletal muscles are metabolically active.
-The more myoglobin content meat contains the darker red it will appear in color.

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RESULTS AND DISCUSSIONS:

1. Acid hydrolysis of intact protein

2. Alkaline hydrolysis of intact protein

COLOR REACTIONS INTACT PROTEIN BASIC HYDROLYSIS

7. Hopkins – Cole test

10. Fohl’s test

11. Test for Amides

12. Pauly’s Test

13. Gluten from wheat flour

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