 Acid Hydrolysis of intact proteins

The purpose of the procedure of acid hydrolysis of the Intact protein is to see that the structure and
size of gluten becomes soluble and turns into an altered protein. The hydrolysate is formed by
hydrolyzed wheat gluten which obtains some emulsifying properties. It breaks down casein into
peptides and amino acids and After autoclaving of the acid hydrolysate, a dark brown-black
solution was formed.

 Myoglobin from beef

The Myoglobin was isolated from the beef by salt-induced precipitation wherein the proteins are
less soluble at salt concentrations (high ionic strength) because the salt ions bind most of the water
molecules. Myoglobin can be isolated by ammonium sulfate precipitation from the buffered
muscle extract.

 Hydrolysis of intact proteins

Made of Hydrolysis Description of the Hydrolysate

Acidic Dark brown solution
Basic Clear solution
Enzymatic Dark brick red solution with no turbidity

For the acidic hydrolysis, the acid catalyst which is the HCl will vaporize and will eventually react
with the protein, thus hastening the hydrolysis. From a pale red turbid solution, the hydrolysate
appeared to be a dark brown solution after autoclaving as shown in table. Meanwhile for the basic
hydrolysis, the only difference is that it uses a basic catalyst in the presence of 4M NaOH and
unlike in acidic hydrolysis, the basic hydrolysate was neutralized with 1M HCl which is acidic.
From a pale red turbid solution, the protein mixture turned into a clear solution, which thus
indicated an alteration in its conformation as well. And in the in enzymatic hydrolysis, a type of
special enzyme was used to hasten the denaturation process of myoglobin under high temperatures.
Basing from the experiment the isolated myoglobin was put in an environment which is almost the
same as the environment inside the human body; with a pH of 7.5, a temperature of 35-40 oC and
with an enzyme catalyst. And so, as expected, the protein was denatured as proved by the change
in appearance of the sample from a pale red turbid solution to a dark brick red solution without
turbidity.
 Separation and Identification of amino acid by thin layer chromatography
In the paper chromatography, results are based on the polarity of the amino acids. The non-polar
Tryptophan moves farthest from the base line since it is hydrophobic. Meanwhile, histidine is a
polar amino acid and moves least from the base line. The mobile phase in the solvent system is the
butanol and acetic acid which means that tryptophan has the highest affinity to the mobile phase
while histidine has a high affinity to the stationary phase which is water. The 1st group of amino
acid, the hydrophobic non-polar amino acids must be seen as the farthest from the base line, while
the 2nd and 3rd group of amino acids must be seen near the base line which are the polar uncharged
and polar charged amino acids respectively. Butanol, a non-polar solvent, carries the non-polar
amino acids up the chromatogram, while the acetic acid, a polar solvent, carries the non-polar
amino acids up the chromatogram but because of their ratio differences in the solvent system
mixture which is 4 parts of butanol for every 1 part of acetic acid, non-polar amino acids are
favored than polar amino acids. The Ninhydrin solution sprayed to the paper chromatogram gave
the amino acids their distinct blue and violet colors except proline which gives a yellow color.
Despite not having the solvent front to fully reach the assigned mark, the spots on the paper
chromatogram are still semi-discernible and can still be accounted for. The acid hydrolysate that
was used produced an orange-yellowish with shades of purple in its spotted form, and because of
the prominence of the yellow spots, it is inferred that the acid hydrolysate of casein contains large
amount of proline. Colors aside from yellow also indicates presence of different amino acids.