Lesson 3.3. Hydrolysis of Intact Proteins

Hydrolysis

carried out by treating intact protein with acid, alkali or proteolytic enzymes

5 mL 6M HCl

Acid Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Cover the test tube with cotton and ___________________.

3. Add _____________ Transfer in a 250- mL beaker.

4. Neutralize the mixture with ____________ using pH paper/pH meter.

5. Use the neutralized mixture for _____________________________

1 = ?

autoclave (15psi, 5 hrs)

Acid Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Cover the test tube with cotton and ___________________.

3. Add _____________ Transfer in a 250- mL beaker.

4. Neutralize the mixture with ____________ using pH paper/pH meter.

5. Use the neutralized mixture for _____________________________

2 = ?

10 mL of Distilled Water

Acid Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Cover the test tube with cotton and ___________________.

3. Add _____________ Transfer in a 250- mL beaker.

4. Neutralize the mixture with ____________ using pH paper/pH meter.

5. Use the neutralized mixture for _____________________________

3 = ?

1M NaOH

Acid Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Cover the test tube with cotton and ___________________.

3. Add _____________ Transfer in a 250- mL beaker.

4. Neutralize the mixture with ____________ using pH paper/pH meter.

5. Use the neutralized mixture for _____________________________

4 = ?

characterization and chromatography

Acid Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Cover the test tube with cotton and ___________________.

3. Add _____________ Transfer in a 250- mL beaker.

4. Neutralize the mixture with ____________ using pH paper/pH meter.

5. Use the neutralized mixture for _____________________________

5 = ?

Acidic and Alkaline/Basic Hydrolysis

causes complete hydrolysis of proteins

Little or no racemization

Advantage of Acidic Hydrolysis

Tryptophan

Disadvantage of Acidic Hydrolysis:

a. _______ is destroyed and is converted to humin (black precipitate)

b. partial destruction of ____________________

c. Hydrolysis of ______________

a = ?

Serine, Threonine, and Cysteine

Disadvantage of Acidic Hydrolysis:

a. _______ is destroyed and is converted to humin (black precipitate)

b. partial destruction of ____________________

c. Hydrolysis of ______________

b = ?

Asparagine to Aspartic Acid and Glutamine to Glutamic Acid

Disadvantage of Acidic Hydrolysis:

a. _______ is destroyed and is converted to humin (black precipitate)

b. partial destruction of ____________________

c. Hydrolysis of ______________

c = ?

10 mL 6M NaOH

Alkali Hydrolysis of Intact Proteins:

1. To the isolated protein, add _____________

2. Cover the test tube with cotton and __________________

3. Add _____________. Transfer in a 250-mL beaker.

4. Neutralize the mixture with ___________.

5. Use the neutralized mixture for ____________________________

1 = ?

autoclave (15psi, 5 hrs)

Alkali Hydrolysis of Intact Proteins:

1. To the isolated protein, add _____________

2. Cover the test tube with cotton and __________________

3. Add _____________. Transfer in a 250-mL beaker.

4. Neutralize the mixture with ___________.

5. Use the neutralized mixture for ____________________________

2 = ?

10 mL of Distilled Water

Alkali Hydrolysis of Intact Proteins:

1. To the isolated protein, add _____________

2. Cover the test tube with cotton and __________________

3. Add _____________. Transfer in a 250-mL beaker.

4. Neutralize the mixture with ___________.

5. Use the neutralized mixture for ____________________________

3 = ?

1M HCl

Alkali Hydrolysis of Intact Proteins:

1. To the isolated protein, add _____________

2. Cover the test tube with cotton and __________________

3. Add _____________. Transfer in a 250-mL beaker.

4. Neutralize the mixture with ___________.

5. Use the neutralized mixture for ____________________________

4 = ?

characterization and chromatography

Alkali Hydrolysis of Intact Proteins:

1. To the isolated protein, add _____________

2. Cover the test tube with cotton and __________________

3. Add _____________. Transfer in a 250-mL beaker.

4. Neutralize the mixture with ___________.

5. Use the neutralized mixture for ____________________________

5 = ?

Tryptophan is stable

Advantage of Alkaline/Basic Hydrolysis

Racemization

Disadvantage of Alkaline/Basic Hydrolysis:

1. ____________ of all amino acids

2. _________ decomposes to ornithine and urea

3. Partial destruction of _____________

1 = ?

Arginine

Disadvantage of Alkaline/Basic Hydrolysis:

1. ____________ of all amino acids

2. _________ decomposes to ornithine and urea

3. Partial destruction of _____________

2 = ?

Cysteine, C-C, Serine, and Threonine

Disadvantage of Alkaline/Basic Hydrolysis:

1. ____________ of all amino acids

2. _________ decomposes to ornithine and urea

3. Partial destruction of _____________

3 = ?

10 mL of 1-2% protein solution in buffer (PB, 7.4)

Enzymatic Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Add ___________________________

3. Incubate the mixture in a _________________________________.

4. Allow the mixture to cool and proceed to ____________________.

1 = ?

5 mL of saturated protease solution (meat tenderizer)

Enzymatic Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Add _________________________, saliva (amylase) is used as a substitute

3. Incubate the mixture in a _________________________________.

4. Allow the mixture to cool and proceed to ____________________.

2 = ?

water bath (35-40 C) for 60 minutes

Enzymatic Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Add ___________________________

3. Incubate the mixture in a _________________________________.

4. Allow the mixture to cool and proceed to ____________________.

3 = ?

qualitative test and TLC

Enzymatic Hydrolysis of Intact Proteins:

1. To the isolated protein, add ______________

2. Add ___________________________

3. Incubate the mixture in a _________________________________.

4. Allow the mixture to cool and proceed to ____________________.

4 = ?

Enzymatic Hydrolysis

proteolytic enzymes causes partial or selective hydrolysis of polypeptide to yield mixture of peptide fragments

Proteases/Peptidases

• enzymes that hydrolyze peptide bonds at specific sites

• can affect either the NH2 and COOH of the amino acid like in Serine, Threonine, Cysteine, aspartic, and glutamine

Amino acids

Advantages of Enzymatic Hydrolysis:

a. ________________ are not affected

b. it requires _________________________ for optimum activity of enzymes.

c. Hydrolysis is _____________________.

a = ?

certain temperature & pH conditions

Advantages of Enzymatic Hydrolysis:

a. ________________ are not affected

b. it requires _________________________ for optimum activity of enzymes.

c. Hydrolysis is _____________________.

b = ?

not complete

Advantages of Enzymatic Hydrolysis:

a. ________________ are not affected

b. it requires _________________________ for optimum activity of enzymes.

c. Hydrolysis is _____________________.

c = ?