Lec 10: Protein Methodologies

Question 1

• A solution used to determine non-protein nitrogenous substances.
• used to prevent errors such as foaming, precipitation, or direct interference

Answer 1

PFF (Protein Free Filtrate)

Question 2

The optimum pH for CHON to precipitate from:
• serum/plasma: ___
• whole blood: ___

Answer 2

• serum/plasma: 5.3
• whole blood: 6.4

Question 3

Physical methods in preparing PFF

Answer 3

Ultrafiltration,
Heat denaturation

Question 4

Physical method wherein it is the change of CHONs from soluble to insoluble form brought about by heat.

Answer 4

Heat denaturation

Question 5

Chemical methods in preparing PFF

Answer 5

Antigen-antibody reaction,
CHON dehydration,
Insoluble formation

Question 6

Precipitation by ___ Method:

Organic solvents lower the solubilities of proteins and precipitate them from highly polar solutions.

Answer 6

Addition of Organic Miscible Solvents

Question 7

The principle effect of ppt by addition of Organic Miscible Solvents.

Answer 7

Reduction of water activity

Question 8

Mechanism of ppt by addition of Organic Miscible Solvents

Answer 8

• molecule of non-aqueous solvent steal water molecules from CHON.
• dielectric constant of aqueous solution is lowered by the addition of non-aqueous miscible solvent.

Question 9

Factors affecting ppt by addition of Organic Miscible Solvents:

Answer 9

pH,
ionic strength,
temperature,
organic solvent concentration

Question 10

PROCEDURE of ppt by addition of Organic Miscible Solvents:

Answer 10

• 1 serum/whole blood + 9 of 70% acetone or 80% ethanol

• 1 whole blood + 85% methanol

• 1 serum + 90% methanol

Question 11

Precipitation by ___:

increase in salt concentration will have salt steal water molecules from CHON, leaving CHON less soluble and dehydrated.

Answer 11

Salting Out

Question 12

Precipitation by ___:

Addition of neutral salt will decrease attraction of CHON for each other, therefore, increasing solubilty.

Answer 12

Salting In

Question 13

Examples of salts with multivalent anions

Answer 13

• Na₂SO₄
• (NH₄)₂SO₄
• Mg salts
• PO₄ and citrates

Question 14

Factors affecting ppt by Salting Out

Answer 14

pH, temperature, salt concentration

Question 15

Precipitation dependent on soluble salt formation:

Principle

Answer 15

• Anionic acid precipitant + cationic CHON form = insoluble CHON salt [alkalinization]

• heavy metal cations + anionic CHON form = insoluble proteinates [acidification]

Question 16

Proteins are ___ charged in acidic solutions, while they are ___ charged in alkaline solutions.

Answer 16

Proteins are positively charged in ___ solutions, while they are negatively charged in ___ solutions.

Question 17

Anionic precipitants

Answer 17

Tungstic acid,
TCA,
Perchloric acid

Question 18

Methods under Tungstic Acid

Answer 18

• Folin and Wu method
• Folin’s modification
• Haden’s modification

Question 19

PROCEDURE of Folin and Wu method

Answer 19

1 blood + 7 distilled water + 1 of 10% Sodium tungstate + 1 of ⅔ Sulfuric Acid

Question 20

Folin and Wu method:
Purposes of Sulfuric Acid

Answer 20

• converts Sodium tungstate to Tungstic acid
• will cause RBC lysis/rupture the RBC which turns solution from dark red to choco brown

Question 21

PROCEDURE of Folin’s modification

Answer 21

8 distilled water + 1 blood + 0.5 of 10% Sodium tungstate + 0.5 of ⅔ Sulfuric Acid

Question 22

PROCEDURE of Haden’s modification

Answer 22

• 1 sample + 8 of N/12 Sulfuric Acid + 1 of 10% Sodium tungstate
• shake then filter

Question 23

Method under Trichloroacetic acid

Answer 23

Green Wald’s method

Question 24

PROCEDURE of Green Wald’s method

Answer 24

Original:
• 1 sample + 9 of 2.5% TCA

Modified:
• 1 sample + 9 of 5% TCA

Question 25

PROCEDURE of the method under Perchloric acid

Answer 25

1 sample + 9 of 1.11M HClO₄

Question 26

Cationic precipitants (heavy metals)

Answer 26

Copper, Zinc

Question 27

Methods under Zinc

Answer 27

• Hagedorn and Jensen
• Somogyi
• Modified Somogyi
• Zinc + Barium (Somogyi)

Question 28

PROCEDURE of Hagedorn and Jensen

Answer 28

ZnSO₄ with NaOH and then heat

Question 29

Hagedorn and Jensen:
Purpose of NaOH

Answer 29

To make the solution alkaline

Question 30

Hagedorn and Jensen:
Purpose of Heat

Answer 30

To destroy other substances and to complete the precipitation

Question 31

PROCEDURE of Somogyi for Zinc

Answer 31

• 1 whole blood + 7 distilled water + 1 of 10% ZnSO₄

• 1 serum/plasma + 8 distilled water + 0.5 of NaOH

Question 32

PROCEDURE of Modified Somogyi under Zinc

Answer 32

1 blood + 1 acid ZnSO4 reagent + 1 of 0.75N NaOH

Question 33

Composition of acid ZnSO₄ reagent

Answer 33

ZnSO₄ + 0.25N Sulfuric Acid in distilled water

Question 34

PROCEDURE of Zinc + Barium (Somogyi)

Answer 34

• whole blood/serum + 15 distilled water + 2 of 0.3N Barium hydroxide
• add 2 of 5% ZnSO₄
• mix, stand for 2 mins, filter

Question 35

Zinc + Barium (Somogyi):
• NaOH was replaced with ____ due to ____

Answer 35

“NaOH was replaced with Barium hydroxide due to adsorptive capacity of Barium sulfate formed.”

What method under Zinc?

Question 36

PROCEDURE of Somogyi under Copper

Answer 36

Whole blood:
• 1 whole blood + 7 distilled water
• add 1 of 10% Sodium Tungstate dihydrate
• shake then centrifuge and filter

Serum/plasma
• 5% CuSO₄ & 6% Sodium Tungstate dihydrate

Question 37

PROCEDURE of Precipitation by Shaking with Chloroform

Answer 37

0.25 Chloroform + 1 amyl alcohol
• shake for 15 to 60 minutes
• centrifuge

Question 38

A Physical method wherein low molecular weight particles pass through the selective semipermeable membrane while the high molecular weight particles are left behind

Answer 38

Ultrafiltration