BIOCHEM LAB F EXAM

5 substrate samples of enzyme

Distilled water

Phenol

Catechol

Guaiac

Pyrogallol

hemoprotein catalyzing the oxidation by hydrogen peroxide of a number of substrates.

Peroxidase (HRP)

in enzyme, what consists test tube A

heated at 37C + potato peroxidase + Hydrogen peroxide

results of test tube A in enzyme

Distilled water = negative = no substrate

Phenol = pink to brown

Catechol = brown

Pyrogallol = brown

Guaiac = green

in enzyme, what consists test tube B

heated at 37C + potato oxidase

results of test tube B in enzyme

Distilled water = negative = no substrate

Phenol = pink to brown

Catechol = brown

Pyrogallol = brown

Guaiac = green

very pale blue liquid, slightly more viscous than water, that appears colorless in dilute solution. It is a weak acid, has strong oxidizing properties

Hydrogen peroxide (H2O2)

In substrate concentration of 4 mL catechol solution

dark brown

In substrate concentration of 2 mL catechol solution + distilled water

light brown

In substrate concentration of 4 mL guaiac solution

dark green

In substrate concentration of 2 mL guaiac solution + distilled water

light green

In enzyme concentration of 1.5 mL oxidase + catechol + heat

dark brown

In enzyme concentration of 0.5 mL oxidase + catechol + heat + distilled water

light brown

In enzyme concentration of 1.5 mL peroxidase + catechol + heat + H2O2

dark brown

In enzyme concentration of 0.5 mL peroxidase + catechol + heat + H2O2 + distilled wate

light brown

make and break intra- and intermolecular bonds, changing the shape of the enzyme and, therefore, its effectiveness.

pH

Pepsin has an optimum pH of ? – at this pH enzyme activity is at its maximum. Beyond this, denaturation occurs.

2

In enzymatic reaction, it has a light color in what pH and buffer

pH 7 Phosphate buffer and 0.1 M Na2CO3

Enzymatic reaction is faster on

acidic medium

Peroxidase is extracted in water. NaF inhibits oxidase, allowing separate extraction.

Potato Peroxidase (no NaF)

Potato Peroxidase (no NaF) observation

Clear filtrate

NaF inhibits peroxidase, isolating oxidase activity.

Potato Oxidase (with 2% NaF)

Potato Oxidase (with 2% NaF) observation

Clear filtrate

Enzymes require specific substrates; water is nonreactive.

2 mL Dist. H2O

2 mL Dist. H2O in test tube A (PEROXIDASE + H₂O₂)

No color change (control)

2 mL Dist. H2O in test tube B (OXIDASE)

No color change (control)

poor substrate for these enzymes.

2 mL Phenol

2 mL Phenol TEST TUBES A (PEROXIDASE + H₂O₂)

No/faint color change

2 mL Phenol TEST TUBES B (OXIDASE)

No color change

Peroxidase rapidly oxidizes ___ with H₂O₂. Oxidase acts slower (uses O₂).

2 mL Catechol

2 mL Catechol TEST TUBES A (PEROXIDASE + H₂O₂)

Dark brown (benzoquinone)

2 mL Catechol TEST TUBES B (OXIDASE)

Light brown/yellow (slower)

___specific to peroxidase (H₂O₂- dependent).

2 mL Guaiac

2 mL Guaiac TEST TUBES A (PEROXIDASE + H₂O₂)

Red-brown (tetraguaiacol)

2 mL Guaiac TEST TUBES B (OXIDASE)

No/faint color

Oxidase directly oxidizes ___ using O₂.

2 mL Pyrogallol

2 mL Pyrogallol TEST TUBES A (PEROXIDASE + H₂O₂)

No/faint color

2 mL Pyrogallol TEST TUBES B (OXIDASE)

Deep orange/purple (purpurogallin)

Higher substrate concentration leads to more enzyme-substrate collisions, producing more product (benzoquinone) until enzyme saturation.

4 mL Catechol

4 mL Catechol substrate conc

Dark brown (intense color)

Dilution reduces substrate concentration, slowing the reaction rate due to fewer collisions between enzyme and substrate.

2 mL Catechol + Dist. Water

2 mL Catechol + Dist. Water substrate conc

Lighter brown (less intense color)

oxidized by peroxidase (if present) to form tetraguaiacol. Higher [substrate] = more product.

4 mL Guaiac

4 mL Guaiac substrate conc

Red-brown (intense color)

Diluted guaiac results in less product formation, demonstrating dependence on substrate concentration.

2 mL Guaiac + Dist. Water

2 mL Guaiac + Dist. Water substrate conc

Pale red-brown (faint color)

Higher enzyme concentration increases reaction rate (more oxidase available to catalyze ___ using O₂).

1.5 mL Potato oxidase extract + Catechol

1.5 mL Potato oxidase extract + Catechol enzyme conc

Moderate-to-dark brown color (slower reaction than peroxidase)

Diluted enzyme reduces reaction rate due to fewer active oxidase molecules available.

0.5 mL Potato oxidase + Dist. Water + Catechol

0.5 mL Potato oxidase + Dist. Water + Catechol enzyme conc

Lighter brown color (slower reaction)

High [peroxidase] + H₂O₂ rapidly oxidizes catechol to benzoquinone (enzyme saturation possible).

1.5 mL Potato peroxidase extract + Catechol + H2O2

1.5 mL Potato peroxidase extract + Catechol + H2O2 enzyme conc

Dark brown color (rapid reaction)

Dilution lowers peroxidase concentration, reducing product formation rate.

0.5 mL Potato peroxidase + Dist. Water + Catechol + H2O2

0.5 mL Potato peroxidase + Dist. Water + Catechol + H2O2 enzyme conc

Light brown color (slower reaction)

Extreme pH denatures enzymes, destroying their active sites.

10 M HCl

10 M HCl TEST TUBE A (+ Catechol + Oxidase)

No reaction (clear/no color)

10 M HCl TEST TUBE B (+ Peroxidase + H2O2)

No reaction (clear/no color)

Suboptimal pH alters enzyme structure, reducing activity. Peroxidase is more sensitive to low pH than oxidase.

pH 4 (Acidic)

pH 4 (Acidic) TEST TUBE A (+ Catechol + Oxidase)

Faint brown (slow reaction)

pH 4 (Acidic) TEST TUBE B (+ Peroxidase + H2O2)

Light brown (weak reaction)

Optimal pH for both enzymes. Active sites are functional, maximizing substrate binding and catalysis.

pH 7 (Neutral)

pH 7 (Neutral) TEST TUBE A (+ Catechol + Oxidase)

Dark brown (fast reaction)

pH 7 (Neutral) TEST TUBE B (+ Peroxidase + H2O2)

Dark brown/red (fast reaction)

Oxidase retains slight activity, but peroxidase is denatured in alkaline conditions.

0.1 M Na₂CO₃

0.1 M Na₂CO₃ TEST TUBE A (+ Catechol + Oxidase)

Faint brown (slow reaction)

0.1 M Na₂CO₃ TEST TUBE B (+ Peroxidase + H2O2)

No reaction (enzyme inactive)

Low temperature reduces molecular motion, decreasing enzymesubstrate collisions. Reactions are too slow for detectable color change.

0°C

0°C TEST TUBE A (+ Oxidase + Catechol

No/Slow reaction (colorless/faint yellow)

0°C TEST TUBE B (+ Peroxidase + Catechol + H2O2)

No/Slow reaction (colorless/faint brown

Near body temperature; enzymes are most active. Optimal kinetic energy for catalysis.

37°C

37°C TEST TUBE A (+ Oxidase + Catechol

Dark brown (rapid reaction)

37°C TEST TUBE B (+ Peroxidase + Catechol + H2O2)

Dark brown/red (rapid reaction)

High temperature breaks hydrogen/ionic bonds in enzymes, permanently destroying their 3D structure.

70°C

70°C TEST TUBE A (+ Oxidase + Catechol

No reaction (enzyme denatured)

70°C TEST TUBE B (+ Peroxidase + Catechol + H2O2)

No reaction (enzyme denatured)

Cu²⁺ reduced to Cu⁺ (Cu₂O) by reducing sugars (glucose). Diabetes mellitus, renal glycosuria.

Fehling’s Test

Fehling’s Test

Green → Brick-red precipitate

Alkaline CuSO₄ reduced by glucose → colored precipitates. Hyperglycemia

Benedict’s Test

Benedict’s Test

Orange/red precipitate

Reduction of bismuth in alkaline medium by glucose. Confirms reducing sugars.

Nylander’s Test

Nylander’s Test

Black precipitate (Bismuth oxide)

Heat denatures proteins; acetic acid dissolves phosphates (distinguishes albumin). Nephrotic syndrome, glomerulonephritis.

Heat + Acetic Acid

Heat + Acetic Acid

Turbidity persists after acid

Proteins precipitate with concentrated HNO₃ (denaturation). Proteinuria

Heller’s Test

Heller’s Test

White ring at urine-HNO₃ interface

MgSO₄ + HNO₃ precipitate albumin. Renal disease.

Robert’s Test

Robert’s Test

White ring at contact zone

Acid precipitates proteins (albumin/globulin). Early detection of proteinuria.

Exton’s Test

Exton’s Test

Turbidity with sulfosalicylic acid

Thermosoluble monoclonal light chains (paraproteins). Multiple myeloma.

Bence Jones Protein

Bence Jones Protein

Turbidity at 45–65°C, clears at 100°C, reappears on cooling

Peroxidase-like activity of hemoglobin oxidizes benzidine. Hematuria, hemoglobinuria.

Benzidine Test

Benzidine Test

Blue/green color

Hemoglobin catalyzes H₂O₂ oxidation of guaiac. Occult blood (e.g., UTI, trauma).

Guaiac Test

Guaiac Test

Blue color

Similar to benzidine; detects heme. Sensitive for blood.

Ortho-tolidin Test

Ortho-tolidin Test

Blue color

HNO₃ oxidizes bilirubin → biliverdin (green), then yellow. Obstructive jaundice, hepatitis.

Gmelin’s Test

Gmelin’s Test

Color rings (green → yellow)