Bio 1511 - Enzymes and Proteins AA

What are the main elements found in amino acids and proteins?

Nitrogen, carbon, oxygen, hydrogen, and sometimes sulfur.

What functional groups are present in amino acids?

A carboxyl group and an amino group.

How do R groups differentiate amino acids?

R groups determine the reactivity and properties of amino acids; they can be charged, polar, non-polar, or aromatic.

What is the role of proteins in biological systems?

Proteins generate and maintain cell shape, catalyze biological reactions as enzymes, and interact with other molecules.

What process links amino acids to form peptide bonds?

Dehydration synthesis.

What is produced during the formation of a peptide bond?

Water is generated.

What is hydrolysis in relation to peptide bonds?

Hydrolysis is the reverse reaction of dehydration synthesis, requiring water to break peptide bonds.

What does the Biuret Test indicate and what is it?

The presence of peptide bonds, indicating the presence of proteins. The reagent is alkaline solution of copper sulfate that reacts with more than two C-N bonds to form a purple-colored product.

What color change indicates a positive Biuret Test and what color change indicates a negative Biuret Test?

The solution turns light purple to deep violet in the presence of peptide bonds.

The solution turns a blue in the presence of free/individual AA/water. 

What is the exception in the Biuret Test for cysteine?

Cysteine reacts with sulfur and turns brown/black.

What does thin-layer chromatography (TLC) separate based on?

Affinity for the stationary phase (TLC paper) and mobile phase (solvent).

How do non-polar molecules behave in TLC?

Non-polar molecules will move with non-polar solvents.

What is the formula for calculating Rf values in TLC?

Rf = (distance traveled by substance) / (distance traveled by solvent).

What is the maximum value for Rf values?

Rf values are always less than 1.0 and have no units.

What is the significance of the origin placement on a TLC sheet?

The origin should be 2 cm above the bottom of the sheet to ensure proper separation.

What should you do before placing the TLC sheet in the chamber?

Place very small spots of the sample, let them dry, and repeat several times.

What is the expected outcome of heating free amino acids in TLC?

Free amino acids will turn colors, which can be used for identification.

What are enzymes?

Biological catalysts that speed up chemical reactions by lowering activation energy.

What is the lock-and-key model in relation to enzymes?

A model describing how the 3-D structure of an enzyme's active site binds to a specific substrate like a key fits into a lock.

How do enzymes reduce activation energy?

By facilitating the binding or separation of molecules at the active site, making reactions occur more easily.

What are the basic units of proteins?

Amino acids.

How are enzymes related to proteins?

Most enzymes are proteins composed of amino acids.

What factors affect enzyme function?

Temperature, pH, substrate concentration, and enzyme concentration.

What happens to enzymes at their optimal conditions?

They function best, often determined by their natural environment.

What is denaturation in enzymes?

The disruption of an enzyme's structure due to factors like temperature or pH, leading to loss of function.

What is the result of sucrose hydrolysis?

Sucrose breaks down into glucose and fructose.

How can you test for the presence of reducing sugars?

Using Benedict's test, which indicates the presence of reducing sugars through color changes.

What does a colorimetric scale measure in enzyme activity?

The amount of reducing sugars produced, ranging from no activity (0) to high activity (4).

What is maximum velocity in enzyme reactions?

The rate at which an enzyme-catalyzed reaction reaches its peak activity under specific conditions.

When concentration reaches saturation point. 

What can cause enzyme denaturation?

Extreme temperature, pH changes, and other environmental factors.

Is denaturation always permanent?

No, it can be reversible if the denaturing conditions are removed.

What does it mean if an enzyme-catalyzed reaction is enzyme-limited?

The reaction rate is limited by the amount of enzyme available.

What does it mean if an enzyme-catalyzed reaction is substrate-limited?

The reaction rate is limited by the amount of substrate available.

What is the significance of enzyme properties for metabolism?

They determine how efficiently metabolic reactions occur in cells and organisms.

Define a ninhydrin test

Causes free amino acids to turn a color when heated. Nitrogen of free amino group interacts with 2 molecules of ninhydrin = colored product.

Exceptions are proline (yellow) because of the substitution of the amino group.

Positive reactions range from purple to pink.

Define TLC thin layer chromatography test

Used to separate substances based on the affinities and polarities between stationary phase (TLC Paper, polar) and mobile phase (solvent front, mainly either nonpolar/polar)

Define ascending chromatography

Action of solvent will travel upwards as a result of capillary action in the stationary phase

What’s solvent front?

Component that’s highly soluble, becoming dissolved in the solvent and travels with leading edge. FARTHEST POINT REACHED by solvent

Define a chromatogram.

Once the sheet chromatography has run and the spots have migrated. Now, you can analyze to identify the solute.

What’s another exception when you heat the ninhydrin with the TLC?

Exceptionally strong AA can be visible even while TLC is in chamber

What must you be careful with upon indicating in the biuret test?

That it ONLY indicates peptide bonds, not if the substance is a polypeptide or a protein.

What and how produces a false biuret test?

Urea produces a positive biuret test, even when it’s not a polypeptide/protein. When heated, two molecules of urea combine and form a total of four C-N bonds forming a “biuret” and reacts like a polypeptide. The C-N bonds are considered to be “amide” bonds and produce a reaction even when it is not a peptide bond.

Presence of free SH group colors

Yellow = some SH

Green = more SH

black = many SH 

What can you experiemental test can you do to assess the relative rate of the enzymatic reaction?

Green = slow/less reduced sugars produced

Yellow = large

Orange = much larger

Red = greatest amt of both rate and reduced sugars produced