BIOL 220: Microscopy & Electrophoresis of Hemoglobin

Use the "learning goals" and questions in the exercises as your study guide

Identify the components of a light microscope

Identify the light path

Illuminator —> condenser lens —> specimen —> objective lens —> tube lens —> ocular lens —> eye

Calculate the limit of resolution

LR = 0.61lambda/NA, where NA is the numerical aperature

A higher NA means a higher resolution, and general a better quality lense

a lower LR means higher performance

How do you properly align your microscope for normal, bright field microscopy?

Through Kohler Illumination, where you try to get the highest intensity and uniform illumination. This is done through 1. focusing the bulb at the plane of the condenser and 2. focusing an image of the iris at the object plane

1. Use 10x to focus on specimen

2. Look at oculars & close field iris until edges are visible (of the hexagon thing)

3. Adjust the substage condenser focus so that the edges of the field iris (hexagon) are as sharp as possible

4. Center the field iris, then open the field iris until the edges are no longer visible

5. Close the condenser iris ¾ the way to adjust (increase) the contrast (don’t want to ruin resolution)

6. Adjust base illuminator (light source) to your liking

7. Switch to 40x and quickly repeat steps to fine-tune the scope

How do you set up phase contrast microscopy? Benefit of phase?

1. Focus w/ 10x, then 40x with good Kohler Illumination

2. Switch condenser to correct phase position - seleect phase annulus ring that matches number on objective

3. Open the condenser all the way and make your brightness all the way

   1. Condenser opened —> the light cone must be wider than the annulus, or no light will pass through

   2. Maximum light —> phase annulus blocks a lot of light

Don’t stain specimen; can see organelles

What does phase microscopy do to the wavelengths of light?

Phase annulus allows a hollow cone oof light on the specimen —> then angled straight upward after passing through the specimen

Phase plate retards diffracted light an additional quarter wavelength, causing diffracted and direct light rays to be out phase by 1/2

Causes destructive interference, creating a dark area and contrast where none actually exists

Limit of Resolution

Smallest distance at which two points can be distinguished as two points and not as one blob

Working Distance

Tells us how far away the lens needs to be from the specimen to properly form an image; higher the NA, smaller working distance

Depth of Field

Tells us thickness of a specimen that will be in focus using given lenses; higher magnification, smaller depth of field

Aberrations (spherical, chromatic)

errors

spherical - arise when light from edge of lens isn’t focusing on same point as light from center of lens

chromatic - when different wavelengths of light focus at slightly different points

Parfocal vs Parcentric Microscopes

Parfocal - specimen will be nearly in focus when you switch objective

Parcentric - specimen will remain in center of view when you switch objective

Total Magnification

Ocular x objective

What are proteins made of? What are amino acids made of?

Polypeptide chains made from amino acids

Amino acids: carboxyl group, amino group, central alpha carbon with R group/side chain

What parts of a polypeptide are ionizable?

First amino group (+) and last carboxyl group (-) in a chain; some R groups are also acidic/basic and can therefore ionize in solutions

Primary vs secondary vs tertiary vs quaternary structures

Primary - sequence of amino acids attached by peptide bonds from the amino terminal end (n-terminus) to the carboxyl terminal (c-terminus)

Secondary - hydrogen bonding between amide (NH-) and carbonyl (CdoublebondO) groups at peptide bonds sites and are regular repeated shapes in short regions along polypeptide chain (eg. alpha helices, beta sheets)

Tertiary - formed by hydrogen bonding, van der Waals, or electrostatic interactions involving the R groups of the many amino acids in polypeptides; the overall 3D shape formed and consists of irregular loops and shapes not classified as secondary structures

Quaternary - when polypeptides are arranged in other particular ways, and are formed in same manner as tertiary structures

In amino acids with hydrophobic R groups, where are the R groups located?

In the center of tertiary/quaternary structures so that they are away from water

Hemaglobin Structure & Function

Oxygen-transporting protein in red blood cells; has a heme group that can allow hemoglobin to bind to oxygen (reverse)

4 polypeptides - adult has 2 alpha and 2 beta subunits - each with a heme group, allowing for 4 oxygen molecules to be carried at a time

Hemoglobin A vs A2 vs F

A - most common form in adults, with 2 alpha and 2 beta subunits (146 amino acids long each)

A2 - accounts for rest of hemoglobin in adults (2-3%), with 2 alpha and 2 delta subunits (10/146 changes)

F - fetal hemoglobin which allows for fetus to get oxygen from mother, with 2 alpha and 2 gamma subunits (39/146 changes)

Hemoglobin S vs C

S - mutation that causes sickle-cell anemia; result from a change in the amino acid at the 6th position of the B chains from glutamic acid (-) to valine (nonpolar)

C - mutation that causes disease (not as bad as sickle-cell); result from a change in the amino acid at the 6th position of the B chains from glutamic acid (-) to lysine (basic/+)

In electrophoresis, where do +/- charges flow?

Negative to + anode, positive to - cathode

What kind of native electrophoresis did we participate in?

We separated proteins in their native (normal) shape and their native (normal) net charge

What factors affect migration of proteins during electrophoresis?

Protein mass, shape, and charge

What does the net charge of a protein depend on?

The protonation status of the terminal amino and carboxyl groups of the polypeptide chains, and that of the acidic/basic amino acids present in the protein

pKa

The pH where 50% of a chemical group are protonated and 50% are deprotonated

pKa values of the following:

• terminal amino group

• terminal carboxyl group

• acidic side chains of aspartate & glutamate

• basic side chains of histidine, lysine, and arginine

• 8

• 3

• 4

• 6, 10.5, 12.5

When pH>pKa…and why? Where do proteins travel to?

There is a bigger percentage of de protonated molecules.

When the pH is greater, there are less H+ available. This causes the system to shift towards making more H+, ie. increasing the percentage of de protonated ions. This doesn’t decrease the pH of the solution because the buffer immediately neutralizes the H+, therefore always leaving more de protonated molecules.

The proteins will travel to the positive anode because they become negatively charged when the buffer takes their H+ away.

When pH<pKa…

There is a greater proportion of protonated molecules

When a molecule is in a substance where the pH is one or two units above its pKa, 90-99% of the molecules will be…

deprotonated (same goes the other way, except if pH is one or two units below)

pI

Isoelectric point; the pH at which the sum of all the negative and positive charges of the various chemical groups in a protein molecule (from amino acids) is 0, where the protein has no net charge

As the pH > pI…

There will be more de-protonation and the protein will have a - net charge, and will migrate to the anode

Why do Hb A, S, and C have distinct pI values, even though they only differ by the amino acids at the sixth position of the B chain?

Because Hb A has glutamate, an acidic negatively charged group…Hb S has valine, a non-polar group…and Hb C has lysine, a basic positively charged group

Carbonic anhydrase

Leads to formation of carbonic acid and appears as a very light band close to the top/point of application

Why does Hb S result in a more severe condition than Hb C? How does that happen?

Hb S has a non polar group (valine) at the 6th position of the amino acid of the beta chain, while Hb C has a basic/+ charged lysine. When there is abundant deoxygenated hemoglobin, a hydrophobic cleft opens on each of the beta subunits. This causes the valine of one hemoglobin molecule to attach to the cleft of the adjacent molecule. As more molecules attach to eachother, they become a long network of fibers that push against the red blood cell, creating a crescent shape. This doesn’t happen with lysine, since it’s positive charge isn’t attracted to the non-polar cleft.