Lab 3 Membrane structure and function

1. Investigate effects of stressful experimental treatments on living membranes2. Investigate concepts about membrane structure3. Learn basic principles of spectrophotometry and gain experience using spectrophotometer

Objectives

The Membrane: Overview

The Membrane: Overview

The Membrane: Overview

Phospholipids

Phospholipids contain hydrophobic and hydrophilic regions (amphipathic)

The heads are polar which makes them hydrophilic (water loving)

The tails are non-polar which makes them hydrophobic (water hating)

This molecular structure is what allows phospholipids to form membranes

Phospholipids form a bilayer

Membrane is embedded with proteins

Membrane contains many proteins

Peripheral proteins are bound to the surface of the membrane

Integral proteins penetrate the hydrophobic core

These proteins provide a wide variety of functions for the cell

Fluid mosaic model The membrane is FLUID

Lateral movement of phospholipids is rapid

Fluidity of the membrane is important to its function

Fluidity changes with temperature

Fluidity depends on the composition of the membrane Ex: some fish live in

extremely cold environments. How do they keep their membranes fluid?

The membrane is a mosaic: many different proteins

Membrane permeability: “Are you on the guest list?” Plasma membranes are selectively permeable Permeable to non-polar molecules

hydrophobic molecules that can enter the lipid bilayer

Ex. O2, CO2

Non permeable to polar molecules (charged molecules) hydrophilic therefore cannot enter the lipid bilayer

and remain in the aqueous environment. Ex. Na+, glucose, amino acids. (there are, however, mechanisms the cell has

developed to allow import/export) what do you think the transmembrane proteins do?

Hydrophilic VS Hydrophobic

Hydrophilic- Polar (charged)- Water loving- H-bonds to H2O

Hydrophobic- Non-polar (non

charged)- Water fearing- Binds to other non-

polar molecules

C – H2.5 2.1

(close - non-polar)

O

H H2.1

3.5

Solvents used today

Methanol 2-propanol

(Ratio of polar : non-polar groups)

Polar Non-Polar

Size

SUMMARY:

Polar molecules are hydrophilic will stay in the water (H-bonds with water) and not enter the lipid bilayer of the membrane Do we expect them to cause damage to a cell

membrane? Non-polar molecules are hydrophobic therefore

will enter the lipid bilayer Do we expect them to cause damage to a cell

membrane? Does the size of a non-polar molecule influence

the extent of damage?

Today’s lab

Investigate how different temperatures and solvents can cause membrane damage

Beta vulgaris

Temperature stressOrganic solvent stress

Measure betacyanin leakage as a wayto quantify membrane damage

Betacyanin: red pigment

• Betacyanin is found in vacuole (enclosed in membrane)

• If vacuole membrane is damaged, betacyaninwill leak out (red pigment)

• How can we measure this?

Betacyanin

Spectrophotometer

525nm(the wavelength absorbed by betacyanin)

• Betacyanin absorbs light at a wavelength of 525nm

• The spec will be set to shine 525nm light on your tubes

• the amount of light being absorbed will be measured.

• The more betacyanin in the tube, the ___?___ absorbance reading.

Spec 20s

Follow the instructions in Appendix F

Betacyanin absorbs light maximally at 525nm, so you need to set the Spec to shine light of 525nm. Make sure filter lever is turned to the right range

Experimental design

Hypotheses Specific, logical

Independent variable This is the part that you are controlling ________ (Temperature)__________

Dependent variable This is the part that you measure _________ (absorbance)___________

Controls Best control is the removal of the

independent variable (ie: the controls for this experiment are room temp OR water)

Hypothesis:

A good hypothesis must: explain how or why: provide a mechanism. be compatible with and based upon the existing

body of evidence. link an effect to a variable. state the expected effect. be testable. have the potential to be refuted.

Hypothesis: make your own!“I hypothesize that …”

“The rationale for the hypothesis is…”

Remember

You will find that certain temperatures and solvents will damage the membrane more than others. MAKE SURE YOU CAN EXPLAIN WHY certain temps/solvents damage the membrane

What is happening to: the phospholipids? the proteins?

Why does this result in betacyanin leakage?

Graphs

Temp treatment is a line graph because the data is continuous Independent variable on the X axis Dependent variable on the Y

Solvent treatment is a bar graph Follow guidelines in Appendix Graph should be full page Include your data AND class data