Lab 3 Membrane structure and function

1. Investigate effects of stressful experimental treatments on living membranes 2. Investigate concepts about membrane structure 3. Learn basic principles of spectrophotometry and gain experience using spectrophotometer Objectives

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. O 2, CO 2  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 – H (close  non-polar) O HH

Solvents used today Methanol 2-propanol (Ratio of polar : non-polar groups) PolarNon-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 stress Organic solvent stress Measure betacyanin leakage as a way to quantify membrane damage

Betacyanin: red pigment Betacyanin is found in vacuole (enclosed in membrane) If vacuole membrane is damaged, betacyanin will 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