Cell Membrane Permeability (lab 3) Flashcards
Fundamentally what is a cell membrane composed of?
- 2 lipid layers
- AKA phospholipids (the lipids contain phosphorous)
- Associated w/ the bilayer are proteins
What is the job of the Lipid Bilayer?
- Forms boundary between contents of a cell & its external environment
- Controls what molecules pass through
- Must remain intact so that cell lives
What is Permeable?
-Molecule can pass through the membrane
What is Impermeable?
-Molecule cannot pass through membrane
Why does this lab use Red Blood cells?
-Plasma membrane of a RBC is simple bc its mostly phospholipids w/ very few proteins
What is the goal for this lab?
-Investigate 3 factors that influence cell membrane permeability
What is the role of diffusion?
- One factor that governs the fate of the solute (X)
- Moves from high concentration to low concentration
- So the tendency is to enter the cells
What must we understand first?
- Diffusion
- Membrane permeability
- Water potential
- Fragility of animal membranes
What is the role of Membrane Permeability?
- If membrane is permeable to solute then the substance will enter cell & increase the solute concentration inside the cell
- If high permeability= fast
- If low permeability= slow
- Impermeable= solute won’t enter at all
What role does Water Potential play?
- As solute concentration increases within cell, it’s water potential goes down
- RBC are highly permeable to water
What role does the Fragility of Animal Membranes play?
- As water enter RBC they swell causing the cells to lyse (burst)
- It is during this time where the mixture containing them turns clear
What does the time to Lysis represent?
- Indirect measurement of membrane permeability
- The shorter the time, the higher the permeability
What did we use to measure the time for Lysis?
- Spectrometer (Spec 20)
- Operates on the wavelength of 400-700 nm
How does the Spec 20 work?
- White light from lamp enters monochromator & split into component wavelengths (colors!) by prism
- Mech in the Spec 20 selects desired wavelength & passes it through the tube containing the mixture
- Light that is NOT absorbed by tube falls on detector & gets converted to electrical signal
- Electrial signal is then amplified & its magnitude is displayed by needle on readout scale
What does the Spec 20 read at first before the cells start to lyse? What about after they lyse?
- It will read it as low transmittance because the mixture is turbid
- After the cells lyse the transmittance value will be higher bc the mixture is now clear
What does the value H50 represent?
- Hemolysis 50
- Time required to reach half the max value indicated by the plot on the computer
- So its our measure of the time to lysis
- The smaller the H50, the greater the permeability is & vise versa
What is the Purpose of the lab?
-How the size of the solute molecule, the net charge of the solute particle & the lipid solubility affect cell membrane permeability
What are the Independent Variables?
- Size of molecule
- The net charge of the solute particle
- Lipid solubility of the solute molecule
What is the Dependent Variable?
-Cell membrane permeability
What is important to note about the weight of a molecule?
- Size of a molecule is generally proportional to its weight
- The higher the molecular weight, the harder it is for it to pass through the cell membrane
What 6 liquids did use to illustrate the size on permeability?
- Water
- Urea
- Glycerol
- Glucose
- Sucrose
What is important to note about the charge of a molecule?
- Uncharged molecules pass through cell membranes more easily than charges ones
- Small uncharged water soluble molecules diffuse between lipid molecules of cell membrane in an uncontrolled fashion
- Larger water soluble molecules & small charged molecules must have specific channel to diffuse
Which molecules would easily pass through cell membrane?
- Lipophillic or non polar
- Since they easily dissolve in the lipids of the cell membrane, the cell membrane cannot control which lipid soluable molecules enter
How can we determine lipid solubility by looking at the chemical structure of a molecule?
-The more CH3, CH2, or CH groups a molecule has the more lipid soluble it will be
How can we determine water soluablity by looking at the chemical structure of a molecule?
-The more OH- (Hydroxyl) groups it has, the more water soluble/ polar it will be
What ratio was used to keep track of soluability?
- CHn/ OH
- Meaning that the higher the ratio, the more lipid soluble a molecule will be
What did we use to determine lipid solubility?
- Methanol
- Propanol
- Butanol
- Pentanol
What else did we use to illustrate the effect of lipid solubility?
- Propanol
- 1,2 Propanediol
- 1,2,3 Peopanetriol (glycerol)
- Since these have more OH- groups they’ll become more water soluable as they increase in weight
How do you callibrate the Spec 20?
- Turn on the knob on it’s left end of the top scale so that it reads 0% transmittance. If you don’t then the Spec 20 won’t be able to the test tube due a shutter speed
- Turn the knob at the right end of the top scale with a blank test tube in place so that it actually reads 100% transmittance
- Make sure the wavelenght is at 620 nm
How do you make a blank test tube?
- Place 3 drops of animal blood in a clean & dry test tube
- Then inject 5ml of distilled water
- wait 1 minute
- Then place it in the Spec 20 after you calibrate the left knob at 0%
How do you calculate H50 on the computer graph?
- Move the horizontal line of the blue cursor at the max value that the plot appears to be approaching
- Move red cursor to lowest value of the plot
- Calculate; Blue cursor TV- Red Cursor Divided by 2 & then add Red cursor TV
