OSMOTIC RELATIONS AND ERYTHROCYTE PERMEABILITY
Biol 303
Introduction
A cell placed in a hypotonic solution will absorb water osmotically and will swell. An erythrocyte (red blood cell, or RBC) in such a situation will burst open, releasing hemoglobin into the solution. This process is called hemolysis. A cell placed in a hypertonic solution will lose water osmotically and shrink. An isotonic solution is one where the cell remains at constant volume, neither swelling nor shrinking. Theoretically, an isotonic solution is one whose osmotic pressure is the same as the osmotic pressure of the cell’s contents, the cytoplasm.
In physical chemistry, the osmotic pressure developed by a solution depends only on the concentration of solute particles according to the Van t’Hoff relation:
= RT C (1)
where is the osmotic pressure and C is the total concentration of all osmotically active particles. Technically, molal concentration (moles/kg of solvent) should be used but we will use molarity (moles/liter of solution) instead. The small resulting error is negligible in comparison with the other problems with this equation described below.
A 0.9% solution of NaCl is isotonic for mammalian RBCs. This corresponds to a 0.15 M solution. Since NaCl dissociates into separate Na+ and Cl- ions, the actual osmotic concentration is 0.3 OsMolar. Therefore any solution that has an osmotic concentration of 0.3 OsMolar should be isotonic for RBCs.
Unfortunately, the theory only applies to a perfect semipermeable membrane, one which is freely permeable to the solvent (water) and completely impermeable to all the solutes. The actual osmotic pressure developed across a real membrane, however, depends on whether the solute is permeable. A particle that moves across the membrane as readily as water will develop no osmotic pressure; a solute that is completely impermeable will develop the full osmotic pressure calculated in Eq. 1. The actual pressure is:
= RT C(2)
where , the “fudge factor” is called the reflection coefficient. The magnitude of depends on its relative permeability. A solute which cannot cross the membrane has = 1, while one which is as permeable as water has = 0.
As described above, all solutions of 0.3 OsMolar concentration should be isotonic for RBCs. However, if the membrane is permeable to the solute, then 1, the solution will be hypotonic
If RBCs are placed in a 0.3 M solution of a non-electrolyte for which 1, the solution will be
hypotonic, and water will enter causing hemolysis. The rate at which water enters depends on the osmotic pressure. Therefore, the length of time it takes for the cells to hemolyze will depend on , which in turn depends on the permeability of the solute. Solutes that are very permeable ( near zero) will cause rapid hydrolysis and solutes that are poorly permeable ( near one) will cause slow hydrolysis. Solutes that are impermeable ( = 1) will not cause hemolysis at all since a 0.3 OsMolar solution is isotonic.
Hemolysis can be detected rather easily. A suspension of RBCs appears turbid; you cannot see through it because the cells scatter light. When hemolysis occurs, the cell “ghosts,” the broken membrane fragments, scatter light much less so that the solution becomes transparent. Of course the hemoglobin from the hemolyzed RBCs still remains in the solution so that the red color remains, but the solution is clear enough to see through.
In this experiment, you will measure the relative permeability of a variety of substances by measuring the time it takes for them to induce hemolysis. The substances can be grouped into different series of differing molecular weight, polarity, lipid solubility, etc., so that various factors that influence membrane permeability may be studied.
Report Work in teams of two. Each team should fill in the blanks on the attached table and turn it in (one sheet per team) at the end of the laboratory period. We will discuss the significance of the data during the laboratory period. No other report is required. However, you may well expect an exam question about the relationship between molecular size, polarity, and lipid solubility and its permeability across a membrane.
Procedure Put 2 ml of the test solution into a test tube. Add 2 drops of the blood suspension.
Mix the tube and start timing. Hold the tube in front of some sharply outlined object, such as the print on this page or the following lines:
______
______
______
The time when the print or the lines first becomes clear can be taken as the hemolysis time.
Run the test solutions in duplicate but don’t run your replicates at the same time. Otherwise you may prejudice your determination of exactly when the end point is reached knowing that the two should hemolyze at the same time.
You don’t have to time each sample exactly. We are only interested in relative comparisons of permeability. So use the following categories of hemolysis time
Immediate – hemolysis is evident as soon as the sample is shaken
Very fast -- a few seconds, perhaps one to five
Fast – about five to 15 seconds
Medium – less than one minute
Slow – several minutes
Very slow – up to a half hour
Never – it hasn’t hemolyzed in a half hour
We will interpret the data in terms of the following “series”
methanol – ethanol – n-propanol – n-butanol
methanol – ethylene glycol – glycerol – erythritol
ethylene glycol – di-ethylene glycol – tri-ethylene glycol
urea – thiourea
NaCl – KCl
In lab, we will discuss the significance of these groupings and why the permeabilities vary within each series.
OSMOTIC RELATIONS AND ERYTHROCYTE PERMEABILITY
Biol 303 Data Sheet
Name ______Date ______
______
Material / Hemolysis Time / Molecular weight / Structural formulawater / xxxxxxxx
NaCl / xxxxxxxx
KCl / xxxxxxxx
urea
thiourea
methanol
ethanol
n-propanol
n-butanol
ethylene glycol
di-ethylene glycol
tri-ethylene glycol
glycerol
erythritol