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Lab #2
I. Introduction.
Materials are continually being exchanged between living organisms and their environments.
At the cellular level this depends on several physiological properties collectively
referred to as the permeability of the cell or plasma membrane. Membrane
permeability is based on properties of surface membranes and driving forces. Cell
membranes are differentially permeable to various materials and show alterations in
permeability under (Fig. 1) different physiological and environmental conditions.
Driving forces are those physical forces that depend on the concentration of a substance
across a cell membrane and on the electrical potential of the cell. These experiments
consider cell permeability in relation to diffusion gradients and the structure of the cell
membrane as revealed by the penetration of solutes with various characteristics.
Phospholipid
Bilayers
Ions
Integral
Protein
Solutes that are permeable or nearly as permeable as water (for example urea) do not
contribute to osmotic pressure. Thus a cell will swell in a urea solution much as it will
in distilled water. Subsequently the plasma membrane may rupture due to excessive
internal pressure.
(2 Drops or
100 ul)
(2 Drops or
100 ul)
Figure 6. Sequence of solution additions to the series of five (5) test tubes.
2. To each appropriate tube add 2 ml of one of the various molar solutions of Sucrose
or NaCl:
NaCl 0.16, 0.12 0.08, 0.04, 0.02, 0.00
Sucrose 0.31, 0.25, 0.15, 0.08, 0.04 0.00
**Note one of your group should check that these values are correct with the osmometer and flame photometer. ***
1 Physiological saline is 0.9% NaCl or 150 mM.
Does the hemolysis occur slowly and steadily or does it occur quickly? Is there a latency
or does the hemolysis seem to start immediately?
Determine which solutions produced hemolysis (Fig. 7) by holding each tube against this
printed page. If the printing clear, hemolysis has occurred.
+ Hemolytic
Compound
=
Microscope
4 5 6 Slide
osmolarity
Figure 8. Schematic for determining the status of RBCs in test tubes in which NO
hemolysis occurred.
or
Observed osmolarity
Activity Coefficient=
Theoretical
osmolarity
In the above example at 0.1 M, the osmotic coefficient is O.188/0.186 = 1.010.
A second consideration, which is fairly obvious, but nevertheless one which students and
others often contrive to forget, is that the osmotic effect of electrolytes depends upon
the number of ions yielded by dissociation in solution. Thus a 0.1 M solution of NaCl
should behave approximately as a 0.2 M sucrose solution, and a 0.1 M Na2S04 should
behave as a 0.3 M sucrose solution. Hence, an appropriate multiplier should be
introduced in the above equation when dealing with dissociating electrolytes.
A third consideration which follows directly from the second is that many electrolytes do not
dissociate completely. Even though strong electrolytes like NaCl, KBr, and Na2S04
have an osmotic effect slightly less than predicted, as if they were not totally
dissociated (even though they are). Some authorities treat the correction for this
separately, but here there seems to be no practical reason for not including it as part of
the previously defined Activity Coefficient. A 0.1 M NaCl solution would be expected
to have a freezing point of -0.372C and in fact has Tf of -0.347, thus an i of 0.95. At
0.01 M and 1 M the i for NaCl is 0.968 and 0.905 respectively.
MCB 403 Fall Page 6 of 13
Red Blood Cell Membrane Permeability. Lab #2
Because of such anomalies, D. A. T. Dick (Water and Living Cells) has proposed that the term
"osmole" be defined as that concentration of any specific solute which has the effect of
one mole of an ideal non-electrolyte in aqueous solution (i.e. the concentration which, for
example, causes a Tf of -1.86 C). Thus, solutions of two different solutes, each of
which was 1 osM would very likely have different molar concentrations. This term, and
Dick's definition of it, are widely used.
IC. Discussion.
1. At what concentrations of NaCl and Sucrose was hemolysis produced?
2. At what concentrations would the cells be isotonic (i.e. undergo no volume change)?
3. In what way does the shape of the cell bring about a difference in the answers between
Questions (l) and (2)?
4. What would be the osmotic pressure exerted at room temperature by the solute in
previously unmolested red blood cells, if the cells were placed in pure water, if its
volume did not change, and if the membrane allowed no diffusion out?
Use
! = cRT
where:
! = osmotic pressure (atm.)
c = concentration (moles/liter)
R = 0.0821 atm./mole
T = temperature (K)
&
Osmotic pressure = cRT
Where
c=0.29M (an RBC has an osmolarity of 290 mosM)
T=298K (25 C)
R=0.0821 atm/Kmole
Thus:
OP=.29*0.0821*298
OP=7.09atm
Recall that one mole of an ideal non-electrolyte in 1 liter H20 exerts 22.4 atm. at
STP.
** Note - other factors, such as partition coefficients, along with size must **
be considered in evaluating the rate at which a given substance enters a cell.
IIA. Procedure.
1. Add 2 drops (or 100 ul) of red blood cell suspension to each of four test tubes containing
respectively 2 ml of 0.3 M solutions of:
Compound Molecular Weight
Urea 60
Ethylene glycol 62
Glycerol 92
Glucose. 180
(2 Drops or
100 ul)
Figure 9. Sequence of solution additions to the series of four (4) test tubes.
Time to
Hemolysis.
Molecular weight.
Figure. 10. Plot of the time (in seconds) that it took solutions of various molecular weight to
cause hemolysis.
2. Since all the solutions used in this part of the experiment are hyperosmotic with respect to
the intracellular fluid, why did hemolysis occur? What is the difference between
isotonic and isosmotic? Is it possible for a solution to be isosmotic, but not isotonic?
How and Why?
(2 Drops or
100 ul)
Figure 11. Sequence of solution additions to the series of four (4) test tubes.
2. Mix each of the tubes and hold them against a printed page.
3. Determine the time required for hemolysis to occur for each tube. If the hemolysis is too
fast to allow an accurate estimate of time, try chilling the cells in ice4 water.
4 Be sure to record in the data the temperature of the "ice water" and the Temp of your solutione when the
experiment was run
MCB 403 Fall Page 10 of 13
Red Blood Cell Membrane Permeability. Lab #2
IIIB. Results and Discussion.
1. Plot hemolysis time against partition coefficient and comparative length of carbon
chain.
Time to Time to
Hemolysis Hemolysis
2. What can be concluded about the effect of carbon chain length on red blood cell membrane
permeability?
IVA. Procedure.
1. Add 2 drops (or 100 ul) of red blood cell suspension to each of 4 test tubes containing
respectively 2 ml of 0.3 M solutions of:
(2 Drops or
100 ul)
Figure 13. Sequence of solution additions to the series of four (4) test tubes.
2. Mix each of the tubes and hold them against a printed page.
3. Determine the time required for hemolysis to occur for each tube.
Time to Time to
Hemolysis Hemolysis
2. What do these data suggest about the electrical condition of the membrane?
3. From the above experiment (Sections I through IV) what can be concluded about the
chemical nature of the cell membrane? Discuss your answer in terms of evidence
collected from these experiments.
A B C D E F G
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H I J K L M N
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O P Q R S T U
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V W X Y Z AA AB
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AC AD AE AF AG AH AI AJ
6
7 Alternative procedure I. Electrolytes/Nonelectrolytes.
8 Absorbance
9 Time 0.16 M 0.12 M 0.08 M 0.04 M 0.02 M 0.00 M
10 (min) NaCl NaCl NaCl NaCl NaCl NaCl
11 1
12 2
13 3
14 4
15 5
16 6
17 7
18 8
19 9
20 10
21 11
22 12
23 13
24 14
25 15
26
27
28 Absorbance
29 Time 0.32 M 025 M .015 M 0.08 M 0.04 M
30 (min) Sucrose Sucrose Sucrose Sucrose Sucrose
31 1
32 2
33 3
34 4
35 5
36 6
37 7
38 8
39 9
40 10
41 11
42 12
43 13
44 15
45
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