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A laboratory exercise focused on understanding the importance of hemoglobin in facilitating gas transport throughout the body and demonstrating the use of a spectrophotometer to study the basic dynamics of the binding of oxygen with hemoglobin. The lab includes an introduction, instructions for constructing an oxygen-hemoglobin dissociation curve, and a post-lab discussion on factors affecting the curve. Key topics covered include the structure of hemoglobin, the cooperative binding of oxygen, the use of a spectrophotometer to measure absorbance, and the effects of parameters like temperature and ph on the oxygen-hemoglobin dissociation curve.
Typology: Summaries
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Road Map
20 min : Introduction
5 min : Oxygen binding curve of hemoglobin and factors
affecting the curve
15 min : Use of spectrophotometer to locate peak
absorbance for colored solution
50 min : Deoxygenation of hemoglobin and constructing
Hb-O
2
binding curve
20 min : Post-lab discussion
Hemoglobin protein is made of 4 polypeptide
chains
Each of these 4 chains contains a heme group
The heme group contains Fe
2+
that can bind O
2
Oxygen-Hemoglobin Dissociation
Curve
(Pages 1-3)
P50: the pressure at
which half of the
hemoglobin is bound
to oxygen
Cooperative
binding: the
(un)binding of one
molecule promotes the
(un)binding of the next
Absorbanc
e
How a spectrophotometer works:
We use absorbance of visible
light to study the Hb-O
2
relationship.
Be sure to set the
spectrophotometer
to Absorbance mode
Zero the
spectrophotometer
with a blank tube
between samples
and different
experimental
conditions
Reminders for using the
spectrophotometer
Part 1 (Page 4-6): finding maximal
absorption (λ max) of a colored (blue)
solution
using spectrophotometer (not directly
related to hemoglobin)
500-700 nm
‘fine-tune’ λ max using a 10nm increment
Blank every time you change
wavelengths.
Part 2 (Page 7-9): Hb-O
2
dissociation curve
Side-arm cuvette
Deoxygenate the
oxygen from PaO
2
760 mmHg in
140mmHg intervals
Measure absorbance
of Hb-O
2
after every
decrement until PaO
2
reaches 0
Spectrophotometer
Using the absorbance data, calculate the % O
2
saturation of
hemoglobin at each step
For example, a 280 mm Hg decrease in pressure: (760–280) =
480 mmHg Calculate the PaO
2
: (0.21 x 480) = 101 mmHg
Construct an oxygen-hemoglobin dissociation curve for the
sample (control and treatment)
% Saturation = (A–B)/(A–C) x 100%
Part 2 (Page 7-9): Hb-O
2
dissociation curve
C = Absorbance before deoxygenation (before removing
oxygen)
B = Absorbance after each deoxygenation step (in
between)
A = Absorbance after complete deoxygenation (after
removing oxygen)
You must memorize this formula for practical
spectrophotometer, wipe each tube with
a Kimwipe.
of spectrophotometer with a dark
cloth to prevent incident light from
affecting measurements.
pump (next to windows) and nitrogen
tubing (next to doors).
and rinse tonometers (NO SOAP) and
Experimental Tips
Post-Lab
Discussion
On whiteboard: draw your Hb-O
2
dissociation curve for
your control group versus treatment group (pH or cold)
Factors Affecting the
Oxygen-Hemoglobin Dissociation Curve
CADET faces right
(↑ in CO
2
, acidity, DPG, Exercise, Temperature)
P
is reached when _______
A) the partial pressure of O2 in blood is 50
mmHg
B) hemoglobin is 50% saturated with
oxygen