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Hemoglobin-Oxygen Binding Lab, Summaries of Business and Labour Law

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

2021/2022

Uploaded on 04/23/2024

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PNB 2265: Lab 2
Log onto HuskyCT and Start the
in-class quiz.
You have five minutes to finish it.
Remain quiet until we start class.
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PNB 2265: Lab 2

Log onto HuskyCT and Start the

in-class quiz.

You have five minutes to finish it.

Remain quiet until we start class.

Lab 2: Hemoglobin-

Oxygen Binding

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

  • This exercise is to get you familiar with

using spectrophotometer (not directly

related to hemoglobin)

  • Two-step plotting:
    • find λ max in the range of wavelengths between

500-700 nm

‘fine-tune’ λ max using a 10nm increment

Blank every time you change

wavelengths.

• PLEASE DO NOT DISCARD BLANK AND
COLORED SOLUTION AT THE END OF LAB

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

  • Before placing tubes into the

spectrophotometer, wipe each tube with

a Kimwipe.

  • Cover the opening

of spectrophotometer with a dark

cloth to prevent incident light from

affecting measurements.

  • Work with your TA for using the vacuum

pump (next to windows) and nitrogen

tubing (next to doors).

  • When finished, dump Hb solution

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