Haemoglobin Binding


*** Advanced Project Notice ***

The following project is an ADVANCED project and may require handling of dangerous materials and/or equipment and is intended to be conducted by adults only!


Purpose

To demonstrate the ability of oxygen and carbon monoxide to bind with haemoglobin.


Additional information

Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of vertebrates, and the tissues of some invertebrates. Oxygen binding to haemoglobin is a reversible reaction. At lungs oxygen binds to haemoglobin to form the oxygen-haemoglobin complex called oxyhaemoglobin. Once these oxyhaemoglobins are transported into tissues they release oxygen for cellular respiration. After this, the haemoglobins are available to bind with oxygen again at lungs. But carbon monoxide binds to haemoglobin irreversibly and 200 times faster than oxygen to form a very bright red compound called carboxyhaemoglobin, meaning that small amounts of CO dramatically reduce hemoglobin's ability to transport oxygen. This incident can even bring death to individuals. When inspired air contains CO levels as low as 0.02%, headache and nausea occur; if the CO concentration is increased to 0.1%, unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked by CO. In similar fashion, hemoglobin also has competitive binding affinity for cyanide (CN-), sulfur monoxide(SO), nitrogen dioxide (NO2), and sulfide(S2-), including hydrogen sulfide (H2S). All of these bind to iron in heme without changing its oxidation state, but they nevertheless inhibit oxygen-binding, causing grave toxicity.

Haemoglobins absorb electromagnetic radiation in visible range. The wave lengths at which a maximum absorbance is showed depends on the bound ligand. By plotting a graph, absorbance vs. wave length we can identify haemoglobin (no bound ligand), oxyhaemoglobin and carboxyhaemoglobin.


Sponsored Links


Required materials

  • Centrifuger
  • Colorimeter (Spectrophotometer-visible range)
  • 15ml Test tubes
  • Glass/rubber tubes
  • 1 ml of human/animal blood sample
  • Sodium citrate buffer (pH 7.0)
  • Deionized water
  • 0.9% NaCl solution
  • Stokes’ reagent –Dissolve 3g of ferrous sulphate in a small quantity of water and add to it in watery solution 2g of tartaric acid. Make up the mixture to 100ml and just before using add NH4OH until the precipitate which at first forms is dissolved.

Estimated Experiment Time

1-2 hours


Step-By-Step Procedure

  • A. Haemoglobin Isolation
    • 1. Collect a blood sample from a human/animal.
    • 2. Transfer 1ml of blood into 5ml of citrate buffer (to prevent clotting).
    • 3. Centrifuge the mixture at 3000g*1 for 5 min.
    • 4. Remove the supernatant plasma.
    • 5. Add 10ml of 0.9% NaCl solution to the bottom cellular sediment and mix gently.
    • 6. Centrifuge the sample again at 3000g for 5min.
    • 7. Remove the supernatant again.
    • 8. Repeat steps 5-7, to wash the red cells until the supernatant is colorless.
    • 9. Resuspend the washed red blood cells in 2 volumes of deionized water and centrifuge at 10000g for 10 min.
    • 10. Decant the red supernatant and use it for the following tests.
  • B. Oxyhaemoglobin spectrum
    • 1. Dilute a sample of haemoglobin using the buffer solution. (The dilution depends on the absorbance values. Dilute the sample until the absorbance values are less than 1.0. You have to figure out the dilution by doing trials).
    • 2. Shake the sample in air for a while (to form oxyhaemoglobin complexes)
    • 3. Measure the absorption spectrum between 400-700nm, using the colorimeter*2. (If necessary dilute the sample again).
    • 4. Plot a graph absorbance vs. wave length.
  • C. Deoxyhaemoglobin (haemoglobin with no bound oxygen)
    • 1. Prepare a diluted sample of haemoglobin.
    • 2. To remove bound oxygen if there any, add 2 drops of freshly prepared Stoke’s reagent to 4ml of haemoglobin solution.
    • 3. Obtain the absorbance spectrum as in procedure B.
  • D. Carboxyhaemoglobin spectrum
    • 1. Get a diluted sample of haemoglobin solution.
    • 2. Bubble CO gas through the sample.
      • i. CO is found in vehicle emission. Use the FIGURE A set to mix vehicle emission with haemoglobin.
      • ii. Attaché the funnel/funnel shaped tube to the exhaustion pipe of a started vehicle.
      • iii. Bubble the haemoglobin solution for 1-2 min.
    • 3. Obtain the absorbance spectrum as in procedure B.

Note

*1 - Relative centrifugal force (RCF) expressed in units of gravity (times gravity or x?g). Many microcentrifuges only have settings for speed (Revolutions per minute, RPM), not relative centrifugal force. Consequently, a formula for conversion is required to ensure that the appropriate setting is used in an experiment. The relationship between RPM and RCF is as follows: g = (1.118 ??10-5) R S2 Where g is the relative centrifugal force, R is the radius of the rotor in centimeters, and S is the speed of the centrifuge in revolutions per minute.

*2 – for colorimetric measurements, use the buffer used to dilute the haemoglobin sample as the zero dilution sample. If you are not familiar with colorimeter better use some guidance from a technician. As this is an expensive instrument, be cautious.

Figures & Illustrations

Figure 1
Figure 1


Observation

You will get absorbance spectra for haemoglobin(deoxyhaemoglobin), oxyhaemoglobin and carboxyhaemoglobin similar to following figure.

Figure 2
Figure 2


Figure 3
Figure 3


Result

See Observation


Sponsored Links


Take a moment to visit our table of Periodic Elements page where you can get an in-depth view of all the elements, complete with the industry first side-by-side element comparisons!


Your email:
Your name:
Recipient email:
Recipient name:
Message:
 

Print this page   Bookmark this page  

Hide/View all projects Hide all projects Hide/View all projects

All Projects List

  • Accelerate Rusting
  • Acids And Bases
  • Additive Colors
  • Ant Microphotography
  • Apple Mummy
  • Balloon Rocket Car
  • Barney Banana
  • Bending Water
  • Bernoulli’s Principle
  • Blind Spot in Vision
  • Boiling Point of Water
  • Build an Electromagnet
  • Build an Inclinometer
  • Caffeine And Typing
  • Candle Race
  • Candy Molecules
  • Capillarity of Soils
  • Carbon in the Atmosphere
  • Checking vs. Savings
  • Chemical Metamorphosis
  • Clean Cleaners
  • Cleaning Oil Spills
  • Climbing Colors
  • Cloud Cover
  • CO2 & Photosynthesis
  • Collecting DNA
  • Colorful Celery
  • Coloring Matter in Food
  • Colors And Temperature
  • Composition of a Shell
  • Computer Passwords
  • Construct a Lung Model
  • Corrosiveness of Soda
  • Create a Heat Detector
  • Create Lightening
  • Cultivate Slime Molds
  • Cup of Lava
  • Dehydrated Potato
  • Desalinate Sea Water
  • Detergents and Plants
  • Dissolving in Liquids
  • Dissolving Solutes
  • Distillation of Water
  • Double Color Flower
  • Egg in a Bottle
  • Enzyme Activity
  • Eroding Away
  • Erosion Simulator
  • Evaportating Liquids
  • Expanding Soap
  • Exploding Ziploc
  • Extracting Starch
  • Fans And Body Temp
  • Fertilizer & Plants
  • Filtration of Water
  • Floating Ball Experiment
  • Floating Balloon
  • Fog Formation
  • Font and Memory
  • Food and Academics
  • Friction And Vibration
  • Fruit Battery Power
  • Full and Low Fat Foods
  • Galileo's Experiment
  • Gas To Liquid
  • Grape Juice & Cleaners
  • Gravity and Plants
  • Green Slime
  • Growing a Crystal
  • Growing Bread Mold
  • Growing Population
  • Haemoglobin Binding
  • Hard vs. Soft Water
  • Homemade Floam
  • Home-made Geodes
  • Home-Made Glue #1
  • Homemade Snowflakes
  • Home-made Stethoscope
  • Homemade Volcano
  • Homemade Windmill
  • Human Battery Power
  • Inertia of an Egg
  • Information and CD’s
  • Invisible Ink
  • Isolation of Bread Mold
  • Isolation of DNA
  • Jar Compass
  • Lemon Floaties
  • Levers And Force
  • Lift an Ice Cube
  • Light Colors and Plants
  • Long Lasting Bubbles
  • Magic Balloons
  • Magnified Light
  • Make a Compost Pile
  • Make a Fuse Model
  • Make a Parallel Circuit
  • Make An Elevator
  • Make Electric Circuits
  • Make Limestone
  • Make Objects Float
  • Make Static Electricity
  • Make your own sundial
  • Matchbox Guitar
  • Math and Gender
  • Mean, Median and Range
  • Measuring Air Pollution
  • Mentos Soda Volcano
  • Microbial Contaminants
  • Milky Plastic
  • Mini Greenhouse
  • Missing Reflection
  • Mixing With Water
  • Molls Experiment
  • Music and Plants
  • Musical Bottles
  • Nocturnal Plants
  • Ocean Life & Oil Spills
  • Ocean Temperature
  • Optical Mice
  • Oral Bacteria
  • Orange Water Volume
  • Organic vs. Inorganic
  • Osmosis
  • Oven Baked Ice Cream
  • Oxygen & Photosynthesis
  • Paper Bridge
  • Paper Marbling
  • Pascal’s Law
  • Play-Doh and Volume
  • Preserve Spider Webs
  • Pressure Volcano
  • Pulse Rates
  • Pythagorean Tuning
  • Refraction in Water
  • Rollercoasters & Loops
  • Rubber Bones
  • Rubber Heat Reaction
  • Rubbery Egg
  • Rust and Moisture
  • Search Engines
  • Secondary Colors
  • Seed Germination
  • Seed Germination II
  • Separate Salt And Pepper
  • Snappy Sounds
  • Soil Erosion
  • Soil vs. Hydroponics
  • Sound Waves
  • Spectrum through Water
  • Speed of Decomposition
  • Speed of Dissolving
  • Spore Prints
  • Star Observer
  • Static Electricity
  • Statistics and M&M’s
  • Stem-less Flowers
  • Super Strength Egg
  • Sweet Erosion
  • Temperature and CPUs
  • Thirsty Rocks
  • Tornado Demonstration
  • Translucent Egg
  • Transpiration in Plants
  • Typing and Speed
  • Vibrating Coin
  • Volcanic Gas
  • Water and Living Things
  • Water Displacement
  • Water Evaporation
  • Water pH
  • Your Planetary Age