Laboratory
Laboratory
In animals that augment their gas exchange systems with a circulatory (or coelomic) system, the rate at which Of can be delivered to the tissues is a function of both the rate of fluid flow past the tissues and the amount of oxygen carried in the fluid. The oxygen carrying capacity of coelomic or circulatory fluid can be greatly increased by the presence of a respiratory pigment dissolved in the fluid or contained in cells. In homo sapiens, for example, the oxygen carrying capacity increases from 0.24 volume percent (in solution only) to over 19.0 volume percent in the presence of red blood cells.
Respiratory pigments function by forming a reversible bond with oxygen allowing them to load up with oxygen under some conditions and to unload under others. A pigment that binds oxygen strongly will be able to capture large amounts of oxygen from the environment, but may have difficulty releasing it to the tissues. A pigment that binds oxygen weakly, on the other hand can release its oxygen to the tissues readily , but may not capture large amounts from the environment.
The evolution of respiratory pigments has been directed by these two opposing factors as well as by the respiratory demands of the organism and the environmental availability of oxygen. Respiratory pigments have evolved independently in several groups of animals and they include hemoglobin, hemocyanin, hemerythrin, and chlorocruorins each with various affinities for oxygen.
The amount of oxygen that will bind to a respiratory pigment depends on several factors. The concentration of oxygen is the most significant. Respiratory pigments will bind more oxygen when the partial pressure of oxygen is high (as in the alveoli of lungs or the branchial filaments of gills) than they will at the lower partial pressures of oxygen found in the tissues. This relationship is illustrated by an oxygen dissociation curve (Figure 05.1). This curve indicates how much oxygen is bound by a respiratory pigment as the partial pressure of oxygen increases and how much is released as the partial pressure decreases. Note that the shape of this curve is sigmoidal. There is a tendency to be either completely loaded or completely unloaded. This tendency results from the phenomenon of cooperativity. Once a single molecule of oxygen binds it makes it easier for additional oxygen molecules to bind. In the tissues with their lower oxygen concentration and higher CO2 concentrations the opposite happens; as one molecule of oxygen is lost it becomes easier for the remaining oxygen to be released.
As aquatic environments vary in the total oxygen available to organisms and animals vary in the total oxygen concentration required by their tissues, respiratory pigments tend to be fine tuned to each particular organism. In this laboratory you will examine the oxygen dissociation curves of two species estuarine animals and draw conclusions as to any correlations with their life styles.
The following technique will allow you to obtain large quantities of crab blood. Quickly cut through the basis of the swimming leg of the crab with a sharp scissors.
This must be done quickly as the crab has the ability to automize the limb and seal the wound if it is severely traumatized by the procedure. Collect as much blood as possible. About 6 ml or more will work. Avoid contamination with sea water from the branchial chamber.
Clot the blood with vigorous stirring and dilute 1:1 with buffer solution. Transfer the blood to a "Corex" centrifuge tube. Caution: Any other test tube will implode on centrifugation. Spin at 15,000 RPM in the Sorval RCB2 centrifuge for 10 minutes.
Careful balance of paired tubes is imperative at this speed. Weigh the sample tube and balance tube and adjust to within 0.1 g. An unbalanced centrifuge is extremely dangerous.
You can pair up with other groups to balance tubes and save time on the centrifuge runs.
Walter I. Hatch
wihatch@smcm.edu
August 12, 2012