Hemoglobin buffering

When the cells enter the region where tissues are producing carbon dioxide from metabolism, the carbon dioxide in the plasma rises. In this example, we chose a rise to a partial pressure of 46 mm Hg. The pH of the plasma dropped to 7.34, carbon dioxide equilibrated rapidly with the cell water, cell pH dropped, hemoglobin buffered the additional protons, more bicarbonate was formed, and some of the bicarbonate formed exchanged with medium chloride. The end... 

An average rate of metabolic activity produces roughly 22,000 mEq acid per day. If all of this acid were dissolved at one time in unbuffered body fluids, their pH would be less than 1. However, the pH of the blood is normally maintained between 7.36 and 7.44, and intracellular pH at approximately 7.1 (between 6.9 and 7.4). The widest range of extracellular pH over which the metabolic functions of the liver, the beating of the heart, and conduction of neural impulses can be maintained is 6.8 to 7.8. Thus, until the acid produced from metabolism can be excreted as CO2 in expired air and as ions in the urine, it needs to be buffered in the body fluids. The major buffer systems in the body are the bicarbonate-carbonic acid buffer system, which operates principally in extracellular fluid the hemoglobin buffer system in red blood cells the phosphate buffer system in all types of cells and the protein buffer system of cells and plasma. 

Figure 6-9. The Bohr effect. Carbon dioxide generated in peripheral tissues combines with water to form carbonic acid, which dissociates into protons and bicarbonate ions. Deoxyhemoglobin acts as a buffer by binding protons and delivering them to the lungs. In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which then is exhaled.

A phosphate buffer system (pH 6.5 - 7.0) Is useful for detecting small quantities of the hemoglobins H and Bart s these variants will both move toward the anode while Hb-A remains at the origin. Figure 3 gives some examples of separations that can be obtained. 

Fig ure 3. Starch gel electrophoresis of hemoglobins. Tris-EDTA-boric acid buffer, pH 9.0. O-Dianisidine stain. 

The stability of hemoglobin In a freshly prepared red cell hemolysate at temperatures varying between 50 and 70 Is distinctly less than that of a normal control. The unstable variant will also show decreased stability at 37 In the presence of the Isopropanol-trls buffer, pH 7.4. 

Detection of Met(Ferrl-)Hemoglobins (Hb-M) Detection of these variants can be made by starch gel electrophoresis of the ferrl-derlvatlves of hemoglobins In red cell hemolysate using a phosphate buffer, pH 7 0 (25) However, some methemoglobln variants can be separated from normal Hb-A at pH 9 0 (40) ... 

The presence of Individual chains In a hemoglobin variant can also be demonstrated by electrophoresis at alkaline pH after the protein has been dissociated Into Its subunits through exposure to 6 M urea In the presence of 3-mercaptoethanol. The buffer is either a barbital buffer or a tris-EDTA-boric acid buffer, pH 8.0 - 8.6, and contains 6 M urea and 3-niercapto-ethanol. Dissociation of the hemoglobin Into subunits Is best accomplished In a mixture of 1 ml 10 g% Hb (or whole hemolysate), 4 ml 6 M urea barbital or tris-EDTA-boric acid buffer, and 1 to 1.5 ml 3-mercaptoethanol. After 30 minutes to 1 hour the sample Is subjected to cellulose acetate or starch gel electrophoresis. Each chain has a specific mobility and an alteration In electrophoretic mobility easily Identifies the abnormal chain. 

Measurement of free t-PA in plasma presents challenges in terms of preventing t-PA from complexing to PAI-1 released from platelets after blood collection. To dissociate any preformed t-PA-PAI-1 complex, the anticoagulant pH has to be close to 3.0. Even if blood is collected with an acidic anticoagulant, the blood pH will rise because of the powerful buffering action of hemoglobin. Thus, the pH of plasma has to be adjusted to 3.0 in order to dissociate the t-PA-PAI-I complex (115). 

FIGURE 8.5 Multiple-sensor respirometry. Representative calibration traces of PNOS (thin line, left ordinate) and PHSS (thick line, right ordinate) operating simultaneously in PBS, pH 7.3 at 37°C, with 50pM DTPA in a closed chamber respirometer. After NO additions were made, the chamber solution was replaced with fresh buffer, to which Na2S stock solutions were then injected in a stepwise manner. The stable POS signal shown at 2 pM 02 demonstrates that the POS does not respond to NO or H2S. Injections of anoxic buffered NO and H2S stocks are shown with concentrations at arrows, as are additions of Lucina pectinata ferric hemoglobin I (metHb I), which stoichiometrically binds to H2S (after [41]). 

The earliest compensatory response is to chemically buffer excess bicarbonate by releasing hydrogen ions from intracellular proteins, phosphates, and hemoglobin. If respiratory alkalosis is prolonged (more than 6 hours), the kidneys attempt to further compensate by increasing bicarbonate elimination. 

Prepare 100 mL (500 mL if the gel is to be run under the buffer) of an electrophoresis buffer that is 20 mM tris-(hydroxymethyl)amino methane (TRIS or THAM), 6 mM sodium acetate, and 1 mM disodium EDTA. Adjust the pH of this solution to 7.9 using concentrated HC1. Also prepare small volumes of solutions of hemoglobin and cytochrome C in the buffer (the concentration is not important) and also a mixture solution of these two solutes. Add a quantity of sucrose to each. 

In your notebook, make a drawing of the observed banding pattern, labeling both the hemoglobin and cytchrome C. Explain, in relevant detail, what you can determine about the isoelectric points of these proteins and what might happen in the gel if the buffer pH were changed from 7.9 to 3.9. 

Figure 8.5 Effect of pH on protein mobility. Hemoglobin A (pi 7.1) and Hemoglobin C (pi 7.4) were electrophoresed in eight of the McLellan native, continuous buffer systems (Table 8.1). The diagram is drawn to scale. Migration is from top to bottom as shown by the vertical arrows. Bands marked A or C indicate the positions of the two hemoglobin variants in each gel representation. The polarities of the voltages applied to the electrophoresis cell are indicated by + and - signs above and below the vertical arrows. Run times are shown below the arrows. Note the polarity change between the gel at pH 7.4 and the one at pH 8.2. This reflects the pis of the two proteins (and was accomplished by reversing the leads of the electrophoresis cell at the power supply).

AmB formulations were dispersed in phosphate-buffered saline (PBS) at different concentrations (0.1 lOOpg/mL) and incubated for five minutes at 37°C. Freshly isolated human erythrocytes were then added to a final hematocrit of 2% and incubated at the same temperature for 30 minutes. After centrifugation, the supernatant was removed and the RBC pellet was lysed with sterile water. The hemoglobin remaining in the pellet was estimated from its absorption at 560 nm recorded with a spectrophotometer. The percentage hemolysis was calculated from the difference between the hemoglobin remaining in the test samples and the control incubated with PBS alone. 

Buffers resist changes in pH. Substances can act as buffers at their pK values. In proteins, the amino acids with ionizable R-groups can act as buffers, altiiough the only amino acid that is useful in maintaining physiologic pH (7.2-7.4) is histidine with an R-group pK near 7. Hemoglobin can act as an intracellular buffer in red blood cells because it contains histidyi residues. 

Due to their high concentration, plasma proteins—and hemoglobin in the erythrocytes in particular—provide about one-quarter of the blood s buffering capacity. The buffering effect of proteins involves contributions from all of the ionizable side chains. At the pH value of blood, the acidic amino acids (Asp, Glu) and histidine are particularly effective. 

---