Hemoglobin preparation

Purification. Hemoglobin is provided by the red blood ceU in highly purified form. However, the red ceU contains many enzymes and other proteins, and red ceU membranes contain many components that could potentially cause toxicity problems. Furthermore, plasma proteins and other components could cause toxic reactions in recipients of hemoglobin preparations. The chemical modification reactions discussed herein are not specific for hemoglobin and may modify other proteins as well. Indeed, multifimctional reagents could actually couple hemoglobin to nonhemoglobin proteins. 

Roth RI, Levin J. Measurement of endotoxin levels in hemoglobin preparations. Meth Enzymol 1994 231 75. 

One of the earliest methods for determining the molar mass of proteins was based on chemical analysis. A hemoglobin preparation produced from red blood cells separated from the blood by the use of a centrifuge was found to contain 0.335% iron, (a) If the hemoglobin molecule contains 1 atom of iron, what is its molar mass (b) If it contains 4 atoms of iron, what is its molar mass ... 

In our studies the ninhydrin-reaction- revealed a number of new fractions, one of which is fluorescent. There are two fractions with a mobility greater than albumin, and two with a mobility lower than y-globulin (Fig. 62b). Some of these are present in many sera, some are rare. They are present in the ultrafiltrate of serum where also a central fraction can be seen, which has the mobility of the y-globulin. They are also present in normal cerebrospinal fluid and were found in hemoglobin preparations. It is not clear if only amino acids are involved, because larger molecules also react with ninhydrin. 

D4. DeRenzo, E. C., Toppolo, C., Aniconi, G., Antonini, E., and Wyman, J., Properties of the a and /3 chains of hemoglobin prepared from their mercuribenzoate... 

Numerous concerns about the safety of hemoglobin preparations have been presented in the literature. Some of the historical safety concerns have been addressed and are no longer issues with respect to the hemoglobin formulations now in advanced clinical trials. For example, effects such as neutrophil and macrophage activation, the formation of microthrombi, and platelet aggregation appear to have been eliminated from these Hb formulations. However, new safety concerns, centering on effects that have been observed clinically, have been raised. Three principal concerns will be discussed here 1) Mb s pressor activity 2) the potential for acellular Hb to exacerbate injury and 3) the potential for Hb to potentiate or exacerbate infection. 

Kang, Y. Luo, R.G. Chromatographic removal of endotoxin from hemoglobin preparations effect of solution conditions on endotoxin removal efficiency and protein recovery. J. Chromatog. A 1998, 809, 13-20. 

Stroma-free hemoglobin preparations have been developed in response to the need for safe, infection-free, sustainable sources of blood in human medicine and to eliminate the need for cross-matching (Table 17.14). Unfortunately, unmodified free hemoglobin has too high an affinity for oxygen, is rapidly eliminated by the kidneys, causes a substantial increase in oncotic pressure and may cause renal injury. For this reason, research has focused on modified polymers of hemoglobin. At the time of writing four products are under development polymerized bovine... 

Decomposition. A hemoglobin preparation (1.00 ml) is decomposed using 1 ml nitric acid (65%) and 0.5 ml hydrogen peroxide (30%). To prevent foaming three drops of octyl alcohol are added and the solution is carefully heated. A clear, light yellow solution is obtained, which is evaporated almost to dryness. To remove excess acid, 1-2 ml water is added and evaporated in vacuo at 90-100°C this is repeated once. The contents of the digestion tube are finally transferred to a volumetric flask and made up to 50 ml with water. 

Decomposition. A hemoglobin preparation (1.00 ml) is decomposed using 1 ml concentrated sulfuric acid and 250 mg potassium sulfate. Mercury or... 

Preparation of HiCN solution. A hemoglobin preparation (1.00 ml) is pipetted into a 250-ml volumetric flask containing about 50 ml diluting solution (200 mg potassium ferricyanide, 50 mg potassium cyanide, and 1.0 g sodium bicarbonate made up to 1 liter with water). After homogenization the volume is made up to 250 ml with the same diluting solution. 

Figure 10. Electrofocusing equipment consisting of electrofocusing column (LKB 8100-10) (110 ml), power supply (LKB 3371), UV monitor (LKB 8300), recorder (Varian G-14), and test tube rack for collecting fractions. The sample is a commercial hemoglobin preparation used as a colored test sample for methodological studies.

Figure 11. Separation of hemoglobin preparation by electrofocusing in the same apparatus shown in Fig. 10 showing a curve from the same run. The curve with the peaks shows the absorption in the eluate at 260 nm. It is obtained from one run in a series of consecutive experiments with the aim of getting a more narrow pH-gradient and a more refined separation of sample compounds in every step. The steadily falling curve is plot of the pH-gradient. The pi values shown at the various peaks were read from the pH-gradient curve. The electrofocusing was run at +4°C as were the pH readings.

Hydrolysis with trypsin of heat-denatured hemoglobin preparations was performed at constant pH using a Radiometer automatic titrator as a pH-stat. The hydrolyses were carried out at 40°C at pH 8.0 in the presence of Ca ion (0.01 M) with an enzyme to hemoglobin ratio of between 1 and 2% by weight (Worthington twice crystallized tr3q)sin). Ninety minutes was allowed for the hydrolysis except in the case of the apes, where the digestion was stopped after sixty minutes. 

Occasionally small amounts (less than 5 percent of the total protein) of material with mobilities different from that of either kind of hemoglobin were observed in these uncrystallized hemoglobin preparations. According to the observations of Stern, Reiner, and Silber (f2) a small amount of a component with a mobility smaller than that of oxyhemoglobin is present in human erythrocyte hemolyzates. 

Fig. 3. The electrophoretic patterns for (o) normal adult human hemoglobin, (fe) sickle-cell-anemia hemoglobin, from the red cells of patients with sicklecell anemia, (c) sickle-cell-trait hemoglobin, which is indicated to be a mixture of normal adult human hemoglobin and sickle-cell-anemia hemoglobin, and (

Just prior to use, the column was washed with 100 ml of 50 mM sodium phosphate pH 7.4, 0.15 NaCl. Table II summarizes the results of one experiment in which a de-hemoglobinized preparation of normal hemolysate was applied to the column and eluted at 20 ml per hour. Before and after each of the buffer washes, two 20... 

Based on data of Roughton (73) obtained on hemoglobin preparations used in velocity experiments. 

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