Hemolysis enzymes

Glucose- 6-phosphate dehydrogenase Low or absent enzyme activity in about 10% of African populations. Hemolysis following intake of a number of drugs which have electrophilic reactive metabolites, but also, carriers of this enzyme deficiency have a partial protection from malaria. 

TBT and TFT are membrane-active molecules, and their mechanism of action appears to be strongly dependent on organotin(IV) lipophilicity. They function as ionophores and produce hemolysis, release Ca(II) from sarcoplasmic reticulum, alter phosphatodylseiine-induced histamine release, alter mitochondrial membrane permeability and perturb membrane enzymes. Organotin(IV) compounds have been shown to affect cell signaling they activate protein kinase and increase free arachidonic acid through the activation of phospholipase... 

Homogenates of MetruUum senile, possibly the world s most common large sea anemone, yield extracts that are powerfully hemolytic for washed mammalian erythrocytes (22). The active substance, metridiolysin, is a protein of molecular weight approximately 80,000. In contrast to the sphingomyelin-inhibitable toxins, metridiolysin is an acidic protein having a pi of about 5. It is thermolabile and is inactivat by proteolytic enzymes. The optimal pH for hemolysis is between 5 and 6, and at pH 8 the lysin is inactive. It can be dissociated into two subunits of unequal size. Besides being cytolytic in vitro, metridiolysin is lethal when injected intravenously into mice. As shown in Table IV erythrocytes from the horse or dog are about a hundred times as sensitive to lysis as those from the mouse, and erythrocytes from other animals tested are intermediate in sensitivity. 

Red blood cells also contain sufficient acid phenylphospha-tase for mild hemolysis to cause false elevations. Therefore, inhibitors such as ethanol, formaldehyde, copper sulfate> and 1-tartrate have been used to inhibit selectively the enzyme of one or more tissues and enhance the specificity of the test (101). Ethanol is unsuitable because it inhibits the enzyme from erythrocytes and prostate simultaneously, and because it yields serum activities which correlate poorly with prostatic disease. Formaldehyde inhibits the erythrocytic enzyme and has been said to yield clinically satisfactory results. The copoper resistant acid phosphatase of serum is elevated by metastatic carcinoma of the breast, as well as by other metastatic cancers, and is also elevated by a wide variety of non-cancerous diseases. 

Hereditary deficiency of phosphoglycerate kinase (PGK) is associated with hereditary hemolytic anemia and often with central nervous system dysfunction and/or myopathy. The first case, reported by Kraus et al. (K24), is a heterozygous female, and the results are not so clear. The second family, reported by Valentine et al. (V3), is a large Chinese family, whose pedigree study indicates that PGK deficiency is compatible with X-linked inheritance. To date, 22 families have been reported (04, T25, Y3). Nine of these have manifested both symptoms five have shown only hemolysis seven have shown the central nervous system dysfunction and/or myopathy but without hemolysis and one case, PGK Munchen, is without clinical symptoms (F5). PGK II is an electrophoretic variant found in New Guinea populations (Y2). Red blood cell enzyme activity, specific activity, and the kinetic properties of this polymorphic variant are normal. 

GSH-S deficiency is a more frequent cause of GSH deficiency (HI7), and more than 20 families with this enzyme deficiency have been reported since the first report by Oort et al. (05). There are two distinct types of GSH-S deficiency with different clinical pictures. In the red blood cell type, the enzyme defect is limited to red blood cells and the only clinical presentation is mild hemolysis. In the generalized type, the deficiency is also found in tissues other than red blood cells, and the patients show not only chronic hemolytic anemia but also metabolic acidosis with marked 5-oxoprolinuria and neurologic manifestations including mental retardation. The precise mechanism of these two different phenotypes remains to be elucidated, because the existence of tissue-specific isozymes is not clear. Seven mutations at the GSH-S locus on six alleles—four missense mutations, two deletions, and one splice site mutation—have been identified (S14). 

In another study, the carrier protein was replaced by an enzyme compatible solid-phase resin (PEGA), and enzyme-catalyzed cyclization was used to probe substrate specificity. This study demonstrated also that oxo-esters are tolerated as substrates for TE domains, and then-preparation in library format served as an excellent tool for substrate specificity studies, as well as for preparation of cyclized peptides. Figure 13.11 shows how the TycA TE showed selectivity for only residues 1 and 9 (colored in red), and changes at all other residues were tolerated [42]. Hydrogen bonding interactions are shown in green. Several compounds made from this series were shown to demonstrate improved therapeutic indices (with respect to hemolysis) while retaining antimicrobial activity. 

Clinical consequence hemolysis (breakdown of circulating red blood cells) from antimalarials, sulfonamides, nitrofurantoin, and other drugs. a-1 Antitrypsin deficiency, due to variants in this circulating plasma protein Frequency 1 /3000 Northern European Caucasians Clinical consequence predisposition to emphysema in early middle age, especially in cigarette smokers, due to failure to protect against trypsinlike enzymes in lung... 

When in the past 10 years the mechanism of drug-induced hemolysis was studied, the implication of an enzyme system in the integrity-maintaining principle of the red cell became evident as did the probability of its interacting in the processes of maturation and senescence (B8, SI). 

Persons with mutations that partially destroy G6PDH activity may develop an acute, episodic hemolysis. Certain mutations affect the stability of G6PDH, and, because erythrocytes cannot synthesize proteins, the enzyme is gradually lost over time and older red blood cells lyse. This process is accelerated by certain drugs and, in a subset of patients, ingestion of fava beans. In the United States, the most likely cause of a hemolytic episode in these patients is overwhelming infection, often pneumonia (viral and bacterial) or infectious hepatitis. 

Creatine phosphokinase activity has been reported to be minimally inhibited by hemolysis. Hemoglobin concentrations of 1.25 g/100 ml inhibit 5% and 2.5 g/100 ml, 12% (N5). However, in methods utilizing adenosine diphosphate in the reaction mixture, hemolysates containing 100 mg of hemoglobin per 100 ml may have apparent activities of 5-100 units/liter. The activity is presumably related to adenylate kinase in the erythrocyte (S33). In methods utilizing adenosine diphosphate in a coupled enzyme reaction with hexokinase and glucose-6-phosphatase, the inhibitory effect can be eliminated by adding sufficient adenosine mono-... 

Plasma should be separated from the blood cells within a few hours. For most amino acids the levels in plasma and red cells are comparable, but glutamate, aspartate, and taurine have extremely high intracellular levels and thus tend to rise in plasma upon hemolysis. A second effect of red cell degradation is the liberation of the enzyme arginase, which will convert arginine into ornithine. 

The enzyme reaction is performed in duplicate. Incubation buffer (200 pi) is mixed with 50 pi diluted hemolysate and incubated at 37°C for 5 min, then 50 pi substrate solution is added and the tubes are kept at 37°C in the dark for a further 60 min. One null value per experiment with hemolysis buffer instead of erythrocytes is treated the same way. 

Biocompatibility of injectable formulations with tissues can be tested by observing microscopic histology of the tissues so exposed, or by using erythrocyte hemolysis as a surrogate for these other tissues. Alternatively, one can measure the level of the cytosolic enzyme creatine phosphokinase that is released from damaged tissues (18). 

The importance of reaction (13) was also shown with stimulated human neutrophils, which generate OJ and are thus able to lyse erythrocytes. This hemolysis was inhibited by exogenous (Cu,Zn)-SOD in contrast with the heat-denatured enzyme and with catalase. When hemoglobin in the erythrocytes was converted into methemoglobin by a nitrite treatment, SOD became ineffective, but exogenous catalase protected. The erythrocyte became resistant to hemolysis when treated with carbon monoxide, whereby the formation of methemoglobin was blocked and reaction (13) avoided... 

---