Hemoglobin proteolysis

Piper and Fenton [10] indicated that extreme acidity or basicity of the gastric juice denaturalize the enzymatic activity of the pepsin, which shows has a higher activity at a pH = 2. At pH = 5 the enzyme starts to deactivate and at pH= 7, the enzyme irreversibly lose its activity. Fig. 3 shows the pepsin UV-visible spectra before and after interaction with the zeolites while Fig 4 shows the enzymatic activity of the denatured hemoglobin proteolysis versus reaction time. 

Figure 4. Enzymatic activity of the denatured hemoglobin proteolysis vs reaction time with selected samples (samples 5 and 6).

In fact, the malaria parasite has a limited capacity for de novo amino acids synthesis, and its survival is dependent upon hemoglobin proteolysis. The amino acids derived from the hydrolysis of globins are incorporated into the parasite s proteins and appear to be available for energy metabolism. This digestion of hemoglobin releases heme, which is... 

While hemoglobin proteolysis yields needed amino acids, it also releases toxic free heme. Fig. (1) Upon proteolysis, heme is released into the digestive vacuole where the iron of the heme moiety is oxidized from the predominantly ferrous (+2) state to the ferric (+3) state. Estimates suggest... 

In the initial experiments, we resolved reticulocyte lysates on DEAE-cellulose into two crude fractions Fraction 1, which contained proteins not adsorbed to the resin, and Fraction 2, which contained all proteins adsorbed to the resin and eluted with high salt. The original aim of this fractionation was to get rid of hemoglobin, which was known to be in Fraction 1, while most non-hemoglobin proteins of reticulocytes were known to be in Fraction 2. We found that neither fraction was active by itself, but ATP-dependent protein degradation could be reconstituted by combination of the two fractions [13]. The active component in Fraction 1 was a small, heat-stable protein we have exploited its stability to heat treatment for its purification to near homogeneity. We termed this protein at that time APF-1, for ATP-dependent Proteolysis Factor 1 [13]. The identity of APF-1 with ubiquitin was established later by Wilkinson et al. [14], subsequent to the discovery in my laboratory of its covalent ligation to protein substrates, as described below. 

The catalytic activity of pepsin is described in general as add-active proteolysis. The pH optimum for hydrolysis of hemoglobin is 1.5 to 2.5, but the pH optimum is quite broad and depends on the isozyme and specific substrate (e.g., milk clotting exhibits an optimum at pH 5.5). Pepsin acts on a wide variety of peptide bonds, including acidic residues. Part of the broad specificity range may reflect the existence of several isozymes. 

Figure 2. Proteolysis of acid-denatured hemoglobin and inactivation of apotryptophan synthase by proteinase A as a function of pH.

Within infected erythrocytes, Plasmodium falciparum (strain responsible for most fatal cases) digest fee host hemoglobin wife the aid of several specific parasite proteases 4, 5). Hemoglobin comprises 93% of fee cytosolic protein of red blood cells smd up to 80% of the 3 mM hemoglobin is degraded by fee parasite (6). This intensive proteolysis releases free heme and amino-acids. These amino-acids are incorporated into parasitic proteins and feis supply is essential for fee survival of Plasmodium, a "true parasite", which has a limited capacity for fee de novo amino-acid synthesis. Only a very limited amoimt of the free heme is metabolized by fee parasite as iron source. 

It has been underlined that the antimalarial activity of quinoline-based drugs (quinine, chloroquine, mefloquine) also depend on their interactions with heme, thus preventing aggregation of toxic heme released during proteolysis of hemoglobin by die parasite. In vitro, efficient quinolines block the aggregation... 

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