On the mechanisms involved in biological heme crystallization.

Blood-feeding organisms digest hemoglobin, releasing large quantities of heme inside their digestive tracts. Free heme is very toxic, and these organisms have evolved several mechanisms to protect against its deleterious effects. One of these adaptations is the crystallization of heme into the dark-brown pigment hemozoin (Hz). Here we review the process of Hz formation, focusing on organisms other than Plasmodium that have contributed to a better understanding of heme crystallization. Hemozoin has been found in several distinct classes of organisms including protozoa, helminths and insects and Hz formation is the predominant form of heme detoxification. The available evidence indicates that amphiphilic structures such as phospholipid membranes and lipid droplets accompanied by specific proteins play a major role in heme crystallization. Because this process is specific to a number of blood-feeding organisms and absent in their hosts, Hz formation is an attractive target for the development of novel drugs to control illnesses associated with these hematophagous organisms.

On the physico-chemical and physiological requirements of hemozoin formation promoted by perimicrovillar membranes in Rhodnius prolixus midgut.

Triatomine insects are obligatory blood-feeders that detoxify most of the hemoglobin-derived heme through its crystallization into hemozoin (Hz). Previous evidence demonstrates the key role of midgut perimicrovillar membranes (PMVM) on heme crystallization in triatomines. Here, we investigated some of the physico-chemical and physiological aspects of heme crystallization induced by Rhodnius prolixus PMVM. Hz formation in vitro proceeded optimally at pH 4.8 and 28 degrees C, apparently involving three kinetically distinct mechanisms along this process. Furthermore, the insect feeding status and age affected PMVM-induced heme crystallization whereas pharmacological blockage of PMVM formation by azadirachtin, reduced hemoglobin digestion and Hz formation in vivo. Mössbauer spectrometry analyses of R. prolixus midgut showed that Hz represents the only measurable iron species found four days after a blood meal. Autocatalytic heme crystallization to Hz is revealed to be an inefficient process and this conversion is further reduced as the Hz concentration increases. Also, PMVM-derived lipids were able to induce rapid Hz formation, regardless of the diet composition. These results indicate that PMVM-driven Hz formation in R. prolixus midgut occurs at physiologically relevant physico-chemical conditions and that lipids derived from this structure play an important role in heme crystallization.

On the pro-oxidant effects of haemozoin.

Haemozoin (Hz) is a haem aggregate produced in some blood-feeding organisms. There is a general belief that Hz formation would be a protective mechanism against haem toxicity. Here we show that when aggregated into Hz, haem is less deleterious than its free form. When haem was added to phosphatidylcholine (PC) liposomes, there was an intense stimulation of oxygen consumption, which did not occur when Hz was incubated with the same preparation. Evaluation of oxygen radical attack to lipids, by measurement of thiobarbituric acid reactive substances (TBARS), showed significantly lower levels of lipid peroxidation in samples containing PC liposomes incubated with Hz than with haem. However, TBARS production induced by Hz was much higher when using 2-deoxyribose (2-DR) as substrate, than with PC liposomes. Spin-trapping analysis by electron paramagnetic resonance (EPR) of Hz and tert-butylhydroperoxide (tert-BuOOH) showed that production of methoxyl and tert-butoxyl radicals was only slightly reduced compared to what was observed with haem. Interestingly, when large Hz crystals were used in 2-DR TBARS assays and tert-BuOOH EPR experiments, the pro-oxidant effects of Hz were strongly reduced. Moreover, increasing concentrations of Hz did not induce erythrocyte lysis, as occurred with haem. Thus, the reduced capacity of Hz to impose radical damage seems to result from steric hindrance of substrates to access the aggregated haem, that becomes less available to participate in redox reactions.