Heme, the prosthetic group of hemoglobin, may be released from its host due to an intrinsic instability of hemoglobin and accumulate in the erythrocytes. Free heme is in the form of hematin (Fe3+ protoporphyrin IX OH) and follows several pathways of biochemical toxicity to tissues, cells, and organelles since it catalyzes the production of reactive oxygen species. To determine concentration of soluble free heme in human erythrocytes, we develop a new method. We lyse the red blood cells and isolate free heme from hemoglobin by dialysis. We use the heme to reconstitute horseradish peroxidase (HRP) from an excess of the apoenzyme and determine the HRP reaction rate from the evolution of the emitted luminescence. We find that in a population of five healthy adults the average free heme concentration in the erythrocytes is 21±2μM, ca. 100× higher than previously determined. Tests suggest that the lower previous value was due to the use of elevated concentrations of NaCl, which drive hematin precipitation and re-association with apoglobin. We show that the found hematin concentration is significantly higher than estimates based on equilibrium release and the known hematin dimerization. The factors that lead to enhanced heme release remain an open question.
Bibliographical noteFunding Information:
We are deeply indebted to R.C. Willson for his help with the procedure for free heme determination and for generous access to the Tecan Microplate Reader, V. Balakotaiah for insights into enzymatic kinetics and autocatalysis, Y. Li for participation in preliminary experiments, K. Waller, RN, and J. Nguyen, RN, from the UH Health Center for competent and expeditious assistance in the collection of blood from healthy donors, A. Linsey and R. Fuller from the Gulf Coast Regional Blood Center for additional blood samples, and John O'Shea from BBI Enzymes for help with apo-HRP. This work was supported by the Norman Hackerman Advanced Research Program (Grant 003652-0078-2009 ), NSF (Grant MCB-1244568 ), and NIH ( U01HL114476 ).
- Enzymatic analysis
- Hematin concentration
- Heme release
- Hemoglobin instability
- Human erythrocytes