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Heme binding to protein nanospheres

Winther, Nicole LU (2017) KBK820 20171
Pure and Applied Biochemistry
Abstract
The studied protein exists naturally in the human body and can self-assemble into nanospheres at certain physiological conditions. Recently, these nanospheres was found able to bind free heme, the highly reactive gas binding component in hemoglobin which can cause severe tissue damage if in free form in the body. Due to prior experiments, the hypothesis was that the protein-heme binding site contained histidines like the hemoglobin-heme binding site. The focus of this project was to investigate the probability of this hypothesis by constructing ten mutants, by replacing the histidines by alanine, one by one. The constructs where analyzed alongside the wildtype using spectrophotometry between 250-800 nm, to evaluate their heme binding... (More)
The studied protein exists naturally in the human body and can self-assemble into nanospheres at certain physiological conditions. Recently, these nanospheres was found able to bind free heme, the highly reactive gas binding component in hemoglobin which can cause severe tissue damage if in free form in the body. Due to prior experiments, the hypothesis was that the protein-heme binding site contained histidines like the hemoglobin-heme binding site. The focus of this project was to investigate the probability of this hypothesis by constructing ten mutants, by replacing the histidines by alanine, one by one. The constructs where analyzed alongside the wildtype using spectrophotometry between 250-800 nm, to evaluate their heme binding characteristics and the reactivity of the heme iron to sodium dithionite and carbon monoxide.
Complementing experiments were performed on the wildtype analyzing the protein-heme bond in 37oC, also trials testing the stability and formation of the bond in serum.
Seemingly, the bond is stable over time in a 1:2 ratio with Kd=2.52 µM. Rather than one specific binding site, the existence of histidine rich pockets on the surface of the particles may be more probable. On their own, none of the histidines seemed critical to the bond. Still, His6 alternatively the presence of six histidines in the middle of the polypeptide, seems to favor the bond. While bound to the protein, the iron ion in heme appears to favor ferric form but can be reduced to ferrous form, and then bind carbon monoxide. It also seems like the bond can form and is stable over time in 25 vol% serum. Additionally, the bond appears stable over time in 37oC, with and without the presence of bovine serum albumin. (Less)
Popular Abstract
May this protein, that is naturally occurring in the human body, be used as treatment of life threatening conditions like e.g. preeclampsia? The protein studied in this project have been found to bind the molecule heme. A molecule which is the cause of several critical conditions, like preeclampsia which frightens all mothers-to-be. By studying the bond between the protein and heme, the future hope is to use it as an easily administered treatment for these conditions.
With results from the project it was concluded that the protein seems to bind heme quite strongly and in a 1:2 ratio, possibly via histidines, similarly to hemoglobin. The experiments performed suggests the bond is stable and able to form in varying degrees of blood-like... (More)
May this protein, that is naturally occurring in the human body, be used as treatment of life threatening conditions like e.g. preeclampsia? The protein studied in this project have been found to bind the molecule heme. A molecule which is the cause of several critical conditions, like preeclampsia which frightens all mothers-to-be. By studying the bond between the protein and heme, the future hope is to use it as an easily administered treatment for these conditions.
With results from the project it was concluded that the protein seems to bind heme quite strongly and in a 1:2 ratio, possibly via histidines, similarly to hemoglobin. The experiments performed suggests the bond is stable and able to form in varying degrees of blood-like conditions using bovine serum and while storing the sample in 37oC.
The appearance of the binding site was examined by creating ten different mutants of the protein., by removing its histidines in different combinations. The results were quite promising, because even though the exact appearance of the binding site is still unclear, a few of the histidines seemed to be more important to the binding than the rest. Still, the results indicated that rather than one specific hemoglobin like binding site, the existence of histidine rich pockets formed on the surface of the protein nanosphere among the closely packed protein molecules may be more probable to consider.
Even though many questions remain which mean further investigation is required, this project helped to answer some important ones. So this may be considered the first milestone towards a future treatment. (Less)
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author
Winther, Nicole LU
supervisor
organization
course
KBK820 20171
year
type
H2 - Master's Degree (Two Years)
subject
keywords
hemoglobin, Nanoparticle, heme, oxygen, applied biochemistry, tillämpad biokemi
language
English
id
8918139
date added to LUP
2017-09-19 14:47:26
date last changed
2017-09-19 14:47:26
@misc{8918139,
  abstract     = {The studied protein exists naturally in the human body and can self-assemble into nanospheres at certain physiological conditions. Recently, these nanospheres was found able to bind free heme, the highly reactive gas binding component in hemoglobin which can cause severe tissue damage if in free form in the body. Due to prior experiments, the hypothesis was that the protein-heme binding site contained histidines like the hemoglobin-heme binding site. The focus of this project was to investigate the probability of this hypothesis by constructing ten mutants, by replacing the histidines by alanine, one by one. The constructs where analyzed alongside the wildtype using spectrophotometry between 250-800 nm, to evaluate their heme binding characteristics and the reactivity of the heme iron to sodium dithionite and carbon monoxide.
Complementing experiments were performed on the wildtype analyzing the protein-heme bond in 37oC, also trials testing the stability and formation of the bond in serum.
Seemingly, the bond is stable over time in a 1:2 ratio with Kd=2.52 µM. Rather than one specific binding site, the existence of histidine rich pockets on the surface of the particles may be more probable. On their own, none of the histidines seemed critical to the bond. Still, His6 alternatively the presence of six histidines in the middle of the polypeptide, seems to favor the bond. While bound to the protein, the iron ion in heme appears to favor ferric form but can be reduced to ferrous form, and then bind carbon monoxide. It also seems like the bond can form and is stable over time in 25 vol% serum. Additionally, the bond appears stable over time in 37oC, with and without the presence of bovine serum albumin.},
  author       = {Winther, Nicole},
  keyword      = {hemoglobin,Nanoparticle,heme,oxygen,applied biochemistry,tillämpad biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Heme binding to protein nanospheres},
  year         = {2017},
}