İNSAN KANINDAN HEMOGLOBİN UZAKLAŞTIRILMASI İÇİN KRİYOJEL KOLONLARININ HAZIRLANMASI
Tez Türü: Doktora
Tezin Yürütüldüğü Kurum: Hacettepe Üniversitesi, Fen Bilimleri Enstitüsü, Kimya A.B.D., Türkiye
Tez Danışmanı: Adil Denizli
Tezin Onay Tarihi: 2013
Tezin Dili: İngilizce
Desteklendiği Program: Diğer
Özet:
Proteins are vital molecules for the living organisms. They carry
out many important physiological metabolic pathways. Therefore they have always
been the study of interest. The term proteome defines the large-scale study of
proteins including the proteins produced by any organism as well as their
modifications made after the synthesis. An evolving field of the proteomics is
to identify disease linked novel proteins within the erythrocyte proteins. As
in the all proteomic studies of the blood plasma, high abundant proteins in
erythrocytes, i.e. hemoglobin and carbonic anhydrase, interfere with the
analysis of low abundant and disease related proteins. Thus it is essential to
remove these proteins before detailed analysis of cytosolic proteins of
erythrocytes. In the previous studies nickel affinity chromatography has been
investigated for its efficiency for hemoglobin depletion and shown to be
effective for increasing the number of detectable proteins.
In this thesis we aimed the preparation of the metal chelate
affinity cryogels for the hemoglobin depletion. PHEMA cryogels are selected as
base matrix because of their blood compatibility, osmotic, chemical and
mechanical stability. Cryogels are also useful when working with the viscous
samples such as blood, thanks to their interconnected macroporous structure.
PHEMA cryogels are prepared at -12°C for 24 hours. IDA, the chelating agent,
was covalently coupled to PHEMA cryogels after activation with the
epichlorohydrin. Then Cu2+, Ni2+, Zn2+ and Co2+ ions were chelated to the
IDA-bound cryogels. Characterization of the adsorbent was performed with
swelling tests, elemental analysis, FTIR, SEM, surface area measurements and
blood compatibility studies. IDA attachment was found as 10.71 mmol/g. Specific
surface area of the cryogels was 24.7 m2/g. Swelling degree and porosity of the
cryogel was 8.49 g H2O/g and 74.1% for PHEMA and 63.5% for PHEMA-IDA cryogels,
respectively. The effect of pH, Hb concentration, and ionic strength was
investigated. The depletion of the Hb from hemolysate was shown by SDS-PAGE.
The FPLC studies confirmed that the prepared adsorbent has a good resolution
values with respect to other blood proteins.