pH- dependent structural and functional studies on haptoglobin
Student name: Ms Bhamidipati Keerti
Guide: Dr Ramakrishnan Sitaraman
Year of completion: 2014
Host Organisation: CSIR- Centre for Cellular and Molecular Biology, Hyderabad
Supervisor (Host Organisation): Dr Ch. Mohan Rao
Abstract: Haptoglobin is known to have a strong binding affinity for hemoglobin. Infection and certain
genetic disorders can cause premature hemolysis of RBC’s thereby resulting in the release of
cytotoxic hemoglobin into the extracellular space. Many bacterial and parasitic infections also
result in inflammation. It is known that under conditions of inflammation local acidosis
occurs and the levels of acute phase protein haptoglobin are up-regulated 3-8 fold. The Hp2-2
phenotype is over represented in the Indian population and its interaction with hemoglobin
has not been studied in detail. Isothermal titration calorimetry studies reveal that haptoglobin
purified using hemoglobin-affinity column binds hemoglobin at pH 7.5 and 6.0 with a binding
affinity constant of 107 M-1. However, at pH 4.5, haptoglobin does not bind hemoglobin. Gel
filtration chromatography studies reveal that haptoglobin forms a 1:1 (w/w) complex with
hemoglobin at pH 7.5 and pH 6.0. Proteinase K degradation assays of haptoglobin,
hemoglobin and haptoglobin-hemoglobin complex reveal that in the pH range 7.5 to 5.5 the
proteins do not undergo degradation under the conditions investigated. But at pH 4.5
(Lysosomal pH), they are degraded by proteinase K. Haptoglobin is known to have antioxidant
property. Copper, a redox active metal ion, is abundantly present in the extracellular space and
can induce the amyloid fibril formation of α-synuclein. Our studies investigating the effect of
haptoglobin on copper-induced amyloid fibril formation of α-synuclein reveal that haptoglobin
prevents the amyloid fibril formation of α-synuclein as observed by suppression in ThT
(thioflavin T) fluorescence. It also suppresses the formation of fibrillar beta-sheet structures of
α-synuclein. Thus, the study reveals that haptoglobin forms complex with hemoglobin even
under conditions relevant to physiological acidosis and they are readily degradable by
proteases at the lysosomal pH (pH 4.5). These results have implications in the protective role
of haptoglobin against hemolysis. It also shows that haptoglobin is also able to prevent the
Cu2+-induced amyloid fibril formation of α-synuclein implying that haptoglobin has general
protective role against oxidative stress and inflammation.
Keywords: hemolysis, cytotoxic, acidosis, anti-oxidant, haptoglobin, hemoglobin,
synuclein and amyloid fibril.
