Purification and characterization of a novel 88 kDa protein from serum and vitreous of patients with Eales' disease.
Eales' disease is a perivasculitis that affects the peripheral retina of young adults and results in recurrent vitreous hemorrhage. Although increased oxidative stress and decreased antioxidant defense have been reported to be associated with Eales' disease, the exact cause for the disease and its pathogenesis are not known. Here is reported the identification, purification and characterization of a new protein from the serum and vitreous of patients with Eales' disease. This protein was purified using preparative electrophoresis and HPLC. The purified protein had a retention time of 9.2 min in RP HPLC. Its molecular weight as determined by gel permeation chromatography was 88 kDa hence, it was termed as 88 kDa protein. Alcian blue and Schiffs staining revealed 88 kDa protein to be a glycoprotein. Proteins purified from both serum and vitreous exhibited anti lipid peroxidation effect on erythrocyte when added during in vitro assay of thiobarbuteric acid reactive substances (TBARS). In addition to this property the protein also has Fe(2+)sequestering effect. The anti TBARS activity of 88 kDa protein was completely inhibited by 0.1 m M concentration of parachlromercuric benzoate (PCMB) and 5,5' dithiobis(2-nitrobenzoic acid) DTNB. The total thiol content (cysteine) of the purified 88 kDa protein was found to be 8% by mass. Eighty eight kDa protein from both the sources namely vitreous and serum are immunologically identical when studied using polyclonal antibodies raised in goat against purified serum protein. The N terminal sequence of 88 kDa protein by automated Edman's degradation chemistry is A D D P N S L S P S A F A E A L A L L R D S X L A R F V. The protein and DNA data base search revealed no match to 88 kDa protein and hence this was considered as unique protein. Further knowledge on the in vivo function of 88 kDa protein is very important to understand its role in the pathogenesis of Eales' disease.
Sulochana
K N
KN
Biochemistry Research Department, Sankara Nethralaya, Vision Research Foundation, 18 College Road, Chennai-600 006, India.
Rajesh
M
M
Ramakrishnan
S
S
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010689:Phlebitis / Q000378:metabolism; D012164:Retinal Diseases / Q000378:metabolism*; D012169:Retinal Vein; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D014822:Vitreous Body / Q000737:chemistry*; D014823:Vitreous Hemorrhage / Q000378:metabolism
D001798:Blood Proteins; D005136:Eye Proteins; D017392:Thiobarbituric Acid Reactive Substances; D007501:Iron