02742nas a2200241 4500008004100000024000700041245013300048210006900181260001700250300001200267490000700279520195900286653002402245653001602269653003602285653002102321100002402342700002502366700002102391700002202412700002602434856004002460 2022 eng d a0500aIsolation and Characterization of Neuroglobin and The Reducing Enzyme Metneuroglobin (Neuroglobin Fe3+) From Bovine Brain Tissue0 aIsolation and Characterization of Neuroglobin and The Reducing E cOctober 2022 a504-5100 v143 a

Background/Aim: The brain uses 20% of the O2 consumed by the body for energy metabolism. In 2000, found a protein that is thought to be a binding O2 in the brain, namely neuroglobin (Ngb). Ngb is a member of the hemoprotein which has a heme group. The iron ion in the haem group can be oxidized, so a reducing enzyme is needed. In this study, the isolation, purification, and characterization of Ngb protein and the reducing enzyme from oxidized neuroglobin (neuroglobin Fe3+) were carried out. Materials and methods: Ngb protein was isolated by fractionation technique using ammonium sulfate 90% saturation, purified by anion exchange chromatography (DEAE Cellulose) and immunoaffinity chromatography, confirmed by SDS-PAGE and Western blot. The metneuroglobin-reducing enzyme was isolated by RIPA lysis buffer, purified by Affi gel blue chromatography, and confirmed by SDS-PAGE. Results: The isolated Ngb obtained has a molecular weight of 17.26 kDa. Spectrum analysis in the wavelength range of 350- 500nm, showed the afternoon peaks of deoxyNgb, oxyNgb, carboxyNgb and metNgb were 415 nm, 405 nm, 405 nm, and 420 nm, respectively. The results of the isolation of the reducing enzymes obtained consisted of 2 parts, namely the matrix-bound eluate (eluate-1) and matrix-bound eluate (eluate-2). SDSPAGE results of eluate-1, eluate-2 and Ngb-free fraction (byproduct of Ngb purification) showed the same 3 bands at a molecular weight of 72.45; 26.84 and 16.33 kDa were suspected as reducing enzymes. Conclusion: The reduction kinetics was tested by reacting the fraction and metNgb and measuring the deoxyNgb uptake formed per unit time. The results of the measurement of the ratio of NgbFe3+ to NgbFe2+ from the free fractions Ngb, eluate-1 and eluate-2, which has the best reducing activity is eluate-1 because it has the best regression value of 0.8769.

10aBovine brain tissue10aNeuroglobin10aNeuroglobin absorption spectrum10aReductase enzyme1 aMudjihartini, Ninik1 aPurba, Dewi, Pratiwi1 aFadilah, Fadilah1 aSadikin, Mohammad1 aJusman, Sri, Widia A. uhttps://www.phcogj.com/article/1862