Intrinsic tryptophan fluorescence and related energy transfer in Leghemoglobin isolated from Arachis hypogea

Abstract

Objective: To explore the structural properties of heme containing leguminous protein Leghemog- lobin (Lb) by means of intrinsic tryptophan (Trp) fluorescence and interaction with fluorescent dye 1-anilinonaphthalene-8-sulphonate (ANS). Hence to compare the melting temperature, quantum yield and energy transfer properties of Lb with other standard globular proteins. Method: Intrinsic Trp fluorescence, extrinsic probe ANS, UV fluorescent signal were used to investigate Forster energy transfer in the proteins. Quantum yield and melting temperature (Tm) were determined to characterize Lb isolated from Arachis hypogea. Result: Binding of ANS with the proteins Bovine serum albumin (BSA), lysozyme, Ovalbumin (Oval) and Cytochrome-C (Cyt-C) manifested differential enhancement of ANS fluorescence re- vealing energy transfer from Trp. Forster equation was used to estimate the efficiency of energy transfer from Trp to ANS. Estimated binding constants were different for 295 nm and 375 nm excitation suggesting the involvement of an additional pathway in Lb via Forster resonance energy transfer (FRET). This is reflected in higher K D value indicating lower binding affinity of ANS-Lb. Conclusion: This is a pioneering endeavor to unfold the structural properties of Lb isolated from Arachis hypogea, since there is no report regarding the spectroscopic properties of this protein, which is of immense agricultural importance. Our work revealed a comparison of thermal stability of heme containing globular proteins which followed the order: Hb > Lb > Cyt-C. Quantum yield and the binding constant for Trp-ANS interaction of Lb were determined. Apparent distance for Trp-ANS energy transfer in Lb and other globular proteins were explored to follow the order: Oval < Lb < BSA < Cyt-C < Lysozyme.