In-SilicoInvestigation of Luminol, Its Analogues and Improved Mechanism of Chemiluminescence for Blood Identification Beyond Forensics

Abstract

This study aimed to discover chemiluminescent analogues of luminol, understand their molecular binding to hemoglobin of bloodstains in the household crime, and the mechanism of chemiluminescence. Similarity and clustering analyses of luminol analogues were conducted, and molecular docking was carried out on hemoglobin from Homo sapiens and other four domestic organism namely Gallus gallus, Drosophila melanogaster, Rattus norvegicus, and Canis familiaris. The results show that the order of overall binding score is D. melanogaster > H. sapiens > C. familiaris > R. norvegicus > G. gallus. Seven compounds namely ZINC16958228, ZINC17023010, ZINC19915427, ZINC34928954, ZINC19915369, ZINC19915444, and ZINC82294978, were found to be consistently stable in binding to diverse hemoglobin and possibly have chemiluminescence than luminol. The amino acid residues involved in the interaction of human hemoglobin with the 30 test compounds, show that His45, Lys61, Asn68, Val73, Met76, Pro77, Ala79, Ala82, Leu83, Pro95, Phe98, Lys99, Ser102,Ser133, Ala134, and Thr134 are significant in the mechanism of action of presumptive test compounds. The improved mechanism of chemiluminescent identification of blood hypothesized that nitrite interact with the Fe(II) heme, with the cleavage of a hydroxide ion and the formation of the nitrosonium cation in peroxidase reaction. It was proposed that degradation of rhombic heme complex to fluorescent products is possibly inhibited by nitric oxide from the test compound luminol. This study provides novel insight on the luminol and its actual mechanism for broader possible applications of luminol with careful development of new methodologies.