Objective: This study aimed at discovering chemiluminescent analogues of luminol,
predict their molecular binding to hemoglobin of bloodstains in household crime, and
expound the mechanism of chemiluminescence of luminol.
Materials and Methods: Similarity and clustering analyses of luminol analogues were conducted,
and molecular docking was carried out using hemoglobin from Homo sapiens and
four domestic organisms namely Gallus gallus, Drosophila melanogaster, Rattus norvegicus,
and Canis familiaris.
Results: The results showed the order of overall binding score as 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 with
diverse hemoglobin and possibly have chemiluminescence than luminol in this in silico
study. The interaction of human hemoglobin with luminol and its analogues, showed that
amino acid residues His45, Lys61, Asn68, Val73, Met76, Pro77, Ala79, Ala82, Leu83,
Pro95, Phe98, Lys99, Ser102, Ser133, Ala134, and Thr134, were possibly significant in the
mechanism of action of presumptive test compounds. It was hypothesized that the improved
mechanism of chemiluminescent for the identification of blood was based on peroxidase-like
reaction, that produces nitric oxide which binds to hemoglobin (Hb) and inhibits Hb degradation
without yielding fluorescent products. The compound 2,3-benzodioxine-1,4,5(6H)-trione
was formed, which possibly emits light.
Conclusion: This study provides novel insight on the luminol and its expanded mechanism
for broader possible applications with careful development of new methodologies.