1,9-Dimethylmethylene blue is a photosensitizer, virus inactivator and hemoglobin oxidant derived from methylene blue. When activated, 1,9-Dimethylmethylene blue generates reactive oxygen species including singlet oxygen, and acts as a metachromatic dye. When activated in monomeric or dimeric form, 1,9-Dimethylmethylene blue induces photoinactivation of R17 phage and vesicular stomatitis virus and oxidizes hemoglobin via non-singlet oxygen reactive oxygen species or singlet oxygen-mediated pathways, respectively. The monomeric form, with higher nucleic acid affinity, achieves virus inactivation under specific conditions without forming methemoglobin. 1,9-Dimethylmethylene blue binds to substances such as glycosaminoglycans to produce color changes. Although it is susceptible to interference from non-glycosaminoglycan components in urine, it is still applicable to spectrophotometric analysis for glycosaminoglycan quantification. With these unique photochemical and binding properties, 1,9-Dimethylmethylene blue is widely used in studies of viral infections and related biochemical analyses [1] [2].
In Vitro, The activated monomeric and dimeric forms of 1,9-Dimethylmethylene blue (0.04-1 μM; 5-10 min) induce photoinactivation of R17 phage primarily via a singlet oxygen-mediated pathway[1]. Activated monomers of 1,9-Dimethylmethylene blue (2.5-30 μM; 10-40 min) induce methemoglobin (Met-Hb) formation in SFH via reactive oxygen species other than singlet oxygen, whereas activated dimers induce Met-Hb formation mainly through a singlet oxygen-mediated pathway[1]. Light treatment with 1,9-Dimethylmethylene blue does not reduce superoxide dismutase or catalase activity in matrix-free human hemoglobin[1]. 1,9-dimethylmethylene blue (0.1% in 99.5% ethanol; 5-6 h MS analysis) reacts with low-molecular-weight inorganic anions (sulfate, pyrophosphate, citrate, phosphate) in equine and human urine, leading to overestimation of urinary glycosaminoglycan levels detected by the DMB dye-binding assay[2]. Experimental procedures for virus inactivation and Met-Hb generation mediated by monomeric 1,9-dimethylmethylene blue (DMMB) (activated at 655 nm) and dimeric DMMB (activated at 580 nm), respectively[1]: Reagents and Materials DMMB, sodium azide, mannitol, SOD, rutin, histidine, D2O; R17 phage, E. coli Hfr C. I. Preparation of Stroma-free hemoglobin (SFH) 1. Add double the volume of 4 °C distilled water to red blood cells for hemolysis, and incubate at 4 °C for 10 min. 2. Centrifuge at 36000×g for 30 min, filter the supernatant through 0.22 μm and 75 nm filter membranes, and adjust Hb to 1.08 mM. 3. Light treatment Add 1 mL of sample to a 35 mm culture dish, and shake at 70 rpm in the dark at room temperature for 15 min. Monomer activation: 655 nm LED, 3.17 mW/cm2; Dimer activation: 580 nm narrow-band light, 0.49 mW/cm2. II. Detection of Virus Inactivation 1. Mix 100 μL of virus solution with 900 μL of PBS containing scavengers, add DMMB, and expose to light. 2. Determine virus titer using the upper-layer agar plaque assay, and calculate the log10 inactivation titer. III. Detection of Met-Hb and Enzyme Activity 1. Measure the content of Met-Hb using the cyanmethemoglobin method. 2. Detect SOD activity with a kit, and determine CAT activity using spectrophotometry.
References:
[1]. Hirayama J, et al. Involvement of reactive oxygen species in hemoglobin oxidation and virus inactivation by 1,9-dimethylmethylene blue phototreatment. Biol Pharm Bull. 2001;24(4):418-421.
[2]. de Lima CR, et al. Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans. Clin Chim Acta. 2007;378(1-2):206-215.
In Vitro, The activated monomeric and dimeric forms of 1,9-Dimethylmethylene blue (0.04-1 μM; 5-10 min) induce photoinactivation of R17 phage primarily via a singlet oxygen-mediated pathway[1]. Activated monomers of 1,9-Dimethylmethylene blue (2.5-30 μM; 10-40 min) induce methemoglobin (Met-Hb) formation in SFH via reactive oxygen species other than singlet oxygen, whereas activated dimers induce Met-Hb formation mainly through a singlet oxygen-mediated pathway[1]. Light treatment with 1,9-Dimethylmethylene blue does not reduce superoxide dismutase or catalase activity in matrix-free human hemoglobin[1]. 1,9-dimethylmethylene blue (0.1% in 99.5% ethanol; 5-6 h MS analysis) reacts with low-molecular-weight inorganic anions (sulfate, pyrophosphate, citrate, phosphate) in equine and human urine, leading to overestimation of urinary glycosaminoglycan levels detected by the DMB dye-binding assay[2]. Experimental procedures for virus inactivation and Met-Hb generation mediated by monomeric 1,9-dimethylmethylene blue (DMMB) (activated at 655 nm) and dimeric DMMB (activated at 580 nm), respectively[1]: Reagents and Materials DMMB, sodium azide, mannitol, SOD, rutin, histidine, D2O; R17 phage, E. coli Hfr C. I. Preparation of Stroma-free hemoglobin (SFH) 1. Add double the volume of 4 °C distilled water to red blood cells for hemolysis, and incubate at 4 °C for 10 min. 2. Centrifuge at 36000×g for 30 min, filter the supernatant through 0.22 μm and 75 nm filter membranes, and adjust Hb to 1.08 mM. 3. Light treatment Add 1 mL of sample to a 35 mm culture dish, and shake at 70 rpm in the dark at room temperature for 15 min. Monomer activation: 655 nm LED, 3.17 mW/cm2; Dimer activation: 580 nm narrow-band light, 0.49 mW/cm2. II. Detection of Virus Inactivation 1. Mix 100 μL of virus solution with 900 μL of PBS containing scavengers, add DMMB, and expose to light. 2. Determine virus titer using the upper-layer agar plaque assay, and calculate the log10 inactivation titer. III. Detection of Met-Hb and Enzyme Activity 1. Measure the content of Met-Hb using the cyanmethemoglobin method. 2. Detect SOD activity with a kit, and determine CAT activity using spectrophotometry.
References:
[1]. Hirayama J, et al. Involvement of reactive oxygen species in hemoglobin oxidation and virus inactivation by 1,9-dimethylmethylene blue phototreatment. Biol Pharm Bull. 2001;24(4):418-421.
[2]. de Lima CR, et al. Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans. Clin Chim Acta. 2007;378(1-2):206-215.