Real-Time Monitoring of Histamine Released from Rat Basophilic Leukemia (RBL-2H3) Cells with a Histamine Microsensor Using Recombinant Histamine Oxidase

• Published in: Analytical biochemistry 2002-05, Vol.304 (2), p.236-243
• Main Authors: Iwaki, Shoko, Ogasawara, Masahito, Kurita, Ryoji, Niwa, Osamu, Tanizawa, Katsuyuki, Ohashi, Yuichi, Maeyama, Kazutaka
• Format: Article
• Language: English
• Subjects: Amine Oxidase (Copper-Containing) - chemistry; Amine Oxidase (Copper-Containing) - isolation & purification; Amine Oxidase (Copper-Containing) - metabolism; amperometry; Animals; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; Calcium-Transporting ATPases - antagonists & inhibitors; Catalysis; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; enzyme-modified electrode; Histamine - analysis; Histamine - metabolism; histamine microsensor; Histamine Release - physiology; Hydrogen Peroxide - chemistry; Leukemia, Basophilic, Acute - physiopathology; Monitoring, Physiologic - instrumentation; Monitoring, Physiologic - methods; Polymerase Chain Reaction; rat basophilic leukemia cells; Rats; recombinant histamine oxidase; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Thapsigargin - pharmacology; Tumor Cells, Cultured
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2002.5598

To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g., putrescine and cadaverine) or Nτ-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 μl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1 × 106) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 μM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen–antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 μM, and 2.7 μM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.