Hannig Christian, Spitzmüller Bettina, Knausenberger Stefan, Hoth-Hannig Wiebke, Hellwig Elmar, Hannig Matthias
Department of Operative Dentistry and Periodontology, University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany.
Arch Oral Biol. 2008 Sep;53(9):849-58. doi: 10.1016/j.archoralbio.2008.03.003. Epub 2008 Apr 18.
Peroxidase is the main salivary antioxidant. The aim of the present study was to detect and to characterise peroxidase in the in situ enamel pellicle.
Bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 and 120 min) on buccal and palatal sites. Pellicle bound peroxidase activity was determined fluorimetrically using 2',7'-dichlorofluorescin as a substrate. The peroxidase molecules present in the pellicle were visualised with the gold-immunolabelling technique and evaluated by TEM. Furthermore, effects of polyphenols and hydrogen peroxide on peroxidase and its enzymatic activity were examined.
All pellicles which were tested revealed peroxidase activity and labelled peroxidase molecules were detected in all samples. The numbers of gold-labelled peroxidase molecules detectable in cross-sections of the pellicles were correlated significantly with the pellicle formation time. After 3 min, 0.50+/-1.01 labelled molecules were detected (30 min: 1.42+/-1.98; 120 min: 4.15+/-4.13, ANOVA, p<0.001). The mean immobilised peroxidase activity exposed at the surface amounted to 24.4+/-27.7 mU/cm2; no continuous increase of activity with formation time was found. Hydrogen peroxide and polyphenolic beverages inactivated peroxidase activity of the pellicle. Despite these inhibiting effects, considerable amounts of peroxidase molecules were still detectable by gold-immunolabelling. After contact with the inhibiting agents in situ, peroxidase activity of the pellicle reconstituted slowly.
Peroxidase activity is present in the pellicle already after 3 min of formation time, but is inhibited by the substrate and polyphenolic beverages.
过氧化物酶是唾液中的主要抗氧化剂。本研究的目的是检测原位牙釉质薄膜中的过氧化物酶并对其进行表征。
将牛牙釉质片固定在上颌夹板上，由6名受试者分别在颊侧和腭侧部位佩戴不同时间（3、30和120分钟）。使用2',7'-二氯荧光素作为底物，通过荧光法测定薄膜结合的过氧化物酶活性。用金免疫标记技术对薄膜中存在的过氧化物酶分子进行可视化，并通过透射电子显微镜进行评估。此外，还研究了多酚和过氧化氢对过氧化物酶及其酶活性的影响。
所有测试的薄膜均显示出过氧化物酶活性，并且在所有样品中均检测到标记的过氧化物酶分子。在薄膜横截面中可检测到的金标记过氧化物酶分子数量与薄膜形成时间显著相关。3分钟后，检测到0.50±1.01个标记分子（30分钟：1.42±1.98；120分钟：4.15±4.13，方差分析，p<0.001）。表面暴露的固定化过氧化物酶平均活性为24.4±27.7 mU/cm2；未发现活性随形成时间持续增加。过氧化氢和多酚饮料使薄膜的过氧化物酶活性失活。尽管有这些抑制作用，但通过金免疫标记仍可检测到大量的过氧化物酶分子。在原位与抑制剂接触后，薄膜的过氧化物酶活性缓慢恢复。
在形成3分钟后，牙釉质薄膜中就已存在过氧化物酶活性，但会受到底物和多酚饮料的抑制。
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