Journal of the Korean Association of Oral and Maxillofacial Surgeons

The Korean Association of Oral and Maxillofacial Surgeons (대한구강악안면외과학회)
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BIOASSAY OF HUMNA TOOTH PROTEIN BLOTTED POLYVINYLIDENE DIFLUORIDE(PVDF)MEMBRANE

사람치아 단백질을 분리 흡착한 PVDF막의 생체반응에 관한 연구

  • Kang, Na-Ra (Department of Oral and Maxillofacial Surgery, College of Dentistry, Seoul National University) ;
  • Hong, Jong-Rak (Department of Oral and Maxillofacial Surgery, College of Dentistry, Seoul National University) ;
  • Choung, Pill-Hoon (Department of Oral and Maxillofacial Surgery, College of Dentistry, Seoul National University)
  • 강나라 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 홍종락 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 정필훈 (서울대학교 치과대학 구강악안면외과학교실)
  • Published : 2004.06.30
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Abstract

Purpose: Human tooth proteins are highly heterogeneous, comprising diverse proteins derived from a number of genes. The attempts to identify protein for activity of tooth matrix proteins have been defied by several factors. First, the amount of proteins within teeth is very small relative to many extracellular matrix proteins of other tissues. Second, the bioassay system is tedious and needed for long time. Therefore we tried to find easy techniques, which increase the product rate, and an assay of small proteins, with which amino acid sequence is possible without additional procedures. Materials and Methods: Total protein were extracted from 300 g enamel removed teeth and 600 g teeth with 4 mol/L guanidine HCl and purified by gel chromatography. Aliquot of proteins was implanted into muscle pouches in Sprague-Dawley rats for bioassay. By SDS-PAGE and membrane blotting, molecular weight of each protein was estimated and a partial amino acid sequence was obtained. Each fraction blotted on the membrane was cut out and inserted in rat ectopic model. Results: In dissociative method, total tooth proteins were obtained 1mg/ml from enamel removed teeth and 3.5 mg/ml from teeth. In SDS-PAGE, four clear bands at the sites corresponding to 66, 40, 20 and 18 kD. Especially The 66 kD band was clearly exhibited. Amino acid sequencing from tooth could be possible using PVDF membrane blotting technique. In amino acid sequencing, 66 kD protein was identified as albumin. Conclusion: Compared with conventional method for extraction of teeth protein and bioassay of proteins, the methods in this study were easy, time-saving and more productive technique. The matured tooth proteins omitting additional procedure of mechanical removal of enamel were simply analyzed using blotted PVDF membrane. This method seems to make a contribution as a technique for bioassay and amino acid sequencing of protein.

Keywords

References

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