THE CHEMICAL NATURE OF THE PROTEINS FROM HUMAN ENAMEL

¹ Department of Biological Chemistry, Georgetown University Dental School, Washington, D. C.

Enamel, from human, freshly extracted, noncarious, erupted permanent molars, has been divided into several parts, the cuticle, the enamel proper, and the total enamel, comprising both the cuticle and the enamel proper. Proteins have been isolated from these three divisions, from crowns with minimal pits and grooves and unstained, also from both stained and unstained crowns with Another sample dried to constant weight in an oven at 110° C. gave a moisture content of 0.699 per cent. The oven-dried sample ashed with a Fisher burner for 4 hours gave an ash content of 96.07 per cent. It was noted that further heating produced additional loss in weight. After thirty hours' heating with the Fisher burner, the weight of the ash remained constant within 0.1 mg. and the new ash value was 95.19 per cent. During the process of ashing, carbon dioxide is lost from the inorganic portion of the enamel. According to Deakins and Volker, this loss ranges from 3.0 to 4.0 per cent.⁶ As an approximation, the organic content of the enamel can be estimated by adding the amounts of carbon dioxide and moisture lost to the ash content and subtracting from 100 per cent. Upon this basis the possible organic content of the enamel is from 0.2 to 1.1 per cent. Determinations of total nitrogen by the Kjeldahl method, upon duplicate 500 mg. samples of total powdered enamel, gave a value of 0.073 per cent nitrogen. If this figure is multiplied by the usual factor 6.25, a possible protein value of 0.46 per cent is obtained. The uncertainty in the actual loss of carbon dioxide during ashing prohibits a close correlation between the two methods of estimation.

The actual amounts of protein isolated by the dialytic determination procedure are given in Table I. It can be seen that from the total enamel with deep pits and grooves (2), 0.39 per cent protein was isolated, while from the total enamel with minimal pits and grooves, (1), 0.30 per cent protein was isolated. When the enamel was stained (3), the per cent protein isolated was even higher, 0.49 per cent. From the enamel proper (4), the yield was 0.22 per cent, and the amount of cuticle (5, a.) isolated from the same enamel was 0.08 per cent. The sum of these two is 0.30 per cent, identical with the value from (1), the total enamel with minimal pits and grooves and unstained. The amount of protein isolated depends upon the character of the enamel studied, the deeper the pits and grooves the higher the protein content, probably because of a higher cuticle content in these areas. When the deep pits and grooves were ground out, the amount of protein isolated approximated that from the crowns with minimal pits and grooves. The protein value calculated from the total nitrogen is 0.46 per cent, this value is probably low since the factor 6.25 is based upon nitrogen content of 16 per cent which may be too low according to results published by Pincus.⁹ However, it is apparent that the amount of protein isolated is within the range established by the nitrogen values. Further studies are contemplated upon the nitrogen content of the isolated proteins.

Cystine, methionine, and phenylalanine.—The fat-free insoluble protein was hydrolyzed with 20 per cent hydrochloride acid in 50-c.c. acetylation flask fitted with a ground glass reflux condenser. The hydrolysis was carried out by partially immersing the flask in a wax bath maintained at 125° C. for a period of six hours. The cystine method of Hess and Sullivan,¹⁵ the modified McCarthy-Sullivan¹⁶ methionine method of Hess and Sullivan,¹⁵ and the phenylalanine method of Hess and Sullivan¹⁷ were employed upon the hydrolysate. All the colorimetric readings were made with a Klett-Summerson photoelectric colorimeter using a 42 filter. The results are given in Table II.