Specific labelling of insoluble elastin with tetraphenylporphin sulphonate

Parotid saliva is a particularly good system to study exocrine secretory processes in man. Various proteins from human parotid saliva that have been previously isolated and partially characterized include glycoproteins, acidic proteins that contain little or no carbohydrate, and smaller acidic peptides rich in histidine or tyrosine (Henkin et al.. 1978). The secretory process in the parotid gland includes the synthesis transport and storage of these materials. The study of these secretory mechanisms requires the isolation and characterization of these proteins. Preparative flat-bed electrofocusing was used in an attempt to simultaneously purify a number of human parotid salivary proteins. Citric. acid (5%)-stimulated parotid saliva was obtained from three volunteers (with their informed consent) by using a Curby cup-collection device (Jenkins, 1978). The saliva was dialysed overnight against 1% glycine and then centrifuged. The dialysed saliva was mixed with the gel slurry and, after evaporation at room temperature, focusing was performed for 17h at 8 W constant power as described previously (Winter et al., 1975). The voltage increased from 400 to 1700V on completion of focusing. Eight precipitates were visible in the focused gel. The addition of 0.01% Tween 20 to the saliva during dialysis and subsequent focusing did not alter the precipitation pattern. The precipitates were removed and the gel was then fractionated. Gel samples were eluted with 0.15 M-NaCI/O. I5 M-Tris/HCI. pH 6.8. The eluates were subsequently analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (Laemmli. 1970) as shown in Fig. I . In contrast with the normal blue staining pattern, some of the proteins stain pink-violet with Coomassie Blue. as indicated by dots in Fig. 1. At least four distinct groups of polypeptides have been isolated by this procedure: precipitates 1 and 2. precipitate 3. precipitate 4 and precipitate 8. We have not yet established if the proteins in the other precipitates are related to, or distinct from, these four groups. The irregular banding pattern observed in precipitates I and 2 appears to be due to incomplete denaturation. Precipitate 4 consists primarily of the major parotid salivary protein, amylase. The background staining observed in precipitates 7 and 8 disappears on prolonged destaining. revealing. more clearly in precipitate 8, seven main polypeptide bands. The identical violet-staining character of these bands suggest that these polypeptides are related. The yield of protein as determined by A,,, was 70.5%. These results indicate that this method enables the simultaneous isolation of several salivary proteins in a single step. In view of the simplicity and high-load capacity of this method, it offers a more convenient purification procedure than the .94