Sodium dodecyl sulfate electrophoretic analysis of the protein in palatine saliva

Sodium dodecyl sulfate electrophoretic the protein ‘in palatine saliva

analysis of

Akihiko Shiba, D.D.S.,* Jiro Yoshida, D.D.S.,** Makoto Nakao, M.D.S.,*** Kiyoko Sano, M.D.S., **** Hiroko Cho,***** and Toshio Hayashi, D.D.S.****** Stiowa University,

School of Dentistry,

Tokyo,

Japan

T

he protein and glycoprotein content of palatine gland saliva may play a role in maxillary complete denture reiention.ls6 The difficulty of sample collection of this very viscous saliva has been recently overcome.7m’o Furthermore our modification of the electrophoretic method of Shiba’ et al.” and Weber and Osborn’* provides an effective analysis of salivary protein. This method is referred to as sodium dodecyl sulfate (SDS) electrophoresis. This study reports on the possible prosthetic significance of the amount and nature of palatine saliva and its variations related to sex and age.

MATERIAL

AND METHODS

‘Fifty-eight healthy subjects, 28 men and 30 women ranging from 22 to 72 years of age, with no history of systemic diseases and with clinically normal oral mucosa were selected. Palatine saliva samples were collected,9 and the homogenization of the saliva, SDS electrophoresis, and the staining of chromatographic gels were carried out.” Palatine salivary proteins with molecular weights greater than 300,000 were analyzed by the method of Liu et a1.,13which is a combination of the method of Peacock and Dingman14 and the use of SDS. Partial modifications included the addition of 0.02 ml of Tris-EDTA, Na2-H,BO, buffer (pH 8.3) containing 0.2% SDS, followed by 0.01 ml of mercaptoethanol and 0.15 ml of H,O to 0.02 ml of a palatine saliva sample. The mixture was reduced for 5 minutes at 80” C, and *Professor, Department of Prosthetic Dentistry. **Assistant Professor, Department of Prosthetic Dentistry. ***Professor, Department of Biochemistry, Tokyo Medical and Dental University, School of Medicine. ****Assistant Professor, Department of Biochemistry, Tokyo Medical and Dental University, School of tiedicine. *****Assistant, Department of Biochemistry, Tokyo Medical and Dental University, School of Medicine. ******Professor, Department of Prosthetic Dentistry, Tokyo Medical and Dental University, School of Dentistry.

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an aliquot of 0.2 ml was charged onto the gel placed in a glass tube of 5 mm diameter. The gel was prepared according to the method of Peacock and Dingman,’ but 2.5% polyacrylamide gel, 0.5% agarose, and 0.2% SDS were added. Electrophoresis was also carried out as described by Peacock and Dingman,14 3 mA per gel column for about 90 min. The protein was stained with Coomassie Brilliant Blue-G-250, the glycoprotein with Schiff reagent, and the sialoglycoprotein with Brilliant Blue-YMC.” Quantitative assay of the protein in each of the bands on the gel was carried out by scanning the column with a densitometer (Model 02-802, Asuka Ind. Co., Tokyo, Japan) through a 510 nm filter. Since reference for high molecular weight protein samples was commercially unavailable, various polymers of phosphorylase a (Sigma Co., St. Louis, MO.) were prepared and used. Phosphorylase a, 1.5 mg, was dissolved in 0.1 ml of 0.15 M phosphate buffer solution (pH 8), and 0.1 ml of 0.02% glutaladehyde (Wako Pure Chemicals Ind., Tokyo, Japan) was added. After standing at room temperature for 5 to 10 minutes, 0.01 ml of mercaptoethanol was added. These treatments enabled preparation of the polymers of this enzyme up to the heptamer.

RESULTS Typical electrophoretic patterns of the proteins in the palatine saliva obtained from six patients are shown in Fig. 1. The number of patients studied had increased to 29 from our previous studylo and we detected two new protein bands, which are numbered as 2, and 3, in Fig. 2 and corresponded to molecular weights of 260,000 and 190,000, respectively. Therefore, 22 protein bands were now detectable. The following eight bands were found in all specimens tested: band No. 1, molecular weights greater than 300,000; No. 6, 125,000; No. 10, 84,000; No. 12, 66,000; No. 13, 60,000; No. 16, 41,000; No. 18, 25,000; and No. 20, 12,000. Band Nos. 14, 55,000, and 11, 76,000, were detected in 98% and 93% of the 0022-3913/W/030246

+06$00.60/O@

1982 The

C. V. Mosby

Co.

SDS ELECTROPHORETIC

ANALYSIS

Fig. 1. Typical SDS electrophoretic subjects.

patterns of palatine saliva collected from ~LX

total subjects, respectively. We concluded that these 10 bands are the major constituents of palatine salivary proteins. A typical densitometric pattern of the protein is shown in Fig. 3. Relative amounts of the protein in each of the bands were then estimated on the palatine saliva specimens obtained from 16 men and 13 women, 22 to 72 years of age. Mean values with standard deviations are given in Fig. 4. The 10 protein bands were confirmed to occupy the largest proportion of the total protein content. The protein fraction of particular interest was band No. 1 which contained proteins with molecular weights greater than 300,000 and was absent in parotid saliva. The relative contents of this protein fraction were 15.3% +- 7% in men, and 24% t 13% in women, both values being the highest of all. Bands Nos. 12, 13. and 14 also gave a value higher than 10% in both men and women. However, these bands coincided with those of major bands for the parotid salivary protein on the gel, indicating an unspecific nature to the palatine saliva. In view of the specific nature of band No. 1 to the palatine saliva, and of the complete absence of sialoglycoprotein in the bands other than No. 1,” subsequent experiments were made only on band No. 1. Fairly large values of the standard deviation for the protein content in each of the bands indicate the presence of considerably large individual variations in the protein content of palatine saliva. Such a variation was most conspicuous for band No. 1. The change of the relative content of the protein in band No. 1 in relation to the age of subjects was first examined, and the results obtained from 28 men and 30 THE JOURNAL

OF PROSTHETIC

DENTISTRY

R.M.

0.0-

#-a

3

BandNo. ___---.-- --- , =._ -.--_ ------------...----___ -.._--..__ ---3 221 -._

-..

0, M.W.(x104)5;qlIQnc*Os(urre-e 30< 30 26 23

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95.2 33.3

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14.3 100.0 4.8

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(20-39yrs)

Fig. 2. Apparent molecul .ar weights and frequency of occurrence of palatine saliva proteins women are shown in Fig. 5. The mean value for a group consisting of 20- to 39-year-old subjects and that for a group of 40- to 70-year-old subjects were calculated separately for men and women. In men, the 20- to 39-year-old group showed the value of 15.3% t7%, and the 40- to 70-year-old group showed 13.6% 2 247

SHIBA

ET AL

Fig. 3. Densitometric pattern of palatine saliva protein bands.

Bend

No.

Male

153t

0.6-

----es__ --__ %_. --‘... -- ._ .I\ --.* . .>. %\ '\ '. 1. '.. +, '\ ‘\'\ '\ \\'\\ I\'. '\ \\ '\ \\ ‘,'\‘\ ','\ \ \ '\ \ ', '\ \ ', ‘\'\, '\ ', \ \ \ '\'\'\ i‘ '\ ' l;\,'\, \\ '

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Fig. 4. Relative protein contents in bands of palatine saliva. 4.8%. In women, the values were 24% -+ 13% and 13.8% + 6.5% for the younger and elder groups, respectively. Significant decline of this value

The protein content of band No. 1 in edentulous and dentulous patients was then compared (Fig. 6). Mean values of the protein were 15.1% + 4.8% for men and 16.6% + 8.1% for women in edentulous patients. The age of these subjects ranged from 45 to 70 years, and since some of these subjects did not wear dentures it was difficult to examine the specific effect of wearing dentures on the protein content of band No. 1. Therefore, subjects without dentures were divided into two groups, 20 to 39 and 40 to 59 years of age; those with dentures (45 to 70 years) were considered as one group. Fig. 5 shows that 43% of the nondenture-wearing subjects of 20 to 39 years of age showed relative protein contents as high as 20% to 30%, while 62.5% of the nondenture-wearing subjects of 40 to 59 years of age presented a protein content of 10% to 20%. Fifty-one percent of the edentulous subjects (45 to 70 years of age) wearing complete maxillary dentures also showed 10% to 20% protein contents. These results were interpreted as a decrease in the protein content in band No. 1 associated with advancing age rather than the influence of denture wearing. However, some influence from denture wearing could account for the observation that 21% of denture-wearing subjects showed a protein content of 20% to 40%. Unfortunately, the number of subjects studied was not statistically significant. Our results indicate that the protein in band No. 1 is abundant in palatine saliva and participates in the maintenance of the high viscosity of the saliva. It is still MARCH

1982

VOLUME

47

NUMBER

3

SDS ELECTROPHORETIC

ANALYSIS

40

50

60

70yrs

60

O2V----

Age

7oycs

Age

Fig. 5. Relative protein content in band No. 1 and age of men (left) and women (right) subjects. Vertical bars represent standard deviations for mean values (open circles) calculated for each generation. to be demonstrated whether this band contains a single protein or not. The electrophoretic method,14 in which the concentration of polyacrylamide gel was decreasingly changed and agarose newly added to maintain the strength of the gel, was applied to band No. 1 to examine its homogeneity. The concentration of 0.5% agarose containing polyacrylamide gel was changed to 3.5%, 3%, and 2.5% and the electrophoretic patterns observed. As shown in Fig. 7, at 2.5% polyacrylamide gel concentration, band No. 1 was separated into three bands which were tentatively named bands 1 a, 1 b, and 7 c from the cathodal end. Bands 7 a and c consistently appeared as well-defined, but band 7 b was rather ill-defined and was observed as a broad band situated between bands 7 a and c. Periodic acid-Schiff (PAS) staining and sialoglycoprotein staining of the 2.5% gel also showed three bands which were similar to those of the protein bands (Fig. 8). To estimate the molecular weight of the protein in each of the three bands, the polymers of phosphorylase were used for reference. However, since the molecular weight of all three proteins was greater than that of the heptamer of this enzyme (> 700,000) (Fig. 9), the molecular weight of these proteins could not be accurately estimated. Their molecular sizes were expressed in terms of the relative mobility to pyronin Y. The relative mobilities of band 7 a were found to be similar under the three staining conditions: 0.009 to 0.027 in protein staining, 0.002 to 0.02 in glycoprotein staining, and 0.005 to 0.015 in sialoglycoprotein staining. Band 1 c also showed similar

mobilities:

OF PROSTHETIC

0

Female

Young Adult (zo-3Dm) Mate 11

4

Fc~lc

10

2

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lo-20

293-m

3wa

@e&l

to-

A

Middtr Female

Adult (W-Sym) 6

EderW&ius~~-mm) Hate 6 Femalr

6

Fig. 6. Distribution of relative protein cantent of band No. 1 in young and middle-aged nondenture wearers and in denture wearels. 1 = 11 men and 10

women, 20 to 39 years of age, without

complete

maxillary denture; 2 = 8 middle-aged wamen, 40 to 59 years of age, nondenture wearer’s; 3 = edenkulous denture wearers 45 to 72 years of age, 6 men and 8 women.

0.086 to 0.120 in

protein staining, 0.069 to 0.11 in glycoprotein staining, and 0.089 to 0.13 in sialoglycoprotein staining. These results suggested that bands a, b, and c contain both glycoprotein and sialoglycoprotein and have highly similar properties. THE JOURNAL

ma Male

DENTISTRY

The relative protein content of each of banas f a, b, and c was then estimated by means of densitometry. As shown in Table I, band 1 a was most densely stained for protein, the relative content being 40.5% + 13.5%, and band 7 c was most darkly stained with PAS, the 249

SHIBA

ET AL

Fig. 7. Electrophoretic patterns of band No. 1 at varied gel concentrations. I = 2.5% gel, 2 = 3%, and 3 = 3.5%.

Fig. 8. Electrophoretic bands of protein contained in band No. 1, stained by three staining methods. 2 = protein staining with Coomassie Brilliant Blue G-250; 2 = PAS staining with Schiff reagent, and 3 = sialoglycoprotein staining with Brilliant Blue YMC.

Table I. Relative amounts of protein,

and sialoglycoprotein

glycoprotein,

in bands 1 a, b, and c

Band

CoomassieBrilliant Blue - C + 250 for protein (IO) PAS for glycoprotein (8) Brilliant Blue YMC for sialoglycoprotein (6)

IC

(o/o)

40.5

It 13.5

28.5

28.5 39.7

rf: 16.1 + 7.7

23.8 + 11.1 26.4 L!Z 10.7

content being 56.8% f 9.5%. By sialoglycoprotein staining, bands 7 a and c showed similar values, namely, 39.7% + 7.7% for 1 a and 33.9% f 11.3% for 7 c. Fairly large standard deviation values for these mean values are indicative of the existence of large individual differences in the protein content. It was therefore inferred that these individual differences may influence the retention of the complete maxillary denture. DISCUSSION It has been reported that palatine saliva may be the optimal contributor to maxillary denture retention.lW4 2.50

lb (%)

la (%I

Stain (No. of subiects)

k 8.8

38.3

k 17.2

56.8 33.9

t 9.5 * 11.3

This study suggests that band No. 1 figures prominently in this role because of the following observations: Band No. 1 was absent in parotid saliva and specifically present in the palatine saliva; it contained the largest amounts of glycoprotein and sialoglycoprotein, which are known to be closely related to the high viscosity of saliva; it presented at the highest concentration in the palatine saliva of men (15.3% + 7%) and also of women (24% + 13%) when compared with other palatine salivary proteins (Fig. 4). The protein content of band No. 1 was found to be higher in women and to significantly declirie with age (p < .Ol), while no such decrease with age was MARCH

1982

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47

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SDS ELECTROPHORETIC

ANALYSIS

observed in men (Fig. 5). The secretion volume of the palatine saliva is known to decrease markedly with age,4 and it has been also reported that, unlike the sexual difference in the secretion of the total saliva,‘5,‘6 women secrete greater amounts of palatine saliva than men do.’ The protein content of band No. 1 did not parallel changes in the secretion volume of the palatine saliva, therefore it is unlikely that the volume of the palatine saliva per se is significantly related to denture retention. The difference in protein compositions in the palatine saliva might he more responsible is probably the contributory factor to denture retention. The protein content of hand No. 1 appeared to be higher in subjects wearing dentures when compared with dentulous subjects (Fig. 6). This suggests some degree of influence from the presence of the denture on the cells of the palatine salivary gland. We also observed that hand No. 1 consisted of three similar proteins, hands 7 a, h, and c (Fig. S), each of which contained glycoprotein and sialoglycoprotein but in varied concentrations (Fig. 9). As shown in Table I, hand 7 a contained the highest amounts of protein and sialoglycoprotein; band 7 c was highest in glycoprotein, and its sialic acid content per protein was also high. Band 7 b showed the lowest content of protein, glycoprotein, and sialoglycoprotein. It is still not clear how the glucides content in the glycoprotein affects the retention of complete maxillary dentures. Further investigations are clearly indicated.

Monotwr

i 6 II I

10.

I\leye~, J., Golden. J. S., Steiner, N.. and Necheles. H.: The ptpaiio content of human saliva in old age. Am J Physiol

Il.

119:G(K~. I’)17 2.


Sahva and denture retention. J PROSTHE]

THE JOURNAL

12.

1960.

3. Y,wwuki. K., and Taizo. H.. The role of saliva in retention of m.lxill.uv complete dentures. J PR~STHET DENT 40:131_ 10’8. 1 iistlund, S. G.: Palatine glands and mucin. Factors influencing thv rctention of complete dentures. Odontol Tidskr 62:l. 10% i l);wr~, (1. and Wood, (Z. M.. The comribution of oral minor mucous gland secretions to the volume of whole saliva in man. ,\rt,h Oral Biol l&337. 1973. 6. Sicher. H.. and Bhaskar. S. N.: Orban’s Oral Histology and FmbrLology. ed 7. SI. Louis, 1972, The C. V. Mosby Co. Kutsc~her. .\. H.. hiandel, 1. D., ,md Zegarelli. E. V.: A teehnique for collecting the secretion of minor salivary glands. I: Uw of capillary tubrs .J Oral Ther Pharmacol 3~391. I ‘)(I:. 8 K.taber. S.. Sodium and potassium content in human palatine gland src‘reuon. Arch Oral Bioi X:529, 1977. 9. Shiba, A.. Saw, K.. Nakao, M., and Hayashi, T.: A new method of collecting saliva from human palatine glands for rlectrophoretir study. Collection of palatine saliva. Arch Oral Biol 25:503. 1980.

OF PROSTHETIC

DENTISTRY

04

06

R M ( Pyronln

Ok-----

10

Y )

Fig. 9. Graph shows approximate molecular weights of proteins in bands 1 a, b, and c. Ordinate scale: Log molecular weight of monomer and polymers of phosphorylase a. Abscissa scale: Relative mobility to pyronin Y.

REFERENCES I

io’2 ! II I

13.

14.

15. 16.

Prtrcrsrn, :\. H.: Some chemical characteristics of human minor salivq gland srrretmns. 42ct;t Odwvol Stand 39:18. 1976 Shiba. .I.. Saw. K., Nakao. Xl., Yoshida. J., (Iho. Ii., and Hayash&, ‘I‘.: Electrophoretir analysis of the protein in palatine saliva. J PROSTHETDENT 43~385, 1980. Weber, K.. and Osborn, M.: Reliability OLmolecular weight determinations by doderyl sulfate polyacryiamidr gel. ,J Biol Chem 244~4406, 1969. Liu, S., Fairbanks, C., and Patek, J.: Spont;wrous, revewble protein cross-linkmg in the human rrythrocytr membrane. Temperature and pH dependence. Biochemistry 16~4066, 1977. Peacock, ;I. C., and Dingman, (:. W Lfolecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Bwhemistry 7:668. 1968. ijstlund, S. G.. and ikesson. N.A.: Inhibition and stimulation of salivation. Acta Odonrol Stand I&183. 1960 Sharry. J. J.: Complete Denture Prosrhodontics. ed I New York, 1902. L.lcGraw--Hill Book Co p 40

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