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The carbohydrates of human submaxillary glycoproteins in secretors and non-secretors of blood group substances
BIOCHIMICAET BIOPHYSICAACTA
157
BBA 85047 T H E C A R B O H Y D R A T E S OF HUMAN S U B M A X I L L A R Y G L Y C O P R O T E I N S IN SECRETORS AND NON-SECRETORS OF BLOOD G R O U P SUBSTANCES
R O B E R T C. C A L D W E L L AND W A R D P I G M A N
Departments of Oral Biology and Biochemistry, University of A labama Medical Center, Birmingham, Ala. (U.S.A.) (Received December I4th, 1964)
SUMMARY
Chemical analyses of human submaxillary salivary gland secretions from individuals of various blood types and blood group secretor statuses revealed a relationship between secretor status and the composition of total submaxillary glycoproteins. The dialyzed secretions from both secretors and non-secretors contained sialic acid, D-galactose, L-fucose, D-glucosamine, D-galactosamine, and protein. Half of the total hexose, one-third of the nitrogen-containing material and minor amounts of hexosamine were dialyzable. None of the sialic acid and fucose components was dialyzable. The submaxillary saliva of secretors had a higher concentration of proteinbound carbohydrates than that of non-secretors and had about three times as much fucose. The ratio of sialic acid/fucose was o.62±O.lO (S.D.) for secretors and 1.874-o.25 (S.D.) for non-secretors. There was a predictable relationship among tile various carbohydrates in each type of secretion, and no major differences were observed among individuals of different blood types.
INTRODUCTION
Since the elucidation of the structure of the sialic acids 1, there has been an accelerating interest in the chemistry of salivary glycoproteins. Much of the methodology was established b y work on the submaxillary gland extracts of various m a m malian species. However, relatively little work has been done on human salivary secretions. In IV[ORGAN'S2 work on blood group substances, which are chemically closely allied to salivary glycoproteins, it was emphasized that pooling secretions from various sources was undesirable. I f materials had to be pooled, care should be taken to pool only secretions from individuals with the same blood phenotype and secretor status. This admonition has not been heeded in studies of the composition of human salivas. The past work has either been on pooled whole saliva from a haphazardly selected group of subjects, or, in the few studies of separated secretions, no mention has been made of the blood group types or secretor status. In addition, the salivary Biochim. Biophys. Acla, ioi (1965) I57-165
158
R . C . CALDWELL, W. PIGMAN
flow rate was usually not recorded, although the composition is affected b y the rate of salivary flow 3. In this study, the chemical composition of human submaxillary secretion has been studied in secretors and non-secretors of various blood types, and the secretions have not been pooled. As predicted b y MORGAN2, it has been found that salivary secretions fronl secretors and non-secretors are chemically different in some important respects. While this study was in progress, data on the composition of human submaxillary secretion were reported b y MANDEL et al. 4. This and the present work are the only known studies of the chemical composition of the glycoproteins of human submaxillary secretion. MATERIALS AND METHODS
Seven apparently healthy male subjects varying in age from 2o to 4o years participated in the study. Secretor status was determined on the subjects' whole salivas b y means of hemagglutination inhibition tests 5 on type O(H) cells using anti-H lectin (Hyland, Los Angeles). Blood group typing was performed at the University of Alabama Blood Bank. For each subject an appliance was constructed for the collection of pure submaxillary saliva, according to the method of SCHNEYER6. Saliva was collected directly from Wharton's ducts, and, after voiding of the first 2 ml, the secretion was allowed to flow into graduated glass cylinders packed in iceL Tile flow of saliva was stimulated b y having the subjects suck lime-flavored candy ("Lifesavers") while the collection apparatus was in place. Since we have observed that concentrations of the components of saliva are flow-rate dependent s , the time taken to collect each sample was measured b y a stop watch, and salivary flow rate was calculated as ml/min. The data presented here cover flow rates of from 1.88 to 3.4 o ml/min for seeretors, and from 1.36 to 3.78 ml/min for non-secretors. The saliva samples were dialyzed against distilled water in seamless regenerated cellulose dialysis tubing for 18 h, the water being stirred constantly and changed once. Throughout the period of collection and dialysis, the temperature was maintained at 2-4 ° . Before chemical analysis, the dialyzed sample was placed in a ground-glass test tube and homogenized gently to ensure an even distribution of material prior to pipetting aliquots for the different analytical methods. This procedure also improved the accuracy of pipetting because the secretion lost much of its viscosity during homogenization. Paper chromatography was performed b y tile method of MASAMUNE AND YOSIZAWAs to identify the carbohydrates present in dialyzed, hydrolyzed saliva. The hydrolysis was carried out as follows. After dialysis, the secretion was lyoptlilized, and Io mg of the dry material was hydrolyzed with 2 ml of 2 N H2SO 4 at IOO° for 5 h. The solution was then brought to p H 5.8 b y addition of saturated Ba(OH)t, and the BaS04 was removed by centrifugation. The supernatant was evaporated to dryness in a desiccator at room temperature, and the sugars were redissolved in two drops of water. Descending chromatography was carried out in n-butanol-pyridine-water (5:3 : 2, v/v) on W h a t m a n No. I filter paper. 8-12/~1 of the sugar solution was applied to the paper, and the same volumes of reference sugars (I mg/ml) were included on the same strip. The known sugars added to the paper Were L-fucose, D-mannose, DBiochim. Biophys. dora, IOI (1965) 157-165
CARBOHYDRATES
OF HUMAN
SUBMAXILLARY
159
GLYCOPROTEINS
glucose, D-galactose, D-glucosamine, and D-galactosamine. The solvent was allowed to drip off the paper, and the total time of each run was 4 ° h. The sugars were detected b y the modified AgNOs-spray method of TREVELYAN et al2. Nitrogen determinations were performed b y a micro-Kjeldahl method 10, which was modified slightly b y digesting 3 ml of the saliva instead of a weighed amount of solid. Since it has been shown that tile protein of salivary-gland mucins contains close to I 6 % nitrogen n, the factor of 6.2 5 was used to convert nitrogen values to protein. Tile nitrogen values in all instances were corrected for the nitrogen content of sialic acid and hexosamine present in tile dialyzed secretion. Hexose was determined b y DISCHE'S1~ cysteine-H~SO, method TM. A galactose standard was used, since paper chromatography revealed that galactose was the only hexose present in appreciable amounts in dialyzed saliva. Fucose was determined b y the cysteine-H~SO 4 reaction after a Io-min heating period, as described b y DISCHE AND SHETTLES13. Total hexosamine was determined b y the Elson-Morgan method as modified by BOAS14, but the method recommended b y BOAS of passing the material to be analyzed through a Dowex-5o ion-exchange column was unnecessary, since no interfering chromogens were present in dialyzed saliva. Hydrolysis of I-ml saliva samples was carried out in an equal volume of HC1 solution under a variety of conditions (see Table I), and subsequently the hydrolysis conditions chosen were 4 N HC1, IOO°, 3 h. TABLE
I
H Y D R O L Y S I S C O N D I T I O N S FOR RELEASE OF H E X O S A M I N E FROM HUMAN S U B M A X I L L A R Y SALIVA Hexosamine is expressed i n d i c a t e d , a t lOO%
Conch.
as t~moles of glucosamine
released
from
ioo ml of saliva, after the times
Hexosamine released
HCl (N) I 2 4 6
Ih
2h
2.5h
3 h
3.5 h
4h
45.3 73.7 12o.7 127. 4
--135-2 124.o
--138.5 I29.1
93.3 -124.6 127.4 -131.8 139-7 141.914o.8--
The standard, glucosamine. HC1, was also heated in 4 N HC1 under the same conditions. Since glucosamine. HC1 was used as a standard, the correction factor of 0.829 was necessary to give the results in terms of the amount of hexosamine present. Sialic acid was determined b y the direct Ehrlich method 15. The values obtained b y this method were reliable and consistently higher than those obtained b y WARREN'STM thiobarbituric acid method. A standard of N-acetylneuraminic acid (Sigma, St. Louis) was used which was 90% pure when assayed b y comparison with a chromatographically homogeneous sample (Calbiochem, Los Angeles) of N-acetylneuraminic acid containing 4.52% nitrogen. RESULTS
Identification of carbohydrate constituents Fig. i shows typical chromatograms of the non-dialyzable sugars found in Biochim. Biophys. Acta, i o i
(i965)
i57-i65
I6O
R . C . CALDWELL, XV. PIGMAN
SEGRI[TOR SALIVA
CONTROL
Fig. I. Paper chromatog:ams of the non-dialyzable carbohydrates of human submaxillary saliva. Secretor and non-secretor salivas both contain L-ftlCOSe, D-galactose, D-glucosamine, and Dgalactosamine. d i a l y z e d s u b m a x i l l a r y saliva from a secretor a n d a non-secretor. Spots were f o u n d c o r r e s p o n d i n g to L-fucose, D-galactose, I)-glucosamine, a n d D-galactosamine. No D-glucose was d e t e c t e d b y this procedure, a n d D-mannose m a y h a v e been p r e s e n t in v e r y small a m o u n t s , since a f a i n t l y d a r k a r e a was occasionally seen on t h e c h r o m a t o g r a m in the a p p r o p r i a t e area. However, for b o t h secretors a n d non-secretors, t h e p r i n c i p a l hexose p r e s e n t in the d i a l y z e d saliva was galactose, a n d fucose occurred in similar a m o u n t s . The c h r o m a t o g r a p h y t i m e was s h o r t e n e d on one occasion to ensure t h a t no f a s t - m o v i n g sugar such as L-rhamnose was r u n n i n g off the paper. Fig. 2 shows the a b s o r p t i o n s p e c t r a of color complexes d e v e l o p e d b y the v a r i o u s c a r b o h y d r a t e s in the a p p r o p r i a t e chemical m e t h o d s . Fig. 2A shows the close s i m i l a r i t y of the s p e c t r u m for h e x o s a m i n e s l i b e r a t e d on h y d r o l y s i s of saliva to the s p e c t r u m for D-glucosamine as o b s e r v e d in BOAS '14 m e t h o d . Fig. 2B c o m p a r e s t h e s p e c t r a for t-fucose a n d saliva in the m e t h o d of DISCHE AND SHETTLES 13, d e m o n s t r a t i n g also the Biochim. Biophys. Mcta, ioi (1965) 157-165
161
CARBOHYDRATES OF HUMAN SUBMAXILLARY GLYCOPROTEINS 0.25
A
~
D
-
glucosamine HCI
I
0"1"~ 0.1( 360 0 510
I
I 530
0.30 < 0.25
I
5~o
I
C acid
N- acetytneuraminic ( 5.16 m g / l O O m l ) /
0.2( i'-
0.20
~
400
420 tose
0.15
0.15 ~
~
0.10
,
0.10 - acid (3.1 mg/100 mr) I
500
380
I
540
I
0.05 I
580
I
370 Wavelength (mju)
390
410
430
Fig. 2. Absorption spectra of the non-dialyzable c a r b o h y d r a t e s of h u m a n submaxillary saliva. A : hexosamines of hydrolyzed saliva; I, I)-glucosamine • HC1 (4.64 m g per ioo ml) ; II, hydrolyzed saliva. B: L-fucose in saliva; I, saliva; 11; L-fucose (o.94 m g per IOO ml); I i I , saliva w i t h o u t cysteine. C: sialic acid in saliva; I, N-acetylneuraminic acid (5.16 m g per ioo ml) ; II, saliva; III, N - a c e t y l n e u r a m i n i c acid (3.1 m g per ioo ml). D: D-galactose in saliva; 1, D-galactose (5.0 m g per ioo ml); 1I, saliva; I I l , L-fucose (O.76 mg per IOO ml).
importance of a non-cysteine blank in this method. Fig. 2C shows the absorption spectra for color complexes developed by N-acetylneuraminic acid and saliva in the direct Ehrlich method 15. It is apparent that the spectra are closely similar. Fig. 2D shows the spectra for samples of D-galactose, L-fucose, and saliva in DISCHE'S1~method for hexose determination. The dichromatic readings at 380 and 414 m/z were equal for L-fucose, and the difference in absorbancies of the saliva sample at these wavelengths was taken to represent the galactose present in the secretion. In all instances, no qualitative differences were observed in the spectra for secretor and non-secretor salivas. T A B L E II RECOVERY
OF CONSTITUENTS
OF DIALYZED
HUMAN
Analytical results
Hexose (as anhydrogalactose) Fucose (as anhydrofucose) I-Iexosamine (as anhydro-N-acetylglucosamine) Sialic acid (as a n h y d r o - N - a c e t y l n e u r a m i n i c acid) P r o t e i n (Kjeldahl N c o r r e c t e d for hexosarnine a n d sialic acid N × 6.25)
SUBMAXILLARY
SALIVA
mg per zoo ml
3.52 1.36 3.75 4.86 I9o.8
Total 204.2 Concentration of non-volatile solids in dialyzed saliva 196 Recovery 104 % Biochim. Biophys. Acta, I 0 1 (1965) I 5 7 - I 6 5
162
R. C. CALD\VELL, W. PIGMAN
Recovery of constituents Table I I shows that, within the limits of experimental error, the total amount of solid material remaining in dialyzed saliva dried to constant weight at IOO° could be accounted for b y the carbohydrate and protein determinations. This represents a complete analysis of dialyzed submaxillary saliva. TABLE III O F DIALYSIS ON THE CARBOHYDRATE AND NITROGEN-CONTAINING CONSTITUENTS OF HUMAN SUBMAXILLARY SALIVA EFFECT
A v e r a g e of 5 d e t e r m i n a t i o n s on n o n - s e c r e t o r s a l i v a a n d i d e t e r m i n a t i o n on s e c re t or s a l i va . H i g h e r a m o u n t s of fucose w o u l d be p r e s e n t in a v e r a g e d v a l u e s for a g r o u p c o m p o s e d m a i n l y of secretors. Values are e x p r e s s e d a s / t m o l e s e x c e p t K j e l d a h l N, w h i c h is e x p r e s s e d as m g % .
Compounds
Undialyzed
Hexose Fucose Sialic acid Hexosamine Kjeldahl N
757 82 135 284 33
Dialyzed 24 h 352 (46%) 90 (11o%) 138 (lO2 %) 257 (91%) 22 (67%)
Effect of dialysis on the composition of submaxillary saliva Table III, representing the average of six separate experiments, shows that approximately half the hexose in tile original submaxillary saliva was dialyzable, either free or bound to a dialyzable component of saliva. About 30% of the nitrogencontaining materials and a small amount of hexosamine were dialyzable. Since submaxillary saliva does not contain much glucose or other reducing sugars iv, the nature of this dialyzable component is uncertain, and further work is necessary to establish its identity. Sialic acid and fucose were non-dialyzable, and the assumption is made that, along with the non-dialyzable galactose and hexosamines, these carbohydrates were protein bound and represent the carbohydrates present in human submaxillary TABLE IV CONCENTRATIONS OF NON-DIALYZABLE CARBOHYDRATES IN HUMAN SUBMAXILLARY SALIVA S a l i v a from sev en different m a l e s u b j e c t s was a n a l y z e d . F o u r were secretors, a n d t h r e e were nonsecr eto rs of blood group s u b s t a n c e s . S a l i v a r y flow r a t e r a n g e d from 1.88 t o 3.4 ° m l / m i n for secretors a n d fro m 1.36 to 3-78 m l / m i n for non-secretors. V a l ue s are e x p r e s s e d a s / , m o l e s , e x c e p t for p r o t e i n , w h i c h is e x p r e s s e d as m g % .
Compounds
Galactose Hexosamine Sialic acid Fucose Protein
AII subjects
Secretors
Range
Range
154-722 132-5Ol 71-263 49-397 67-3o 4
Mean ± S.D. 348 262 165 148 168
:]2 ± ~ -~
173 ioi 51 iii 65
Biochim. Biophys. ~tcta, i o i (1965) 157-165
255-722 186-5Ol 76-227 136-397 72-247
Non-secretors Mean -~ S.D. 545 367 182 295 177
~: ~2 212 2~ ~:
Range
Mean
:~ S.D. 174 114 57 57 74
154-416 132-331 71-263 49-147 67-304
264 217 158 85 164
i i :[2 ± ±
81 5° 49 26 66
163
CARBOHYDRATES OF HUMAN SUBMAXILLARY GLYCOPROTEINS
salivary glycoproteins. No differences were observed between secretors and nonsecretors in respect to the effect of dialysis on submaxillary secretion.
Concentrations of protein-bound carbohydrates The data reported were obtained from over IOO chemical determinations, and Table IV shows the ranges of concentrations, the arithmetic mean values, and standard deviations for tile protein and protein-bound carbohydrates in the seven subjects studied. The ranges are wide, but certain patterns could be dependably predicted. In different subjects or in tile same subjects on repeated occasions, hexose was always in the highest molar concentration, followed b y hexosamine and then either fucose or sialic acid. In secretors the fucose always was in higher concentration than sialic acid, whereas sialic acid was in higher concentration in non-secretor saliva. For secretors the sialic acid/fucose molar ratio ranged from 0.54 to 0.80 and averaged o,624-O.lO (S.D.). For non-secretors tile sialic acid/fucose molar ratio ranged from 1.45 to 2.44 and averaged 1.874-o.25 (S.D.). Table IV shows that, under the conditions of this study, dialyzed secretor saliva contained, on the average, more hexose and hexosamine than did dialyzed nonsecretor saliva. The sialic acid concentrations were similar, but tile secretor saliva had about three times as much fucose as non-secretor saliva. DISCUSSION
Purified mucins from salivary glands have been obtained for bovine submaxillary TM and sublinguaP 9 glands, canine submaxillary glands 2°, porcine submaxillary glands zl, and ovine submaxillary glands 2~. The general structure seems to be that of a high-molecular-weight protein to which are attached oligosaccharide subchains, some of which are attached b y 0-glycosidic linkages to the hydroxyl groups of serine and tllreonine 23. Since the same sugar components are present in the dialyzed submaxillary saliva as in the known mucins and blood-group substances, the non-dialyzable components TABLE V COMPARISON WITH
THE
OF THE
MOLAR
CARBOHYDRATE
RATIOS
OF THE
COMPONENTS
NON-DIALYZABLE
OF SALIVARY
MUCINS
CARBOHYDRATES FROM
OTHER
OF HUMAN
SALIVA
SPECIES
Molar r a t i o s b a s e d on h e x o s a m i n e as 2 moles.
Hexosamine
Bovine sublingual Bovine submaxillary Canine submaxillary Porcine submaxillary Ovine submaxillary Human submaxillary (non-secretor) Human submaxillary (secretor)
Galac- Sialic tose acid
Fucose
Total carbohydrate
Sialic acid/ fucose
(Sialic acid+ [ucose) ] hexosamine
2.00 2.00 2.00 2.0o 2.oo
2.00 o.36 1.58 1.3o o.o 4
0.97 1.57 0.53 1.16 1.94
0.72 0.20 1.3o 0.79 0.06
5.69 4.13 5.41 5.25 4.04
1.35 7.85 o.41 1.47 32.3
o.85 0.89 0.92 0.98 i .oo
2.00
2.43
1.46
o.78
6.67
1.87
1.12
2.oo
2.97
0.99
1.61
7.57
o.61
1.3 °
Biochim. Biophys. Acta, i o i (1965) 157-165
16 4
R.C.
C A L D W E L L , W. P I G M A N
of the human submaxillary secretion are probably glycoproteins also. This similarity is shown in Table V, which compares the composition of the purified mucins with that of the human submaxillary secretions of secretors and non-secretors of blood group substances. A comparison of the human non-secretor material wittl mucins from other species suggests a resemblance to bovine sublingual and porcine submaxillary mucins. I extra mole of galactose is present for each 4 moles of hexosamine in comparison with the bovine sublingual mucin, or I extra mole of galactose for each 2 moles of hexosamine as compared witll tile porcine material. Since tile glucosamine/galactosamine ratio is near unity for the human secretion ~, the h u m a n submaxillary mucin resembles the bovine sublingual mucin more than any of tile others which have been studied, the bovine material having a ratio glucosamine/galactosamine of 1:1.2 (ref. 19), while in porcine mucin tile ratio is I :IO (ref. 21). Tile total human secretor submaxillary mucins seem to be similar to canine submaxillary mucin, both substances having sialic acid/fucose ratios well below unity. Also, at least an additional mole of galactose is present per 2 moles of hexosamine. These comparisons can only be considered qualitatively, since the mucins of the human submaxillary saliva apparently consist of a number of components with variable sialic acid/fucose ratios. Variation in the sialic acid/fucose ratios has been noted for canine submaxillary saliva by DISCHE et al. 0-4 for different types of stimulation. An important finding in this study was that, although the range of carbohydrate concentrations varied widely, a predictable pattern of composition was observed. In all subjects tile hexose was in highest concentration, followed b y hexosamine. Thus, a high or low value for one of these components was accompanied by a correspondingly altered concentration of the other. The sialic acid/fucose ratios of secretors and non-secretors were obviously different, with no overlap existing in the observed ratios. A matter requiring further study is the greater variability of the ratio in non-secretors. The fact that submaxillary salivary secretions have different chemical composition in secretors and non-secretors indicates the inadvisability of pooling salivary samples from different subjects when the composition of the secretions is being studied. TABLE
VI
COMPARISON OF THE MOLAR RATIOS OF THE NON-DIALYZABLE CARBOHYDRATES IN SUBMAXILLARY SALIVA FROM INDIVIDUALS OF DIFFERENT BLOOD TYPES Molar ratios based on hexosamine as 2 moles.
Secretor status and blood type
Salivary Hexosflow rate amine (ml/min)
Galaclose
Sialic acid
Fucose
Sialic acid plus fucose
3.78 2.22 2.48
2.00 2.oo 2.o0
2.77 2.36 2.84
1.49 1.43 1.65
0.75 o.76 0.98
2.24 2.19 2.63
1.88 1.92 2.73 2.88
2.00 2.00 2. oo 2.oo
2.74 2.92 2.89 3.o 4
0.82 0.77 I.O 9 I.O6
1.46 1.42 I. 7 ° 1.06
2.28 2.19 2.70 2.72
N o n - secretors A O O
Secretovs A A A I3 O
Biochim. Biophys. Acta, l o l (1965) 1 5 7 - i 6 5
CARBOHYDRATES OF HUMAN SUBMAXILLARY GLYCOPROTEINS
165
The limited number of subjects in this study does not allow a detailed analysis of the relationship between salivary composition and specific blood types. Table VI demonstrates a similarity in carbohydrate composition oi the dialyzed submaxillary secretions in seven different subjects of various blood types. Relating the other carbohydrates to hexosamine, it seems that individuals of various blood types had similar amounts of galactose, and tile sum of sialic acid and fucose was similar. Secretors and non-secretors varied in the relative amounts of sialic acid or fucose, but within the secretor group or non-secretor group, the blood type seemed to have little effect on composition. Due to the limited number of individuals in the study and tile absence of Type-B subjects, we can only propose on tlle basis of our data that no major differences in salivary glycoprotein composition occur in individuals of various blood types. The most obvious factor influencing the composition of human submaxillary secretion is the blood group secretor status of the individual. ACKNOWLEDGEMENTS
This investigation was supported by U.S. Public Health Service Research Grant D E 01858 and carried out during the tenure of a U.S. Public Health Service Research Career Development Award (GM-K3-I528o) to R.C.C. This work is included in a dissertation, "The Glycoproteins of Human Submaxillary Secretion," submitted by one of the authors (R.C.C.) in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry at tile University of Alabama Medical Center. The authors are indebted to the individuals who took part in the study and to Mrs. F. W. WELLS for skillful technical assistance. REFERENCES I A. GOTTSCHALK, The Chemistry and Biology of Sialic Acids and Related Substances, Cambridge University Press, London, 196o. 2 W. T. J. MORGAN, Ciba Found. Syrup., Human Genet., I959, Little Brown, Boston, Mass., 1959, p. 193. 3 R. C. CALDWELL AND V~T. PIGMAN, J . Dental Res., 43 (1964) 754. 4 I. D. MANDEL, R. H. THOMPSON, JR. AND S. A. ELLISON, Intern. Assoc. Dental Res., Abstr. 41st Gen. Meeting, Pittsburgh. Pa., 1963, No. 349 (1963) 122. 5 E. A. KABAT, Blood Group Substances, Academic Press, New York, 1956, p. 56. 6 L. H. SCHNEYER, J. Dental. Res., 34 (1955) 257. 7 M. DEAKINS, J. Dental Res., 20 (1941) 129. 8 t~. ]!¢[ASAMUNEAND Z. YOSIZAWA, Tohoku J. Exptl. Med., 59 (1953) i. 9 X~r. E. TREVELYAN, D P PROCTER AND j. s. HARRISON, Nature 166 (195 o) 444. IO E. P. CLARK, Semimicro Quantitative Organic Analysis, Academic Press, New York, 1943, p. 37. Ii Y. IIASI.IIMOTOAND W. PIGMAN, Ann. N . Y . Acad. Sci., 93 (1962) 541. 12 Z. DISCHE, Methods Biochem. Anal., 2 (1955) 327. 13 Z. Dxscl.iE AND L. B. SHETTLES, J. Biol. Chem., 175 (1948) 595. 14 N. F. BOAS, J. Biol. Chem., 204 (1953) 553. 15 W. PIGMAN, W. L. HAWKINS, M. G. BLAIR AND H. L. HOLLEY, Arthritis Rheumal., t (1958) 151. 16 L. ~VARREN, J. Biol. Chem., 234 (1959) 1971. 17 W. PIGMAN ANn W. L. HAWKI~IS, J. Dental. t?es., 37 (1958) 688. 18 S. TsuIKI, Y. HASHIMOTO AND W. PIGMAN, Nature, 189 (1961) 39919 S. TSOIK! AND ~V. PIGMAN, Arch. Oral. Biol., 2 (196o) I. 20 Y. HASHIMOTO, S. TSlJIKI, G. QUINTARELLI AND W. PIGMAN, Bioehim. t?iophys. Acta, 48 (1961) 404 . 21 Y. HASHIMOTO, S. I-IASHIMOTO AND W. PIG.MAN, Arch. Biochem. Biophys., lO 4 (1964) 282. 22 E. R. B. GRAHAM AND A. GOTTSCI-IALK, Biochim. Biophys. Acta, 38 (196o) 513 . 23 K. TANAKA, M. BERTOLINI AND W. PIGMAN, Biochem. Biophys. Res. Commun., 16 (1964) 404 . 24 Z. DISCHIE, C. PALLAVlCINI, H. KAVASAK1, ~q'. SMIRNO~,V, L. J. CIZEK AND S. CI.IIEN, Arch. Biochem. Biophys., 97 (1962) 459.
Biochim. Biophys. Acta, t o i (1965) 157-165
157
BBA 85047 T H E C A R B O H Y D R A T E S OF HUMAN S U B M A X I L L A R Y G L Y C O P R O T E I N S IN SECRETORS AND NON-SECRETORS OF BLOOD G R O U P SUBSTANCES
R O B E R T C. C A L D W E L L AND W A R D P I G M A N
Departments of Oral Biology and Biochemistry, University of A labama Medical Center, Birmingham, Ala. (U.S.A.) (Received December I4th, 1964)
SUMMARY
Chemical analyses of human submaxillary salivary gland secretions from individuals of various blood types and blood group secretor statuses revealed a relationship between secretor status and the composition of total submaxillary glycoproteins. The dialyzed secretions from both secretors and non-secretors contained sialic acid, D-galactose, L-fucose, D-glucosamine, D-galactosamine, and protein. Half of the total hexose, one-third of the nitrogen-containing material and minor amounts of hexosamine were dialyzable. None of the sialic acid and fucose components was dialyzable. The submaxillary saliva of secretors had a higher concentration of proteinbound carbohydrates than that of non-secretors and had about three times as much fucose. The ratio of sialic acid/fucose was o.62±O.lO (S.D.) for secretors and 1.874-o.25 (S.D.) for non-secretors. There was a predictable relationship among tile various carbohydrates in each type of secretion, and no major differences were observed among individuals of different blood types.
INTRODUCTION
Since the elucidation of the structure of the sialic acids 1, there has been an accelerating interest in the chemistry of salivary glycoproteins. Much of the methodology was established b y work on the submaxillary gland extracts of various m a m malian species. However, relatively little work has been done on human salivary secretions. In IV[ORGAN'S2 work on blood group substances, which are chemically closely allied to salivary glycoproteins, it was emphasized that pooling secretions from various sources was undesirable. I f materials had to be pooled, care should be taken to pool only secretions from individuals with the same blood phenotype and secretor status. This admonition has not been heeded in studies of the composition of human salivas. The past work has either been on pooled whole saliva from a haphazardly selected group of subjects, or, in the few studies of separated secretions, no mention has been made of the blood group types or secretor status. In addition, the salivary Biochim. Biophys. Acla, ioi (1965) I57-165
158
R . C . CALDWELL, W. PIGMAN
flow rate was usually not recorded, although the composition is affected b y the rate of salivary flow 3. In this study, the chemical composition of human submaxillary secretion has been studied in secretors and non-secretors of various blood types, and the secretions have not been pooled. As predicted b y MORGAN2, it has been found that salivary secretions fronl secretors and non-secretors are chemically different in some important respects. While this study was in progress, data on the composition of human submaxillary secretion were reported b y MANDEL et al. 4. This and the present work are the only known studies of the chemical composition of the glycoproteins of human submaxillary secretion. MATERIALS AND METHODS
Seven apparently healthy male subjects varying in age from 2o to 4o years participated in the study. Secretor status was determined on the subjects' whole salivas b y means of hemagglutination inhibition tests 5 on type O(H) cells using anti-H lectin (Hyland, Los Angeles). Blood group typing was performed at the University of Alabama Blood Bank. For each subject an appliance was constructed for the collection of pure submaxillary saliva, according to the method of SCHNEYER6. Saliva was collected directly from Wharton's ducts, and, after voiding of the first 2 ml, the secretion was allowed to flow into graduated glass cylinders packed in iceL Tile flow of saliva was stimulated b y having the subjects suck lime-flavored candy ("Lifesavers") while the collection apparatus was in place. Since we have observed that concentrations of the components of saliva are flow-rate dependent s , the time taken to collect each sample was measured b y a stop watch, and salivary flow rate was calculated as ml/min. The data presented here cover flow rates of from 1.88 to 3.4 o ml/min for seeretors, and from 1.36 to 3.78 ml/min for non-secretors. The saliva samples were dialyzed against distilled water in seamless regenerated cellulose dialysis tubing for 18 h, the water being stirred constantly and changed once. Throughout the period of collection and dialysis, the temperature was maintained at 2-4 ° . Before chemical analysis, the dialyzed sample was placed in a ground-glass test tube and homogenized gently to ensure an even distribution of material prior to pipetting aliquots for the different analytical methods. This procedure also improved the accuracy of pipetting because the secretion lost much of its viscosity during homogenization. Paper chromatography was performed b y tile method of MASAMUNE AND YOSIZAWAs to identify the carbohydrates present in dialyzed, hydrolyzed saliva. The hydrolysis was carried out as follows. After dialysis, the secretion was lyoptlilized, and Io mg of the dry material was hydrolyzed with 2 ml of 2 N H2SO 4 at IOO° for 5 h. The solution was then brought to p H 5.8 b y addition of saturated Ba(OH)t, and the BaS04 was removed by centrifugation. The supernatant was evaporated to dryness in a desiccator at room temperature, and the sugars were redissolved in two drops of water. Descending chromatography was carried out in n-butanol-pyridine-water (5:3 : 2, v/v) on W h a t m a n No. I filter paper. 8-12/~1 of the sugar solution was applied to the paper, and the same volumes of reference sugars (I mg/ml) were included on the same strip. The known sugars added to the paper Were L-fucose, D-mannose, DBiochim. Biophys. dora, IOI (1965) 157-165
CARBOHYDRATES
OF HUMAN
SUBMAXILLARY
159
GLYCOPROTEINS
glucose, D-galactose, D-glucosamine, and D-galactosamine. The solvent was allowed to drip off the paper, and the total time of each run was 4 ° h. The sugars were detected b y the modified AgNOs-spray method of TREVELYAN et al2. Nitrogen determinations were performed b y a micro-Kjeldahl method 10, which was modified slightly b y digesting 3 ml of the saliva instead of a weighed amount of solid. Since it has been shown that tile protein of salivary-gland mucins contains close to I 6 % nitrogen n, the factor of 6.2 5 was used to convert nitrogen values to protein. Tile nitrogen values in all instances were corrected for the nitrogen content of sialic acid and hexosamine present in tile dialyzed secretion. Hexose was determined b y DISCHE'S1~ cysteine-H~SO, method TM. A galactose standard was used, since paper chromatography revealed that galactose was the only hexose present in appreciable amounts in dialyzed saliva. Fucose was determined b y the cysteine-H~SO 4 reaction after a Io-min heating period, as described b y DISCHE AND SHETTLES13. Total hexosamine was determined b y the Elson-Morgan method as modified by BOAS14, but the method recommended b y BOAS of passing the material to be analyzed through a Dowex-5o ion-exchange column was unnecessary, since no interfering chromogens were present in dialyzed saliva. Hydrolysis of I-ml saliva samples was carried out in an equal volume of HC1 solution under a variety of conditions (see Table I), and subsequently the hydrolysis conditions chosen were 4 N HC1, IOO°, 3 h. TABLE
I
H Y D R O L Y S I S C O N D I T I O N S FOR RELEASE OF H E X O S A M I N E FROM HUMAN S U B M A X I L L A R Y SALIVA Hexosamine is expressed i n d i c a t e d , a t lOO%
Conch.
as t~moles of glucosamine
released
from
ioo ml of saliva, after the times
Hexosamine released
HCl (N) I 2 4 6
Ih
2h
2.5h
3 h
3.5 h
4h
45.3 73.7 12o.7 127. 4
--135-2 124.o
--138.5 I29.1
93.3 -124.6 127.4 -131.8 139-7 141.914o.8--
The standard, glucosamine. HC1, was also heated in 4 N HC1 under the same conditions. Since glucosamine. HC1 was used as a standard, the correction factor of 0.829 was necessary to give the results in terms of the amount of hexosamine present. Sialic acid was determined b y the direct Ehrlich method 15. The values obtained b y this method were reliable and consistently higher than those obtained b y WARREN'STM thiobarbituric acid method. A standard of N-acetylneuraminic acid (Sigma, St. Louis) was used which was 90% pure when assayed b y comparison with a chromatographically homogeneous sample (Calbiochem, Los Angeles) of N-acetylneuraminic acid containing 4.52% nitrogen. RESULTS
Identification of carbohydrate constituents Fig. i shows typical chromatograms of the non-dialyzable sugars found in Biochim. Biophys. Acta, i o i
(i965)
i57-i65
I6O
R . C . CALDWELL, XV. PIGMAN
SEGRI[TOR SALIVA
CONTROL
Fig. I. Paper chromatog:ams of the non-dialyzable carbohydrates of human submaxillary saliva. Secretor and non-secretor salivas both contain L-ftlCOSe, D-galactose, D-glucosamine, and Dgalactosamine. d i a l y z e d s u b m a x i l l a r y saliva from a secretor a n d a non-secretor. Spots were f o u n d c o r r e s p o n d i n g to L-fucose, D-galactose, I)-glucosamine, a n d D-galactosamine. No D-glucose was d e t e c t e d b y this procedure, a n d D-mannose m a y h a v e been p r e s e n t in v e r y small a m o u n t s , since a f a i n t l y d a r k a r e a was occasionally seen on t h e c h r o m a t o g r a m in the a p p r o p r i a t e area. However, for b o t h secretors a n d non-secretors, t h e p r i n c i p a l hexose p r e s e n t in the d i a l y z e d saliva was galactose, a n d fucose occurred in similar a m o u n t s . The c h r o m a t o g r a p h y t i m e was s h o r t e n e d on one occasion to ensure t h a t no f a s t - m o v i n g sugar such as L-rhamnose was r u n n i n g off the paper. Fig. 2 shows the a b s o r p t i o n s p e c t r a of color complexes d e v e l o p e d b y the v a r i o u s c a r b o h y d r a t e s in the a p p r o p r i a t e chemical m e t h o d s . Fig. 2A shows the close s i m i l a r i t y of the s p e c t r u m for h e x o s a m i n e s l i b e r a t e d on h y d r o l y s i s of saliva to the s p e c t r u m for D-glucosamine as o b s e r v e d in BOAS '14 m e t h o d . Fig. 2B c o m p a r e s t h e s p e c t r a for t-fucose a n d saliva in the m e t h o d of DISCHE AND SHETTLES 13, d e m o n s t r a t i n g also the Biochim. Biophys. Mcta, ioi (1965) 157-165
161
CARBOHYDRATES OF HUMAN SUBMAXILLARY GLYCOPROTEINS 0.25
A
~
D
-
glucosamine HCI
I
0"1"~ 0.1( 360 0 510
I
I 530
0.30 < 0.25
I
5~o
I
C acid
N- acetytneuraminic ( 5.16 m g / l O O m l ) /
0.2( i'-
0.20
~
400
420 tose
0.15
0.15 ~
~
0.10
,
0.10 - acid (3.1 mg/100 mr) I
500
380
I
540
I
0.05 I
580
I
370 Wavelength (mju)
390
410
430
Fig. 2. Absorption spectra of the non-dialyzable c a r b o h y d r a t e s of h u m a n submaxillary saliva. A : hexosamines of hydrolyzed saliva; I, I)-glucosamine • HC1 (4.64 m g per ioo ml) ; II, hydrolyzed saliva. B: L-fucose in saliva; I, saliva; 11; L-fucose (o.94 m g per IOO ml); I i I , saliva w i t h o u t cysteine. C: sialic acid in saliva; I, N-acetylneuraminic acid (5.16 m g per ioo ml) ; II, saliva; III, N - a c e t y l n e u r a m i n i c acid (3.1 m g per ioo ml). D: D-galactose in saliva; 1, D-galactose (5.0 m g per ioo ml); 1I, saliva; I I l , L-fucose (O.76 mg per IOO ml).
importance of a non-cysteine blank in this method. Fig. 2C shows the absorption spectra for color complexes developed by N-acetylneuraminic acid and saliva in the direct Ehrlich method 15. It is apparent that the spectra are closely similar. Fig. 2D shows the spectra for samples of D-galactose, L-fucose, and saliva in DISCHE'S1~method for hexose determination. The dichromatic readings at 380 and 414 m/z were equal for L-fucose, and the difference in absorbancies of the saliva sample at these wavelengths was taken to represent the galactose present in the secretion. In all instances, no qualitative differences were observed in the spectra for secretor and non-secretor salivas. T A B L E II RECOVERY
OF CONSTITUENTS
OF DIALYZED
HUMAN
Analytical results
Hexose (as anhydrogalactose) Fucose (as anhydrofucose) I-Iexosamine (as anhydro-N-acetylglucosamine) Sialic acid (as a n h y d r o - N - a c e t y l n e u r a m i n i c acid) P r o t e i n (Kjeldahl N c o r r e c t e d for hexosarnine a n d sialic acid N × 6.25)
SUBMAXILLARY
SALIVA
mg per zoo ml
3.52 1.36 3.75 4.86 I9o.8
Total 204.2 Concentration of non-volatile solids in dialyzed saliva 196 Recovery 104 % Biochim. Biophys. Acta, I 0 1 (1965) I 5 7 - I 6 5
162
R. C. CALD\VELL, W. PIGMAN
Recovery of constituents Table I I shows that, within the limits of experimental error, the total amount of solid material remaining in dialyzed saliva dried to constant weight at IOO° could be accounted for b y the carbohydrate and protein determinations. This represents a complete analysis of dialyzed submaxillary saliva. TABLE III O F DIALYSIS ON THE CARBOHYDRATE AND NITROGEN-CONTAINING CONSTITUENTS OF HUMAN SUBMAXILLARY SALIVA EFFECT
A v e r a g e of 5 d e t e r m i n a t i o n s on n o n - s e c r e t o r s a l i v a a n d i d e t e r m i n a t i o n on s e c re t or s a l i va . H i g h e r a m o u n t s of fucose w o u l d be p r e s e n t in a v e r a g e d v a l u e s for a g r o u p c o m p o s e d m a i n l y of secretors. Values are e x p r e s s e d a s / t m o l e s e x c e p t K j e l d a h l N, w h i c h is e x p r e s s e d as m g % .
Compounds
Undialyzed
Hexose Fucose Sialic acid Hexosamine Kjeldahl N
757 82 135 284 33
Dialyzed 24 h 352 (46%) 90 (11o%) 138 (lO2 %) 257 (91%) 22 (67%)
Effect of dialysis on the composition of submaxillary saliva Table III, representing the average of six separate experiments, shows that approximately half the hexose in tile original submaxillary saliva was dialyzable, either free or bound to a dialyzable component of saliva. About 30% of the nitrogencontaining materials and a small amount of hexosamine were dialyzable. Since submaxillary saliva does not contain much glucose or other reducing sugars iv, the nature of this dialyzable component is uncertain, and further work is necessary to establish its identity. Sialic acid and fucose were non-dialyzable, and the assumption is made that, along with the non-dialyzable galactose and hexosamines, these carbohydrates were protein bound and represent the carbohydrates present in human submaxillary TABLE IV CONCENTRATIONS OF NON-DIALYZABLE CARBOHYDRATES IN HUMAN SUBMAXILLARY SALIVA S a l i v a from sev en different m a l e s u b j e c t s was a n a l y z e d . F o u r were secretors, a n d t h r e e were nonsecr eto rs of blood group s u b s t a n c e s . S a l i v a r y flow r a t e r a n g e d from 1.88 t o 3.4 ° m l / m i n for secretors a n d fro m 1.36 to 3-78 m l / m i n for non-secretors. V a l ue s are e x p r e s s e d a s / , m o l e s , e x c e p t for p r o t e i n , w h i c h is e x p r e s s e d as m g % .
Compounds
Galactose Hexosamine Sialic acid Fucose Protein
AII subjects
Secretors
Range
Range
154-722 132-5Ol 71-263 49-397 67-3o 4
Mean ± S.D. 348 262 165 148 168
:]2 ± ~ -~
173 ioi 51 iii 65
Biochim. Biophys. ~tcta, i o i (1965) 157-165
255-722 186-5Ol 76-227 136-397 72-247
Non-secretors Mean -~ S.D. 545 367 182 295 177
~: ~2 212 2~ ~:
Range
Mean
:~ S.D. 174 114 57 57 74
154-416 132-331 71-263 49-147 67-304
264 217 158 85 164
i i :[2 ± ±
81 5° 49 26 66
163
CARBOHYDRATES OF HUMAN SUBMAXILLARY GLYCOPROTEINS
salivary glycoproteins. No differences were observed between secretors and nonsecretors in respect to the effect of dialysis on submaxillary secretion.
Concentrations of protein-bound carbohydrates The data reported were obtained from over IOO chemical determinations, and Table IV shows the ranges of concentrations, the arithmetic mean values, and standard deviations for tile protein and protein-bound carbohydrates in the seven subjects studied. The ranges are wide, but certain patterns could be dependably predicted. In different subjects or in tile same subjects on repeated occasions, hexose was always in the highest molar concentration, followed b y hexosamine and then either fucose or sialic acid. In secretors the fucose always was in higher concentration than sialic acid, whereas sialic acid was in higher concentration in non-secretor saliva. For secretors the sialic acid/fucose molar ratio ranged from 0.54 to 0.80 and averaged o,624-O.lO (S.D.). For non-secretors tile sialic acid/fucose molar ratio ranged from 1.45 to 2.44 and averaged 1.874-o.25 (S.D.). Table IV shows that, under the conditions of this study, dialyzed secretor saliva contained, on the average, more hexose and hexosamine than did dialyzed nonsecretor saliva. The sialic acid concentrations were similar, but tile secretor saliva had about three times as much fucose as non-secretor saliva. DISCUSSION
Purified mucins from salivary glands have been obtained for bovine submaxillary TM and sublinguaP 9 glands, canine submaxillary glands 2°, porcine submaxillary glands zl, and ovine submaxillary glands 2~. The general structure seems to be that of a high-molecular-weight protein to which are attached oligosaccharide subchains, some of which are attached b y 0-glycosidic linkages to the hydroxyl groups of serine and tllreonine 23. Since the same sugar components are present in the dialyzed submaxillary saliva as in the known mucins and blood-group substances, the non-dialyzable components TABLE V COMPARISON WITH
THE
OF THE
MOLAR
CARBOHYDRATE
RATIOS
OF THE
COMPONENTS
NON-DIALYZABLE
OF SALIVARY
MUCINS
CARBOHYDRATES FROM
OTHER
OF HUMAN
SALIVA
SPECIES
Molar r a t i o s b a s e d on h e x o s a m i n e as 2 moles.
Hexosamine
Bovine sublingual Bovine submaxillary Canine submaxillary Porcine submaxillary Ovine submaxillary Human submaxillary (non-secretor) Human submaxillary (secretor)
Galac- Sialic tose acid
Fucose
Total carbohydrate
Sialic acid/ fucose
(Sialic acid+ [ucose) ] hexosamine
2.00 2.00 2.00 2.0o 2.oo
2.00 o.36 1.58 1.3o o.o 4
0.97 1.57 0.53 1.16 1.94
0.72 0.20 1.3o 0.79 0.06
5.69 4.13 5.41 5.25 4.04
1.35 7.85 o.41 1.47 32.3
o.85 0.89 0.92 0.98 i .oo
2.00
2.43
1.46
o.78
6.67
1.87
1.12
2.oo
2.97
0.99
1.61
7.57
o.61
1.3 °
Biochim. Biophys. Acta, i o i (1965) 157-165
16 4
R.C.
C A L D W E L L , W. P I G M A N
of the human submaxillary secretion are probably glycoproteins also. This similarity is shown in Table V, which compares the composition of the purified mucins with that of the human submaxillary secretions of secretors and non-secretors of blood group substances. A comparison of the human non-secretor material wittl mucins from other species suggests a resemblance to bovine sublingual and porcine submaxillary mucins. I extra mole of galactose is present for each 4 moles of hexosamine in comparison with the bovine sublingual mucin, or I extra mole of galactose for each 2 moles of hexosamine as compared witll tile porcine material. Since tile glucosamine/galactosamine ratio is near unity for the human secretion ~, the h u m a n submaxillary mucin resembles the bovine sublingual mucin more than any of tile others which have been studied, the bovine material having a ratio glucosamine/galactosamine of 1:1.2 (ref. 19), while in porcine mucin tile ratio is I :IO (ref. 21). Tile total human secretor submaxillary mucins seem to be similar to canine submaxillary mucin, both substances having sialic acid/fucose ratios well below unity. Also, at least an additional mole of galactose is present per 2 moles of hexosamine. These comparisons can only be considered qualitatively, since the mucins of the human submaxillary saliva apparently consist of a number of components with variable sialic acid/fucose ratios. Variation in the sialic acid/fucose ratios has been noted for canine submaxillary saliva by DISCHE et al. 0-4 for different types of stimulation. An important finding in this study was that, although the range of carbohydrate concentrations varied widely, a predictable pattern of composition was observed. In all subjects tile hexose was in highest concentration, followed b y hexosamine. Thus, a high or low value for one of these components was accompanied by a correspondingly altered concentration of the other. The sialic acid/fucose ratios of secretors and non-secretors were obviously different, with no overlap existing in the observed ratios. A matter requiring further study is the greater variability of the ratio in non-secretors. The fact that submaxillary salivary secretions have different chemical composition in secretors and non-secretors indicates the inadvisability of pooling salivary samples from different subjects when the composition of the secretions is being studied. TABLE
VI
COMPARISON OF THE MOLAR RATIOS OF THE NON-DIALYZABLE CARBOHYDRATES IN SUBMAXILLARY SALIVA FROM INDIVIDUALS OF DIFFERENT BLOOD TYPES Molar ratios based on hexosamine as 2 moles.
Secretor status and blood type
Salivary Hexosflow rate amine (ml/min)
Galaclose
Sialic acid
Fucose
Sialic acid plus fucose
3.78 2.22 2.48
2.00 2.oo 2.o0
2.77 2.36 2.84
1.49 1.43 1.65
0.75 o.76 0.98
2.24 2.19 2.63
1.88 1.92 2.73 2.88
2.00 2.00 2. oo 2.oo
2.74 2.92 2.89 3.o 4
0.82 0.77 I.O 9 I.O6
1.46 1.42 I. 7 ° 1.06
2.28 2.19 2.70 2.72
N o n - secretors A O O
Secretovs A A A I3 O
Biochim. Biophys. Acta, l o l (1965) 1 5 7 - i 6 5
CARBOHYDRATES OF HUMAN SUBMAXILLARY GLYCOPROTEINS
165
The limited number of subjects in this study does not allow a detailed analysis of the relationship between salivary composition and specific blood types. Table VI demonstrates a similarity in carbohydrate composition oi the dialyzed submaxillary secretions in seven different subjects of various blood types. Relating the other carbohydrates to hexosamine, it seems that individuals of various blood types had similar amounts of galactose, and tile sum of sialic acid and fucose was similar. Secretors and non-secretors varied in the relative amounts of sialic acid or fucose, but within the secretor group or non-secretor group, the blood type seemed to have little effect on composition. Due to the limited number of individuals in the study and tile absence of Type-B subjects, we can only propose on tlle basis of our data that no major differences in salivary glycoprotein composition occur in individuals of various blood types. The most obvious factor influencing the composition of human submaxillary secretion is the blood group secretor status of the individual. ACKNOWLEDGEMENTS
This investigation was supported by U.S. Public Health Service Research Grant D E 01858 and carried out during the tenure of a U.S. Public Health Service Research Career Development Award (GM-K3-I528o) to R.C.C. This work is included in a dissertation, "The Glycoproteins of Human Submaxillary Secretion," submitted by one of the authors (R.C.C.) in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry at tile University of Alabama Medical Center. The authors are indebted to the individuals who took part in the study and to Mrs. F. W. WELLS for skillful technical assistance. REFERENCES I A. GOTTSCHALK, The Chemistry and Biology of Sialic Acids and Related Substances, Cambridge University Press, London, 196o. 2 W. T. J. MORGAN, Ciba Found. Syrup., Human Genet., I959, Little Brown, Boston, Mass., 1959, p. 193. 3 R. C. CALDWELL AND V~T. PIGMAN, J . Dental Res., 43 (1964) 754. 4 I. D. MANDEL, R. H. THOMPSON, JR. AND S. A. ELLISON, Intern. Assoc. Dental Res., Abstr. 41st Gen. Meeting, Pittsburgh. Pa., 1963, No. 349 (1963) 122. 5 E. A. KABAT, Blood Group Substances, Academic Press, New York, 1956, p. 56. 6 L. H. SCHNEYER, J. Dental. Res., 34 (1955) 257. 7 M. DEAKINS, J. Dental Res., 20 (1941) 129. 8 t~. ]!¢[ASAMUNEAND Z. YOSIZAWA, Tohoku J. Exptl. Med., 59 (1953) i. 9 X~r. E. TREVELYAN, D P PROCTER AND j. s. HARRISON, Nature 166 (195 o) 444. IO E. P. CLARK, Semimicro Quantitative Organic Analysis, Academic Press, New York, 1943, p. 37. Ii Y. IIASI.IIMOTOAND W. PIGMAN, Ann. N . Y . Acad. Sci., 93 (1962) 541. 12 Z. DISCHE, Methods Biochem. Anal., 2 (1955) 327. 13 Z. Dxscl.iE AND L. B. SHETTLES, J. Biol. Chem., 175 (1948) 595. 14 N. F. BOAS, J. Biol. Chem., 204 (1953) 553. 15 W. PIGMAN, W. L. HAWKINS, M. G. BLAIR AND H. L. HOLLEY, Arthritis Rheumal., t (1958) 151. 16 L. ~VARREN, J. Biol. Chem., 234 (1959) 1971. 17 W. PIGMAN ANn W. L. HAWKI~IS, J. Dental. t?es., 37 (1958) 688. 18 S. TsuIKI, Y. HASHIMOTO AND W. PIGMAN, Nature, 189 (1961) 39919 S. TSOIK! AND ~V. PIGMAN, Arch. Oral. Biol., 2 (196o) I. 20 Y. HASHIMOTO, S. TSlJIKI, G. QUINTARELLI AND W. PIGMAN, Bioehim. t?iophys. Acta, 48 (1961) 404 . 21 Y. HASHIMOTO, S. I-IASHIMOTO AND W. PIG.MAN, Arch. Biochem. Biophys., lO 4 (1964) 282. 22 E. R. B. GRAHAM AND A. GOTTSCI-IALK, Biochim. Biophys. Acta, 38 (196o) 513 . 23 K. TANAKA, M. BERTOLINI AND W. PIGMAN, Biochem. Biophys. Res. Commun., 16 (1964) 404 . 24 Z. DISCHIE, C. PALLAVlCINI, H. KAVASAK1, ~q'. SMIRNO~,V, L. J. CIZEK AND S. CI.IIEN, Arch. Biochem. Biophys., 97 (1962) 459.
Biochim. Biophys. Acta, t o i (1965) 157-165