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The vitamin B12-binding proteins of saliva and tears and their relationship to other vitamin B12 binders
BIOCHIMICAET BIOPHYSICAACTA
747
BBA 36091 T H E V I T A M I N B12-BINDING P R O T E I N S OF SALIVA AND T E A R S AND T H E I R R E L A T I O N S H I P TO O T H E R VITAMIN B12 B I N D E R S
RALPH CARMEL MRC Experimental Haematology Unit, St. Mary's Hospital Medical School, London (Great Britain)
(Received December 6th, 1971)
SUMMARY A study of the vitamin B12 binder in saliva was prompted by different reported molecular weights, some suggesting it was unique among the otherwise identical group of vitamin B1,z binders called R binders. Tears, extremely rich in this protein, were also examined. Gel filtration and sucrose density gradient ultracentrifugation showed saliva binder to be identical in size with other R binders, serum third binder and transcobalamin I. The discrepancy with previous reports is unexplained. It is apparent that vitamin BI~ binders are anomalous proteins, and past estimates of molecular weight m a y be incorrect. Saliva binder resembled leucocyte binder and serum third binder, but not transcobalamin I, in electrophoretic mobility. However, some heterogeneity was obvious, even among different specimens from the same subject. Tear binder was intermediate between R binder and transcobalamin I on electrophoresis. These results support identity of all the R binders except for some heterogeneity, probably acquired, in isoelectric points. It also appears that third binder is the R binder in serum.
INTRODUCTION A single type of vitamin Bi2-binding protein (as determined by immunologic and chromatographic behaviour and molecular sizing) appears in most of the body fluids of man and in leucocytes and erythrocytesl, ~, and is called " R binder"l, 3. The sole exception in the similarity of all R binders to each other has been saliva binder, for which different molecular weights have been reported 4-n. Although R binder is present in serum a, it is uncertain which serum binder corresponds to it due to the variability of separation techniques. The non-identity of R binder with transcobalamin I I has been summarized 7. Transcobalamin I differs from R binder electrophoreticallyS, 9, and serum R binder does not carry endogenous vitamin B12 (ref. I). The recently described third serum binder 1°-13, however, resembles R binder in all tested characteristics1°, 13. Biochim. Biophys. Acta, 263 (1972) 747-752
748
R. CARMEL
The present study was undertaken to further examine saliva binder and its possible resemblance to third binder of serum, and to determine whether it indeed differs in molecular size from other R binders. Tear vitamin Bl~-binding protein was similarly studied. Tears have exceptionally high vitamin B~2-binding capacity, yet the binder has not been characterized apart from determination of electrophoretic mobility at p H 8.6 (ref. 14). MATERIALS AND METHODS Saliva specimens were collected by unstimulated spitting from four fasting, normal subjects and one fasting subject with pernicious anaemia in remission. Spontaneous tears were collected by eye-dropper from two healthy children and pooled. Samples were centrifuged to remove debris, but were otherwise untreated. Testing was done immediately or after storage at --20 °C, with no difference in results. Polycythaemia vera serum was used as the source of third binder. Sephadex G-2oo gel (Pharmacia Fine Chemicals, Uppsala, Sweden) filtration was done at room temperature in 0. 9 cm × 60 cm columns, using I M NaCI-o.I M Tris buffer (pH 8.5) containing 0.02% NaN 3. Samples of O.l-O.3 ml were incubated with excess E~7Colvitamin Blo~ (The Radiochemical Centre, Amersham) specific activity of 15.2/~Ci//~g, for 30 min at room temperature prior to application. Aliquots of IO drops were collected b y gravity. Radioactivity was counted in a well-type scintillation counter. Sephadex G-75 gel filtration was similarly performed, using the above buffer or o.I M sodium phosphate buffer (pH 6.3). Both gel columns had been calibrated with high-molecular weight Blue Dextran, E57Colvitamin B12-1abelled leucocyte extract 9, and various E57Colvitamin B12-1abelled human sera. Protein concentration was determined at 250 nm during flow. Three of the saliva specimens were filtered through Sephadex G-2oo or G-75 without added E57Co]vitamin B12. Filtrate tubes were then each incubated with I ng EsvColvitamin B~2 for 30 rain at room temperature, and the unbound E57Co]vitamin B12 was removed with haemoglobin- and plasma-coated charcoaP 5 prior to counting. Linear sucrose density gradient ultracentrifugation of diluted saliva and serum, labelled with E57Co]vitamin B12, specific activity of 118 #Ci//~g, was done as described b y Martin and Ames ~6, spinning 17. 5 h at 38 ooo rev./min with a 3 × 6.5 swing-out rotor in a Superspeed 65 centrifuge (Measuring and Scientific Equipment, London) at 7 °C. Transferrin, IgG and human serum albumin, all labelled with ~2~i, were used as internal standards. Cellulose acetate electrophoresis was done at pH 8.6 on specimens previously incubated with EaVColvitamin BI~, specific activity of 118/,Ci/#g. Serum labelled with 59Fe was used for comparison. The unstained strips were incubated for 3 weeks with no-grid X-ray film, and stained subsequently for comparison with the radioautographs. Pevikon (Shandon Scientific, London, U.K.) block electrophoresis at pH 4.5 was done on three separate occasions as described elsewhere 9. RESULTS Five saliva samples from three subjects had identical elution patterns on Biochim. Biophys. dcta, 263 (1972) 747-752
749
SALIVA AND TEAR VITAMIN B12 BINDERS
Sephadex G-200 gel filtration, whether excess or subsaturating amounts of [57Colvitamin B12 were used. The binder peak coincided with serum third binder and transcobalamin I peaks and with leucocyte binder peak, and eluted before serum albumin (Fig. I). Saliva filtered without added [SvCo]vitamin B12 behaved identically, though about half the vitamin B]2-binding protein was lost or destroyed during filtration (Fig. I). Filtration through Sephadex G-75 resulted in identical elution relationships, but the R binder and transcobalamin I peak always eluted two tubes after the void fraction.
"--"
A
(0.1ml)
B
(0.3ml)
ALBUMIN OEXTRAN (2xl06mw) TC I; " • THIRD BINt]ER TC II
VITAMIN B12
m
o
3~
l
I
'
i i
i ;
;
I
i
/ Io
tube
7O
8O
9~)
Fig. I. S e p h a d e x G-200 gel filtration of saliva. (A) Saliva filtered after s a t u r a t i o n w i t h [57Colv i t a m i n B12 ; (B) saliva filtered before a d d i n g [57Co~vitanlin B12. T e a r a n d l e u c o c y t e b i n d e r p e a k s coincided w i t h t h e t r a n s c o b a l a m i n I - t h i r d b i n d e r peak. T h e p e a k a t T u b e 7 1 is u n b o u n d [57Co]vitamin B12. TC = t r a n s c o b a l a m i n ; m w = molecular weight.
Assuming a partial specific volume (~) of o.725 cm 3. g-1 (ref. 16), saliva binder, serum third binder and transcobalamin I had a mean sedimentation coefficient of 4.6 =L 0.3 (S.D.) when compared with the standards. Using these figures, calculations as outlined by Siegel and Monty 17 give an estimated molecular weight of 96 ooo for the three binders. However, assumed ~7may be substantially incorrect. Electrophoresis at pH 8.6 showed varying patterns. Specimens from all subjects had fl-globulin mobility or just a bit faster. However, a second sample from one of the subjects showed a wide band of radioactivity encompassing a2- and fl-globulin regions, whereas a second from another subject had a2-globulin mobility. In neither case was there significant quantitative change in vitamin B12 binding. At pH 4.5, binder migration of two saliva specimens was minimal, resembling Biochim. Biophys. Acta, 263 (1972) 747-752
750
R. CARMEL
serum third binder and leucocyte binder, but not transcobalamin I (Fig. 2). Once in three electrophoreses, both salivas had a small cathodal "shoulder" in the same region as transcobalamin II, though the bulk of radioactivity remained at the origin. ..... -
Saliva
|
sooo
i Tears Tc
T/ It::
i
T¢ Ii:
i
/
r~
u
T
. ..d"
i, i ;J jE
"-C
0
A
~
Fig. 2. P e v i k o n b l o c k e l e c t r o p h o r e s i s a t p H 4.5. E a c h p o i n t r e p r e s e n t s a i - c m section. C = c a t h o d e ; O = o rig in; A ~ anode. M i g r a t i o n of e a c h of t h e t h r e e s e r u m b i n d e r s is i n d i c a t e d ( t r a n s c o b a l a m i n II, R a n d t r a n s c o b a l a m i n I); l e u c o c y t e b i n d e r p e a k c oi nc i de d w i t h t h a t of s a l i v a i.
Tear binder was identical to saliva binder on gel filtration. However, electrophoresis at pH 8.6 showed diffuse al-a2 globulin mobility, and at p H 4.5 showed anodal migration intermediate between that of third binder (and leucocyte and saliva binders) and that of transcobalamin I (Fig. 2). DISCUSSION
Filtration characteristics of proteins on gel reflect molecular radius rather than molecular weight, and estimates of the latter from gel filtration m a y be incorrect 17. Overestimation m a y occur particularly with the less globular glycoproteins is, such as vitamin B12 binders. Setting this important consideration aside, gel filtration results correspond to an estimated weight of 12o ooo for all R binders and transcobalamin I (refs 2, 8, Io, ~3 and I9). Using a diffusion cell technique, Gr~tsbeck4 reported such a figure for saliva binder. However, he subsequently calculated from gel filtration and ultracentrifugation data a molecular weight of 60 ooo for the protein a, making it unique among all R Biochim. Biophys. /Icta, 263 (1972) 747 752
SALIVA AND TEAR VITAMIN
B12 BINDERS
751
binders tested in his laboratory z. Hurlimann and Zuber 6, using gel filtration alone, reported a weight of 60 ooo also. However, they also found leucocytic, gastric and serum vitamin B12 binders to behave thus (and found only one serum binder molecular size), all eluting with or after albumin, which is at variance with all other reportsS,10-13,19-~l.
The present data show that saliva binder is identical in molecular size and sedimentation characteristics to other R binders and transcobalamin I. Sucrose density gradient ultracentrifugation of saliva binder and transcobalamin I here gave a mean sedimentation coefficient of 4.6, compared with a value of 3.5 given the former by Gr~isbeck and Visuri 5. These values, however, and calculation of molecular weight from them, assume a ~ which m a y be incorrect. Therefore, the exact molecular weight of all vitamin B12 binders still awaits definition, but appears to be smaller than current estimates, a fact also suggested here and previously 2° by their slight retardation on Sephadex G-75 gel. The reason for the discrepancy with the previous studies of saliva binder size by gel filtration is unclear. The protein m a y exist as a dimer, as m a y indeed other R binders. However, saliva binder elution was identical with and without [57Co]vitamin BI2, indicating that this binding process, unlike vitamin B12 binding by intrinsic factor S, does not itself cause dimerization. Alternatively, the extensive preparatory manipulation and chromatographic purification in the previous studiesS, 6 m a y have altered the protein in size (as noted with transcobalamin I I b y Puutula and Gritsbeck 21) or in shape. Vitamin B12 binders are susceptible fo alteration s, perhaps more so when not protected by being bound to the vitaminS2, z3 or when exposed to very dilute solutions s, both of which occurred in one of the studies s. Therefore, manipulation of samples was kept to a minimum in the present study. In several instances, testing was done directly within minutes of collection. While saliva binder behaves as a single protein in molecular size, binding curve 24, and immunodiffusion studies *v, some heterogeneity obviously existsLS,6. Charge variation m a y explain why the bulk of saliva binder, unlike gastric R binder, was eluted with the a-globulin fraction on rapid DEAE-cellulose chromatography ~*. Particularly noteworthy were the different electrophoretic patterns on different days in the same subject here, suggesting that the differences are due to acquired change, rather than to different genetic forms. Heterogeneity is not confined to saliva binder, and is demonstrable in ]eucocyte binder s and tear binder. The latter had been found to have predominantly a2-globulin mobility 14 but here migrated in a broad band covering the a l - a 2 globulin area. Its lower isoelectric point was further indicated by electrophoresis at p H 4-5 (Fig. 2). It thus appears that R binders from different sources share all known characteristics save minor variations in charge, which, as has been suggested 1,s m a y result from different sialic acid content. Transcobalamin I m a y be a more marked example of this variation, which m a y even play a role in its carrying endogenous vitamin B12. However, the evidence indicates that serum third binder, not transcobalamin I, is the R binder in serum. ACKNOWLEDGMENT
This study was supported by a Fellowship grant from the Wellcome Trust. Biochim. Biophys. Acta, 263 (1972) 747-752
752
6. CARMEL
REFERENCES I 2 3 4 5 6 7 8 9 IO Ii 12 13 I4 15 16 17 18 19 20 21
22 23 24 25
K. Simons, Soe. Sci. Fenn. Comment. Biol., 27 (1964) Suppl. 5. R. Gr/~sbeck, Scand. J. Clin. Lab. lnvest., 19, Suppl. 95 (1967) 7. R. Gr~isbeck, K. S i m o n s a n d I. S i n k k o n e n , Ann. Med. Exp. Fenn., 4 ° (1962) Suppl. 6. R. Griisbeck, Acta Med. Scand. Suppl., 314 (1956) i. R. GrAsbeck a n d K. Visuri, Seand. J. Clin. Lab. Invest. Suppl., IOI (1968) 13. J. H u r l i m a n n a n d C. Zuber, Clin. Exp. Immunol., 4 (1969) 125. K. Simons, Prog. Gastroent., i (1968) 195. U - H . S t e n m a n , K. S i m o n s a n d R. Gr~sbeck, Scand. J. Clin. Lab. Invest., 21 (1968) 202. R. Carmel a n d V. H e r b e r t , Blood, in t h e press. C. A. Hall a n d A. E. Finkler, J. Lab. Clin. Med., 73 (1969) 6o. C. Lawrence, Blood, 33 (1969) 899. E. J. Gizis, M. F. Dietrich, G. Choi a n d L. M. Meyer, J. Lab. Clin. Med., 75 (197 °) 673R. Carmel, Br. J. Haematol., 22 (1972) 53. R. Gr/isbeck a n d I-T. T a k k i - L u u k k a i n e n , Aeta Ophthalmol., 36 (1958) 860. R. Carmel a n d C. A. C o l t m a n , Jr, J. Lab. Clin. Med., 74 (1969) 967. R. G. M a r t i n a n d B. N. A m e s , J. Biol. Chem., 236 (1961) 1372. L. M. Siegel a n d K. J. M o n t y , Bioehim. Biophys. Acta, 112 (1966) 346. P. A n d r e w s , Biochem. J., 91 (1964) 222. ]3. Horn a n d H. Olesen, Scand. J. Clin. Lab. Invest., 19 (1967) 69. B. L. Horn, Biochim. Biophys. Mcta, 175 (1969) 20. A. E. Finkler, P. D. Green a n d C. A. Hall, Bioehim. Biophys. Aeta, 200 (197 o) 151. F. P. Retief, C. V~r. Gottlieb, S. K o c h w a , P. W. P r a t t a n d V. H e r b e r t , Blood, 29 (1967) 5Ol. M. E. G r e g o r y a n d E. S. H o l d s w o r t h , Bioehem. J., 72 (1959) 549. R. w . Bertcher, L. M. Meyer a n d I. F. Miller, Proc. Soc. Exp. Biol. Med., 99 (1958) 513 • K. Simons, T. Weber, M. Stiel a n d R. Gr/~sbeck, Acta Med. Scand. Suppl., 412 (1964) 257.
Bioehim. Biophys. Aeta, 263 (1972) 747-752
747
BBA 36091 T H E V I T A M I N B12-BINDING P R O T E I N S OF SALIVA AND T E A R S AND T H E I R R E L A T I O N S H I P TO O T H E R VITAMIN B12 B I N D E R S
RALPH CARMEL MRC Experimental Haematology Unit, St. Mary's Hospital Medical School, London (Great Britain)
(Received December 6th, 1971)
SUMMARY A study of the vitamin B12 binder in saliva was prompted by different reported molecular weights, some suggesting it was unique among the otherwise identical group of vitamin B1,z binders called R binders. Tears, extremely rich in this protein, were also examined. Gel filtration and sucrose density gradient ultracentrifugation showed saliva binder to be identical in size with other R binders, serum third binder and transcobalamin I. The discrepancy with previous reports is unexplained. It is apparent that vitamin BI~ binders are anomalous proteins, and past estimates of molecular weight m a y be incorrect. Saliva binder resembled leucocyte binder and serum third binder, but not transcobalamin I, in electrophoretic mobility. However, some heterogeneity was obvious, even among different specimens from the same subject. Tear binder was intermediate between R binder and transcobalamin I on electrophoresis. These results support identity of all the R binders except for some heterogeneity, probably acquired, in isoelectric points. It also appears that third binder is the R binder in serum.
INTRODUCTION A single type of vitamin Bi2-binding protein (as determined by immunologic and chromatographic behaviour and molecular sizing) appears in most of the body fluids of man and in leucocytes and erythrocytesl, ~, and is called " R binder"l, 3. The sole exception in the similarity of all R binders to each other has been saliva binder, for which different molecular weights have been reported 4-n. Although R binder is present in serum a, it is uncertain which serum binder corresponds to it due to the variability of separation techniques. The non-identity of R binder with transcobalamin I I has been summarized 7. Transcobalamin I differs from R binder electrophoreticallyS, 9, and serum R binder does not carry endogenous vitamin B12 (ref. I). The recently described third serum binder 1°-13, however, resembles R binder in all tested characteristics1°, 13. Biochim. Biophys. Acta, 263 (1972) 747-752
748
R. CARMEL
The present study was undertaken to further examine saliva binder and its possible resemblance to third binder of serum, and to determine whether it indeed differs in molecular size from other R binders. Tear vitamin Bl~-binding protein was similarly studied. Tears have exceptionally high vitamin B~2-binding capacity, yet the binder has not been characterized apart from determination of electrophoretic mobility at p H 8.6 (ref. 14). MATERIALS AND METHODS Saliva specimens were collected by unstimulated spitting from four fasting, normal subjects and one fasting subject with pernicious anaemia in remission. Spontaneous tears were collected by eye-dropper from two healthy children and pooled. Samples were centrifuged to remove debris, but were otherwise untreated. Testing was done immediately or after storage at --20 °C, with no difference in results. Polycythaemia vera serum was used as the source of third binder. Sephadex G-2oo gel (Pharmacia Fine Chemicals, Uppsala, Sweden) filtration was done at room temperature in 0. 9 cm × 60 cm columns, using I M NaCI-o.I M Tris buffer (pH 8.5) containing 0.02% NaN 3. Samples of O.l-O.3 ml were incubated with excess E~7Colvitamin Blo~ (The Radiochemical Centre, Amersham) specific activity of 15.2/~Ci//~g, for 30 min at room temperature prior to application. Aliquots of IO drops were collected b y gravity. Radioactivity was counted in a well-type scintillation counter. Sephadex G-75 gel filtration was similarly performed, using the above buffer or o.I M sodium phosphate buffer (pH 6.3). Both gel columns had been calibrated with high-molecular weight Blue Dextran, E57Colvitamin B12-1abelled leucocyte extract 9, and various E57Colvitamin B12-1abelled human sera. Protein concentration was determined at 250 nm during flow. Three of the saliva specimens were filtered through Sephadex G-2oo or G-75 without added E57Co]vitamin B12. Filtrate tubes were then each incubated with I ng EsvColvitamin B~2 for 30 rain at room temperature, and the unbound E57Co]vitamin B12 was removed with haemoglobin- and plasma-coated charcoaP 5 prior to counting. Linear sucrose density gradient ultracentrifugation of diluted saliva and serum, labelled with E57Co]vitamin B12, specific activity of 118 #Ci//~g, was done as described b y Martin and Ames ~6, spinning 17. 5 h at 38 ooo rev./min with a 3 × 6.5 swing-out rotor in a Superspeed 65 centrifuge (Measuring and Scientific Equipment, London) at 7 °C. Transferrin, IgG and human serum albumin, all labelled with ~2~i, were used as internal standards. Cellulose acetate electrophoresis was done at pH 8.6 on specimens previously incubated with EaVColvitamin BI~, specific activity of 118/,Ci/#g. Serum labelled with 59Fe was used for comparison. The unstained strips were incubated for 3 weeks with no-grid X-ray film, and stained subsequently for comparison with the radioautographs. Pevikon (Shandon Scientific, London, U.K.) block electrophoresis at pH 4.5 was done on three separate occasions as described elsewhere 9. RESULTS Five saliva samples from three subjects had identical elution patterns on Biochim. Biophys. dcta, 263 (1972) 747-752
749
SALIVA AND TEAR VITAMIN B12 BINDERS
Sephadex G-200 gel filtration, whether excess or subsaturating amounts of [57Colvitamin B12 were used. The binder peak coincided with serum third binder and transcobalamin I peaks and with leucocyte binder peak, and eluted before serum albumin (Fig. I). Saliva filtered without added [SvCo]vitamin B12 behaved identically, though about half the vitamin B]2-binding protein was lost or destroyed during filtration (Fig. I). Filtration through Sephadex G-75 resulted in identical elution relationships, but the R binder and transcobalamin I peak always eluted two tubes after the void fraction.
"--"
A
(0.1ml)
B
(0.3ml)
ALBUMIN OEXTRAN (2xl06mw) TC I; " • THIRD BINt]ER TC II
VITAMIN B12
m
o
3~
l
I
'
i i
i ;
;
I
i
/ Io
tube
7O
8O
9~)
Fig. I. S e p h a d e x G-200 gel filtration of saliva. (A) Saliva filtered after s a t u r a t i o n w i t h [57Colv i t a m i n B12 ; (B) saliva filtered before a d d i n g [57Co~vitanlin B12. T e a r a n d l e u c o c y t e b i n d e r p e a k s coincided w i t h t h e t r a n s c o b a l a m i n I - t h i r d b i n d e r peak. T h e p e a k a t T u b e 7 1 is u n b o u n d [57Co]vitamin B12. TC = t r a n s c o b a l a m i n ; m w = molecular weight.
Assuming a partial specific volume (~) of o.725 cm 3. g-1 (ref. 16), saliva binder, serum third binder and transcobalamin I had a mean sedimentation coefficient of 4.6 =L 0.3 (S.D.) when compared with the standards. Using these figures, calculations as outlined by Siegel and Monty 17 give an estimated molecular weight of 96 ooo for the three binders. However, assumed ~7may be substantially incorrect. Electrophoresis at pH 8.6 showed varying patterns. Specimens from all subjects had fl-globulin mobility or just a bit faster. However, a second sample from one of the subjects showed a wide band of radioactivity encompassing a2- and fl-globulin regions, whereas a second from another subject had a2-globulin mobility. In neither case was there significant quantitative change in vitamin B12 binding. At pH 4.5, binder migration of two saliva specimens was minimal, resembling Biochim. Biophys. Acta, 263 (1972) 747-752
750
R. CARMEL
serum third binder and leucocyte binder, but not transcobalamin I (Fig. 2). Once in three electrophoreses, both salivas had a small cathodal "shoulder" in the same region as transcobalamin II, though the bulk of radioactivity remained at the origin. ..... -
Saliva
|
sooo
i Tears Tc
T/ It::
i
T¢ Ii:
i
/
r~
u
T
. ..d"
i, i ;J jE
"-C
0
A
~
Fig. 2. P e v i k o n b l o c k e l e c t r o p h o r e s i s a t p H 4.5. E a c h p o i n t r e p r e s e n t s a i - c m section. C = c a t h o d e ; O = o rig in; A ~ anode. M i g r a t i o n of e a c h of t h e t h r e e s e r u m b i n d e r s is i n d i c a t e d ( t r a n s c o b a l a m i n II, R a n d t r a n s c o b a l a m i n I); l e u c o c y t e b i n d e r p e a k c oi nc i de d w i t h t h a t of s a l i v a i.
Tear binder was identical to saliva binder on gel filtration. However, electrophoresis at pH 8.6 showed diffuse al-a2 globulin mobility, and at p H 4.5 showed anodal migration intermediate between that of third binder (and leucocyte and saliva binders) and that of transcobalamin I (Fig. 2). DISCUSSION
Filtration characteristics of proteins on gel reflect molecular radius rather than molecular weight, and estimates of the latter from gel filtration m a y be incorrect 17. Overestimation m a y occur particularly with the less globular glycoproteins is, such as vitamin B12 binders. Setting this important consideration aside, gel filtration results correspond to an estimated weight of 12o ooo for all R binders and transcobalamin I (refs 2, 8, Io, ~3 and I9). Using a diffusion cell technique, Gr~tsbeck4 reported such a figure for saliva binder. However, he subsequently calculated from gel filtration and ultracentrifugation data a molecular weight of 60 ooo for the protein a, making it unique among all R Biochim. Biophys. /Icta, 263 (1972) 747 752
SALIVA AND TEAR VITAMIN
B12 BINDERS
751
binders tested in his laboratory z. Hurlimann and Zuber 6, using gel filtration alone, reported a weight of 60 ooo also. However, they also found leucocytic, gastric and serum vitamin B12 binders to behave thus (and found only one serum binder molecular size), all eluting with or after albumin, which is at variance with all other reportsS,10-13,19-~l.
The present data show that saliva binder is identical in molecular size and sedimentation characteristics to other R binders and transcobalamin I. Sucrose density gradient ultracentrifugation of saliva binder and transcobalamin I here gave a mean sedimentation coefficient of 4.6, compared with a value of 3.5 given the former by Gr~isbeck and Visuri 5. These values, however, and calculation of molecular weight from them, assume a ~ which m a y be incorrect. Therefore, the exact molecular weight of all vitamin B12 binders still awaits definition, but appears to be smaller than current estimates, a fact also suggested here and previously 2° by their slight retardation on Sephadex G-75 gel. The reason for the discrepancy with the previous studies of saliva binder size by gel filtration is unclear. The protein m a y exist as a dimer, as m a y indeed other R binders. However, saliva binder elution was identical with and without [57Co]vitamin BI2, indicating that this binding process, unlike vitamin B12 binding by intrinsic factor S, does not itself cause dimerization. Alternatively, the extensive preparatory manipulation and chromatographic purification in the previous studiesS, 6 m a y have altered the protein in size (as noted with transcobalamin I I b y Puutula and Gritsbeck 21) or in shape. Vitamin B12 binders are susceptible fo alteration s, perhaps more so when not protected by being bound to the vitaminS2, z3 or when exposed to very dilute solutions s, both of which occurred in one of the studies s. Therefore, manipulation of samples was kept to a minimum in the present study. In several instances, testing was done directly within minutes of collection. While saliva binder behaves as a single protein in molecular size, binding curve 24, and immunodiffusion studies *v, some heterogeneity obviously existsLS,6. Charge variation m a y explain why the bulk of saliva binder, unlike gastric R binder, was eluted with the a-globulin fraction on rapid DEAE-cellulose chromatography ~*. Particularly noteworthy were the different electrophoretic patterns on different days in the same subject here, suggesting that the differences are due to acquired change, rather than to different genetic forms. Heterogeneity is not confined to saliva binder, and is demonstrable in ]eucocyte binder s and tear binder. The latter had been found to have predominantly a2-globulin mobility 14 but here migrated in a broad band covering the a l - a 2 globulin area. Its lower isoelectric point was further indicated by electrophoresis at p H 4-5 (Fig. 2). It thus appears that R binders from different sources share all known characteristics save minor variations in charge, which, as has been suggested 1,s m a y result from different sialic acid content. Transcobalamin I m a y be a more marked example of this variation, which m a y even play a role in its carrying endogenous vitamin B12. However, the evidence indicates that serum third binder, not transcobalamin I, is the R binder in serum. ACKNOWLEDGMENT
This study was supported by a Fellowship grant from the Wellcome Trust. Biochim. Biophys. Acta, 263 (1972) 747-752
752
6. CARMEL
REFERENCES I 2 3 4 5 6 7 8 9 IO Ii 12 13 I4 15 16 17 18 19 20 21
22 23 24 25
K. Simons, Soe. Sci. Fenn. Comment. Biol., 27 (1964) Suppl. 5. R. Gr/~sbeck, Scand. J. Clin. Lab. lnvest., 19, Suppl. 95 (1967) 7. R. Gr~isbeck, K. S i m o n s a n d I. S i n k k o n e n , Ann. Med. Exp. Fenn., 4 ° (1962) Suppl. 6. R. Griisbeck, Acta Med. Scand. Suppl., 314 (1956) i. R. GrAsbeck a n d K. Visuri, Seand. J. Clin. Lab. Invest. Suppl., IOI (1968) 13. J. H u r l i m a n n a n d C. Zuber, Clin. Exp. Immunol., 4 (1969) 125. K. Simons, Prog. Gastroent., i (1968) 195. U - H . S t e n m a n , K. S i m o n s a n d R. Gr~sbeck, Scand. J. Clin. Lab. Invest., 21 (1968) 202. R. Carmel a n d V. H e r b e r t , Blood, in t h e press. C. A. Hall a n d A. E. Finkler, J. Lab. Clin. Med., 73 (1969) 6o. C. Lawrence, Blood, 33 (1969) 899. E. J. Gizis, M. F. Dietrich, G. Choi a n d L. M. Meyer, J. Lab. Clin. Med., 75 (197 °) 673R. Carmel, Br. J. Haematol., 22 (1972) 53. R. Gr/isbeck a n d I-T. T a k k i - L u u k k a i n e n , Aeta Ophthalmol., 36 (1958) 860. R. Carmel a n d C. A. C o l t m a n , Jr, J. Lab. Clin. Med., 74 (1969) 967. R. G. M a r t i n a n d B. N. A m e s , J. Biol. Chem., 236 (1961) 1372. L. M. Siegel a n d K. J. M o n t y , Bioehim. Biophys. Acta, 112 (1966) 346. P. A n d r e w s , Biochem. J., 91 (1964) 222. ]3. Horn a n d H. Olesen, Scand. J. Clin. Lab. Invest., 19 (1967) 69. B. L. Horn, Biochim. Biophys. Mcta, 175 (1969) 20. A. E. Finkler, P. D. Green a n d C. A. Hall, Bioehim. Biophys. Aeta, 200 (197 o) 151. F. P. Retief, C. V~r. Gottlieb, S. K o c h w a , P. W. P r a t t a n d V. H e r b e r t , Blood, 29 (1967) 5Ol. M. E. G r e g o r y a n d E. S. H o l d s w o r t h , Bioehem. J., 72 (1959) 549. R. w . Bertcher, L. M. Meyer a n d I. F. Miller, Proc. Soc. Exp. Biol. Med., 99 (1958) 513 • K. Simons, T. Weber, M. Stiel a n d R. Gr/~sbeck, Acta Med. Scand. Suppl., 412 (1964) 257.
Bioehim. Biophys. Aeta, 263 (1972) 747-752