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Studies on certain peptide fractions isolated from human dentine
Archs
oral
Bid.
Vol.
14, pp. 503-511,
1969. Pergamon
Press.
Printed
in Gt. Britain.
STUDIES ON CERTAIN PEPTIDE FRACTIONS FROM HUMAN DENTINE
ISOLATED
A. G. LEAVER Department
of Dental Sciences, School of Dental Surgery, University of Liverpool, England
Summary-The acid-soluble nitrogenous components of human dentine were fractionated by gel filtration and ion-exchange chromatography. The fraction believed to be in the molecular weight range 750-5OU0, but later found to contain some free amino acids, comprized approximately O-08 per cent of dentine and contained 20 per cent glycine, 25 per cent dicarboxylic amino acids and 15 per cent basic amino acids which included ornithine. Highvoltage paper electrophoresis was used to obtain nine fractions which were then analysed. Five of the peptide fractions were predominantly acidic and were also rich in hydroxy amino acids, while three, though containing dicarboxylic amino acids, were richer in basic amino acids. One highly basic fraction was also found. The peptide fractions were considered to be comparable to fractions previously isolated from ox bone and, although clearly not fragments of the main tropocollagen chains, were thought likely to be either associated with collagen at physiological pH or possibly to have been originally associated with glycoprotein or mucopolysaccharide.
THE PRESENCE of acid-soluble peptides in human dentine was reported by LEAVER, EASTOEand HARTLES(1960). It was then considered that the peptides were associated with the citric acid of the tissue. That such an association, in the case of similar material isolated from ox bone, was probably fortuitous was demonstrated by LEAVER, SHUTTLEWORTH and TRIFFITT (1965) who separated citric acid, peptides, glycoproteins and traces of soluble collagen by gel filtration and ion-exchange chromatography. LEAVERand SHUTTLEWORTH (1967) carried out an extensive study of the acid-soluble nitrogen (ASN) of bone and dentine from which it was concluded that from 3-5 per cent of the total nitrogen of bone and dentine went into solution in 1 N HCl at 2”C, that from 0.1 to O-25 of such ASN consisted of a mixture of peptides of molecular weight < 5000 and that comparable amounts of apparently similar peptides were released from these hard tissues by demineralization with EDTA at neutral pH. A previous study (LEAVERand SHUTTLEWORTH, 1966) had given qualitative information concerning the nature of the peptides from bone and dentine. Both tissues contained peptides rich in acidic and/or basic amino acids usually with substantial amounts of glycine and serine. Organic phosphate (presumably attached to the serine residues) was present in several fractions. A series of quantitative investigations, involving most of the sub-fractions of bone ASN, has recently been reported, (LEAVERand SHUTTLEWORTH,1968). One major aspect of this work was the use of preparative high-voltage paper electrophoresis to separate individual components of the total peptide fraction in the 750-5000 molecular 503
504
A. G. LEAVER
weight range. Twenty-one such sub-fractions, which appeared to be electrophoretically homogeneous, were hydrolysed and their amino acid composition determined on the Technicon autoanalyser. The results indicated that the sub-fractions were either mixtures of very closely related peptides or fractions in which one peptide predominated but was contaminated by traces of others. Nevertheless the results were of considerable interest as they revealed the presence of peptides of widely varying amino acid composition including very basic, strongly acidic, neutral non-polar (rich in glycine and serine) and “amphoteric” peptides containing both basic and dicarboxylic amino acids in similar proportions. The absence of hydroxyproline and the widely varying levels of glycine indicated that they could not have been derived from the main chains of the tropocollagen molecule. It was, however, considered possible that they might be attached to collagen at physiological pH and released at pH extremes, as was postulated by STEVENand TRISTRAM(1962) in the case of rather similar peptides isolated from calf skin collagen. Whereas the investigations into the bone ASN fractions were very extensive, it was decided to restrict the present study of dentine ASN to the peptide fraction of molecular weight 750-5000, to isolate sub-fractions by high voltage electrophoresis and to compare their amino acid composition with that of the corresponding fractions previously isolated from ox bone (LEAVERand SHUTTLEWORTH, 1968).
EXPERIMENTAL The acid-soluble nitrogenous fraction (ASN) of human dentine was isolated from teeth collected from the extraction departments of the Liverpool Dental Hospital. The preparation of material and the method of demineralization were precisely the same as previously described (LEAVERand SHUTTLEWORTH, 1967). The direct fractionation of the ASN described in this earlier work was preferred to the unnecessarily complex scheme used in the investigations of bone ASN. It is summarized in Fig. 1. The present study was confined to the peptide fraction 2Bl (that excluded by Sephadex G-10 after having been retarded by Sephadex G-25). It was found, however, that this fraction occupied a larger elution volume than had been found in the case of bone and it was considered likely to include both excluded and retarded material. The retarded fraction 2B2 was correspondingly smaller and was discarded. After evaporating fraction 2Bl to dryness in vucuo, it was dissolved in 2 .O ml of water, 0.4 ml of the resulting solution being retained for analysis of nitrogen, phosphorus and amino acids. Separation of peptide fractions from 2Bl by preparative high-voltage paper electrophoresis
In the separation of the bone peptide fractions two successive runs at pH 5.4 and pH 2.0 were carried out. Despite this, amino acid analysis indicated that no absolutely separate entity had been obtained. In view of this only the run at low pH was carried out in the present work, although the electrophoretic conditions were altered with a view to achieving improved separation. Fraction 2Bl was separated into sub-fractions by high-voltage electrophoresis on
STUDIES
ON
CERTAIN
PEPTIDE
FRACTIONS
ISOLATED
FROM
HUMAN
DENTINE
505
Total ASN 1 Amberliie
in 0.5N
HCI
IR-120
I
re-applied in aqueous solution
AKH‘$
I
/Tji[;“.ctio”
Aqueous eluate “Ammonia eluate (discarded) 2” combined with “Ammonia eluate I”
(discarded) I Sephadex G-IO /l Excluded Retarded fraction 281 fraction 282 (discarded) I H.KE.
FIG.
1.
Fractionation
of dentine ASN.
Whatman 3MM paper (78 x 10 cm) using a Locarte apparatus. A pH 1.98 buffer (O-58 M formic acid/O*21 M acetic acid) was used and 4 kV applied to the paper for 2 hr. In each run two strips loaded with 0.1 ml of fraction 2Bl were separated by a third unloaded strip. The three strips were placed on top of each other so that the central unloaded strip adsorbed peptide material during the run and acted as a marker. This was later stained with ninhydrin after drying and the areas of the loaded strips corresponding to the ninhydrin-positive bands cut out and eluted with distilled water overnight. Eight such runs were required to deal with the 1.6 ml of solution. In each run 10 sub-fractions were obtained and combined according to the distances run, which under the electrophoretic conditions used, varied from 18 * 1 to 18 -9 cm for fraction A to 48 a5-49 a9 cm for fraction J. Fraction H was rejected and the nine remaining fractions subjected to amino acid analysis. Amino acid analysis of hydrolysed and unhydrolysed fractions In the previous work on bone, peptide fractions retarded on Sephadex G-10 were found to contain both free and peptide bound amino acids, the former comprising 15.91 per cent and the latter 84.09 per cent of the total retarded material. The possibility that the excluded fraction might contain some free amino acids was not considered. In the present study, the excluded fraction 2Bl was somewhat disperse; hence each fraction was examined for free and peptide bound amino acids. One half of each fraction was hydrolysed in 6~ HCl in a sealed evacuated tube at 105°C for 24 hr, taken to dryness in a vacuum desiccator and finally dissolved in 1 ml of nor leucine solution. The remaining half of each fraction was evaporated without further treatment and re-dissolved in 1 ml of nor leucine solution. Each hydrolysed and unhydrolysed fraction was then individually analysed on the Technicon autoanalyser. One ml of the
A. G. LEAVER
506
final solution in nor leucine was applied to the resin column, carried out using the normal microbead
ion-exchange
Technicon
automatic
analysis being
20 hr run on a 150 cm column
resin. In all hydrolysate
chromatograms
of Type A
the amino
acid
levels were calculated on the basis of a standard amino acid mixture separated on the autoanalyser under identical conditions.
Analysis of the total fraction 2B1 One fifth of the total material in fraction 2Bl was set aside for analysis of nitrogen and phosphorus and for the determination of the overall amino acid composition. Nitrogen was determined by the ninhydrin method of JACOBS(1962) and phosphorus by the method of BERENBLUMand CHAIN (1938). The amino acid composition of the fraction
was determined
before
and after hydrolysis
as described
for the individual
separated fractions. RESULTS
Composition of the total fraction 281 Fraction
2Bl contained
9.24 mg of nitrogen and 0.67
mg of phosphorus.
related to the initial weight of dentine (66 g) the nitrogen content is 0.014 which is rather less than that previously
When per cent
isolated from dentine under similar conditions
(LEAVER and SHUTTLEWORTH,1967). It suggests a peptide content of about O-08 per cent of total tissue or 0.4 per cent of matrix. The ratio of nitrogen to phosphorus is approximately 14:l and is very closely similar to that found in the comparable tions from bone (LEAVER and SHUTTLEWORTH,1968). TABLE1. AMINOACID
COMPOSITION
OF TOTAL
FRACTION
2Bl
(EXPRESSED AS
frac-
MOLES PERCENT) -
Amino acid Hydroxyproline Aspartic Acid Serine Threonine Glutamic Acid Proline Glycine Alanine Cystine Valine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Ornithine Lysine Histidine Arginine
2Bl (peptide bound) 15.35 11.01 3.19 10.22 2.06 20.41 9.56 1.08 2.51 trace 1.06 5.59 2.32 trace 3.94 4.87 2.55 4.29
2Bl (free)
23.36 20.42 trace 18.42 20.02 8.86 trace -
4.88 4.04 -
STUDIES
ON
CERTAIN
PEPTIDE
FRACTIONS
ISOLATED
FROM HUMAN DENTINE
507
The fraction was found to consist of 75 *35 per cent peptide-bound and 24.65 per cent free amino acids. The percentage composition of the peptide-bound and free amino acids is given in Table 1. The overall composition of the bound amino acids in the fraction is quite comparable to that of the corresponding fraction found in ox bone (LEAVER and SHUTTLEWORTH, 1968). The chief difference is an increase in the level of aspartic acid at the expense of glycine. The amount of dicarboxylic acids is therefore correspondingly greater in the dentine peptide fraction whereas the level of basic amino acids is almost identical. A small amount of cystine was present while only a trace of phenylalanine was found. This is in contrast to the bone peptides which contained no cystine and in which the level of phenylalanine exceeded that of tyrosine. The presence of about 25 per cent of free amino acids was clearly due to the extended elution volume of this fraction 2B1. As the present study was confined to the peptide fraction no attempt was made to study the retarded fraction 2B2 which was, in any case, minimal. The determination of free amino acids, both in this total unseparated fraction and in the fractions resolved by electrophoresis, was carried out merely to ensure the precise determination of the composition of the true peptide fractions which was obtained by difference of the total and free amino acids. Amino acid composition of the fractions obtained by high-voltage electrophoresis of fraction 2Bl All of the nine fractions analysed were found to contain a proportion of free amino acids which varied from approximately 2-40 per cent. In each case a single amino acid accounted for at least two-thirds of those present in the free state. The percentage composition of peptide-bound amino acids occurring in the nine fractions is shown in Table 2. Fractions A, B, C and D fall into an obvious group. All are predominantly acidic and are also rich in the hydroxy amino acids. The glycine level is almost constant as is the total of alanine and leucine. A and B are unusual in containing a substantial proportion of tyrosine. Fraction E is again predominantly acidic but does TABLE 2. AMINO ACID COMPOSITIONOF SEPARATEDPEPTIDEFRACTIONS(EXPRESSEDAS MOLES PER CENT) Amino
acid
Asparatic Acid Threonine Serine Glutamatic Acid Glycine Alanine Valine Iso-leucine Leucine Tyrosine Ornithine Lysine Histidine Arginine
A
B
C
D
E
F
18.55 5.50 11.93 15.90 20.80 14.07
12.50 5.36 14.92 16.20 21.43 10.71 -
18.05 5.66 23.89 13.36 20.09 12.92 -
13.85 14.98 16.35 11.24 21.00 9.65 -
13.80 4.37 15.22 10.82 19*77 11.11 10.21 -
8.40 4.98 10.19 9.80 16.02 21.70 -
6.12 7.14 -
10.97 7.91
6.02 -
12.94 -
6.62 4.11 3.93 -
4.20 2.33 7.15 7.15 8.09
-
G
I
J
4.55 2.87 11.00 5.98 14.11 9.33 -
-
6.50 3.38 9.75 15.50 10.63 7.63 3.38 2.13 5.13 -
4.80 6.24 4.68 8.64
15.00 8.38 6.00 6.63
17.22 24.12 3.83 6.98
5.34 4.20 8.16 8.28 42.24 7.48 -
-
-
508
A. G. LEAVER
contain some basic amino acids. It is also notable for its high valine content. Fractions F and G fall into the category of “amphoteric” peptides in which both dicarboxylic and basic amino acids are present, although the latter are present in greater amounts. Peptide fraction J contains 52 per cent of basic amino acids and is highly basic whereas I, though including 36 per cent of these, contains 22 per cent of dicarboxylic acids and is, therefore, more comparable to F and G. It is also unusual in possessing the full range of aliphatic amino acids. All the fractions containing basic amino acids included ornithine. A final point is that all nine fractions contained an excess of ammonia far greater than could have been derived from the breakdown of amide groups in glutamine and asparagine or from the loss of small proportions of arginine and hydroxyacids during hydrolysis. It appears likely that this was due to incomplete removal of the ammonia used in the elution of the IR-120 column. There is little information available concerning the molecular weight of the peptides although, as they were obtained in association with free amino acids, quite small peptides might be expected to be present. These dentine peptide fractions, although obtained from only a single electrophoretic run, were obtained under better and more stringent conditions than were those isolated from bone and are likely to be no more heterogeneous. They are likely to represent either mixtures of closely related peptides or fractions in which one peptide predominates but is contaminated by traces of others within the group.
DISCUSSION
A major purpose of the study was to compare the nature of the peptide fractions in the molecular weight range 750-5000 with those isolated from bone. As suggested earlier, the presence of free amino acids in the fractions did indicate the possibility that peptides of molecular weight < 750 might also be present. Although only nine peptide fractions were obtained from dentine compared with twenty-one from bone, several overall similarities may be noted. Glycine is consistently the most abundant amino acid, proline and hydroxyproline are totally absent and ornithine is invariably present in fractions containing basic amino acids. The majority of peptides from both tissues are rich in hydroxyacids (serine and threonine). On the other hand the acidic dentine peptides tend to have rather more aspartic acid than glutamatic and two such peptides contain tyrosine which only appeared in highly basic fractions isolated from bone. The composition of the dentine peptides, although showing considerable individual variations, does not reveal the very wide variation between fractions shown in the case of bone. Thus, except for the very high glycine content of G (42.24 per cent), no individual fraction contained more than 25 per cent of any single amino acid, whereas three of the bone fractions contained over 50 per cent of a single amino acid. In the light of the results of the present study it seems possible, though by no means certain, that those bone fractions containing very high proportions of a single amino acid might have included a substantial amount of that amino acid in the free form or in the form of a simple dipeptide. It also seems possible that one fraction containing 43 per cent glycine and 41 per cent alanine may have consisted predominantly of glycylalanine.
STUDIES
ON
CERTAIN
PEPTIDE
FRACTIONS
ISOLATED
FROM
HUMAN
DENTINE
509
With these various reservations in mind, it seems reasonable to conclude that the dentine peptide fractions are essentially similar to those isolated from bone. If this is so, then it is appropriate to consider the peptides in the light of the differences between the two tissues. In the first place, dentine, once laid down, is not subject to the processes of resorption and remodelling, which would tend to support the view that they are unlikely to be involved in calcium binding or transport. As reported recently (LEAVER, 1968), preliminary studies using various bone peptide fractions have revealed little calcium binding ability. Secondly, as the odontoblast layer of the dentine was removed prior to demineralization, it would seem unlikely that the dentine peptides (and, by implication, those of bone) could have been derived from the cellular fraction. A possible association between the mucopolysaccharide or glycoprotein fractions of bone and the peptides was previously suggested. Very little is known of the non-collagenous nitrogen of dentine (EASTOE,1967) although it seems unlikely that any single entity is present at anything like the level of the “mucoprotein II” described by HERRING(1964) which is probably similar to the “mucopolysaccharideprotein complex” described by EASTOEand EASTOE(1954). Any attempt to gain further information concerning a possible relationship between the peptide fractions and the mucopolysaccharide and glycoproteins will have to be confined to bone (and in particular to very young or embryonic bone) until more information is available concerning the minor organic fractions of dentine. The most positive suggestion made by LEAVERand SHUTTLEWORTH (1968) was that the bone peptides might be closely related to the fractions isolated from acid soluble calf-skin collagen by STEVENand TRISTRAM(1962). These workers considered that the peptides were associated firmly with collagen at physiological pH and released at pH extremes. LEAVERand SHUTTLEWORTH (1968) suggested that a regular arrangement of peptides, bound to collagen at physiological pH and labile to pH changes, might have a function in the mineralization process. Dentine collagen, however, is unusual in its very high content of organic phosphate which has been shown to be due in the main to the presence of a phosphoprotein linked to collagen through an oligosaccharide and hydroxylysine (VEISand PERRY,1967). As both bone and dentine have been shown to contain phosphate-containing peptides in similar amounts it is unlikely that those from dentine are in any way related to this phosphoprotein. GL~MCHERand LEVINE (1966) described soluble protein and peptide fractions isolated from bovine dental enamel. These fractions were obtained by gel-filtration only and were clearly much more heterogeneous than either the peptide fractions from bone or those dentine fractions described in this present study. It is, therefore, not strictly valid to attempt any precise comparison between our bone and dentine fractions and those from bovine enamel. Only three of the twenty-one bone peptide fractions were considered to resemble the enamel fractions of GLIMCHERand LEVINE(1966) whereas four of the dentine fractions (A, B, C and D) could be placed in this category. Two fractions (A and B) were characterized by the presence of tyrosine, which was absent from all the bone fractions except two highly basic ones. Recently GLIMCHER(1968) has described the isolation of two tripeptides after partial acid hydrolysis of soluble proteins from embryonic bovine enamel. These were identified as yglutamyl-o-
510
A. G.
LEAVER
phosphoseryl-tyrosine and yglutamyl-o-phosphoseryl-leucine. No fraction approximating to these peptides has been found in either bone or dentine, although degradation of fractions such as A might conceivably give rise to such compounds. At the present state of our knowledge of the peptides of adult ox bone and human dentine, we can conclude that they are probably extra-cellular, that they are unlikely to play a part in calcium transport or mobilization and that they are more likely to be associated with collagen than with any non-collagenous fraction. The choice would appear to be between a continuing association with collagen or a previous relationship with mucopolysaccharide or glycoprotein, which seems more feasible in bone than in dentine. A final possibility, of course, is that they represent vestigial material trapped during the mineralization process and possessing no continuing function. We have recently isolated a number of peptide fractions from very young (veal) bone and also from articular cartilage. It is hoped later to investigate any similar fractions which might occur in ossifying epiphyseal cartilage. Analysis of all such fractions may well shed further light on the significance and function of peptides occurring in mineralized tissues. Acknowledgement-I assistance.
should like to thank Miss G. RIXOMfor skilled technical
R&urn&Les composes azotes acido-solubles de la dentine humaine sont separes par filtration sur gel et par chromatographie avec echangeurs d’ions. La fraction, de poids mol6culaire suppose de 750-5000, qui s’est aver&e contenir ulterieurement des acides amines libres, comprend environ 0,OS pour cent de dentine et contient 20 pour cent de glycine, 25 pour cent d’acides amines dicarboxyliques et 15 pour cent d’acides amines basiques, comprenant de l’ornithine. L’electrophorese sur papier, effectuee sous tension Blev&+ a permis d’isoler neuf fractions, qui ont et6 analysees. Cinq des fractions peptidiques sont surtout acides et riches en acides amines hydroxyles, alors que trois sont plus riches en acides amines basiques, bien que contenant des acides amines dicarboxyliques. Une fraction hautement basique a aussi Cte isolde. Les fractions peptidiques semblent identiques a des fractions isolees anterieurement &partir d’os de boeufs. Bien que ne faisant pas partie des chaines principales de tropocollagene, ces fractions semblent associees soit au collagene a des pH physiologiques, soit aux glycoproteines ou aux muco-polysaccharides. Zusammenfassung-Die siureloslichen Stickstoffanteile des menschlichen Dentins wurden durch Gelfiltration und Ionenaustauschchromatographie fraktioniert. Die Fraktion schien sich im Bereich eines Molekulargewichts von 750-5000 zu halten, enthielt splter aber einige freie Aminosauren, umfabte etwa 0,08 Prozent des Dentins und enthielt 20 Prozent Glycin, 25 Prozent Aminodikarbonsauren und 15 Prozent basische Aminosauren einschlieblich Ornithin. Mit Hilfe der Hochspannungspapierelektrophorese wurden 9 Fraktionen analysiert. Fiinf von den Peptidfraktionen waren vorwiegend sauer und reich an Hydroxyaminsauren, wlhrend 3 trotz ihres Gehaltes an Aminodikarbons%uren reicher an basischen Aminosluren waren. Auch eine hochgradig basische Fraktion wurde gefunden. Die Peptidfraktionen 1ieRen sich mit frtiher isolierten Fraktionen vom Rinderknochen vergleichen. Obwohl keinerlei Fragmente der Tropokollagenketten vorhanden waren, schienen sie entweder bei physiologischem pH mit Kollagen zusammenzuh&igen oder mbglicherweise ursprtinglich mit Glykoprotein oder Mucopolysacchariden verbunden gewesen zu sein.
STUDIES ON CERTAIN PEPTIDE FRACTIONSISOLATEDFROM HUMANDENTINE
511
REFERENCES BERENBLUM, I and CHAIN, E. 1938. An improved method for the calorimetric determination of phosphate. Biochem. J. 32,295-298. EASTOE, J. E. 1967. In: StructuraI and Chemical Organisation of Teeth. (Edited by MILES, A. E. W.) Vol. 2, pp. 279-315, Academic, New York. EASTOE, J. E. and EASTOE, B. 1954. The organic constituents of mammalian compact bone. Biochem. J.
57,453-459. GLIMCHER, M. J. 1968. Studies of the early mineral deposits in bone and enamel, and of the organic matrix of enamel. Calc. Tiss. Rex 2, Suppl. 1. GLIMCHER, M. J. and LEVINE, P. T. 1966. Studies of the proteins, peptides and free amino acids of mature bovine enamel. Biochem. J. 98, 742-753. HERRING, G. M. 1964. Chemistry of the bone matrix, Clin. Orthopd. 36, 169-183. JACOBS, S. 1962. The quantitative determination of nitrogen by a modification of the indanetrione hydrate method. Analyst, Lond. 87, 53-57. LEAVER, A. G. 1968. The nature and possible significance of peptides isolated from bone and dentine. Calc. Tiss. Res. 2, Suppl. 85. LEAVER, A. G., EASTOE, J. E. and HARTLES, R. L. 1960. Citrate in mineralized tissues II. The isolation from human dentine of a complex containing citric acid and a peptide. Archs oral BioI. 2,12&126. LEAVER, A. G. and SHUTTLEWORTH, C. A. 1966. The isolation from human dentine and ox bone of phosphate-containing peptides. Archs oral Biol. 11, 1209-1211. LEAVER, A. G. and SHUTTLEWORTH,C. A. 1967. Fractionation of the acid-soluble nitrogen of bone and dentine. Archs oral Biol. 12,947-958. LEAVER, A. G. and SHUTTLEWORTH, C. A. 1968. Studies on the peptides, free amino acids and certain related compounds isolated from ox bone. Archs oral Biol. 13, 509-525. LEAVER, A. G., SHUTTLEWORTH,C. A. and TRIFFIT~, J. T. 1965. The separation of citric acid from other bone constituents by a series of chromatographic procedures. J. dent. Res. 44, 1177-l 178. STEVEN, F. S. and TRISTRAM, G. R. 1962. The presence of non-protein nitrogen in acetic-acid soluble calf-skin collagen. Biochem. J. 83,240-244. VEIS, A. and PERRY, A. 1967. The phosphoprotein of the dentine matrix. Biochemistry 6, 2409-2415.
oral
Bid.
Vol.
14, pp. 503-511,
1969. Pergamon
Press.
Printed
in Gt. Britain.
STUDIES ON CERTAIN PEPTIDE FRACTIONS FROM HUMAN DENTINE
ISOLATED
A. G. LEAVER Department
of Dental Sciences, School of Dental Surgery, University of Liverpool, England
Summary-The acid-soluble nitrogenous components of human dentine were fractionated by gel filtration and ion-exchange chromatography. The fraction believed to be in the molecular weight range 750-5OU0, but later found to contain some free amino acids, comprized approximately O-08 per cent of dentine and contained 20 per cent glycine, 25 per cent dicarboxylic amino acids and 15 per cent basic amino acids which included ornithine. Highvoltage paper electrophoresis was used to obtain nine fractions which were then analysed. Five of the peptide fractions were predominantly acidic and were also rich in hydroxy amino acids, while three, though containing dicarboxylic amino acids, were richer in basic amino acids. One highly basic fraction was also found. The peptide fractions were considered to be comparable to fractions previously isolated from ox bone and, although clearly not fragments of the main tropocollagen chains, were thought likely to be either associated with collagen at physiological pH or possibly to have been originally associated with glycoprotein or mucopolysaccharide.
THE PRESENCE of acid-soluble peptides in human dentine was reported by LEAVER, EASTOEand HARTLES(1960). It was then considered that the peptides were associated with the citric acid of the tissue. That such an association, in the case of similar material isolated from ox bone, was probably fortuitous was demonstrated by LEAVER, SHUTTLEWORTH and TRIFFITT (1965) who separated citric acid, peptides, glycoproteins and traces of soluble collagen by gel filtration and ion-exchange chromatography. LEAVERand SHUTTLEWORTH (1967) carried out an extensive study of the acid-soluble nitrogen (ASN) of bone and dentine from which it was concluded that from 3-5 per cent of the total nitrogen of bone and dentine went into solution in 1 N HCl at 2”C, that from 0.1 to O-25 of such ASN consisted of a mixture of peptides of molecular weight < 5000 and that comparable amounts of apparently similar peptides were released from these hard tissues by demineralization with EDTA at neutral pH. A previous study (LEAVERand SHUTTLEWORTH, 1966) had given qualitative information concerning the nature of the peptides from bone and dentine. Both tissues contained peptides rich in acidic and/or basic amino acids usually with substantial amounts of glycine and serine. Organic phosphate (presumably attached to the serine residues) was present in several fractions. A series of quantitative investigations, involving most of the sub-fractions of bone ASN, has recently been reported, (LEAVERand SHUTTLEWORTH,1968). One major aspect of this work was the use of preparative high-voltage paper electrophoresis to separate individual components of the total peptide fraction in the 750-5000 molecular 503
504
A. G. LEAVER
weight range. Twenty-one such sub-fractions, which appeared to be electrophoretically homogeneous, were hydrolysed and their amino acid composition determined on the Technicon autoanalyser. The results indicated that the sub-fractions were either mixtures of very closely related peptides or fractions in which one peptide predominated but was contaminated by traces of others. Nevertheless the results were of considerable interest as they revealed the presence of peptides of widely varying amino acid composition including very basic, strongly acidic, neutral non-polar (rich in glycine and serine) and “amphoteric” peptides containing both basic and dicarboxylic amino acids in similar proportions. The absence of hydroxyproline and the widely varying levels of glycine indicated that they could not have been derived from the main chains of the tropocollagen molecule. It was, however, considered possible that they might be attached to collagen at physiological pH and released at pH extremes, as was postulated by STEVENand TRISTRAM(1962) in the case of rather similar peptides isolated from calf skin collagen. Whereas the investigations into the bone ASN fractions were very extensive, it was decided to restrict the present study of dentine ASN to the peptide fraction of molecular weight 750-5000, to isolate sub-fractions by high voltage electrophoresis and to compare their amino acid composition with that of the corresponding fractions previously isolated from ox bone (LEAVERand SHUTTLEWORTH, 1968).
EXPERIMENTAL The acid-soluble nitrogenous fraction (ASN) of human dentine was isolated from teeth collected from the extraction departments of the Liverpool Dental Hospital. The preparation of material and the method of demineralization were precisely the same as previously described (LEAVERand SHUTTLEWORTH, 1967). The direct fractionation of the ASN described in this earlier work was preferred to the unnecessarily complex scheme used in the investigations of bone ASN. It is summarized in Fig. 1. The present study was confined to the peptide fraction 2Bl (that excluded by Sephadex G-10 after having been retarded by Sephadex G-25). It was found, however, that this fraction occupied a larger elution volume than had been found in the case of bone and it was considered likely to include both excluded and retarded material. The retarded fraction 2B2 was correspondingly smaller and was discarded. After evaporating fraction 2Bl to dryness in vucuo, it was dissolved in 2 .O ml of water, 0.4 ml of the resulting solution being retained for analysis of nitrogen, phosphorus and amino acids. Separation of peptide fractions from 2Bl by preparative high-voltage paper electrophoresis
In the separation of the bone peptide fractions two successive runs at pH 5.4 and pH 2.0 were carried out. Despite this, amino acid analysis indicated that no absolutely separate entity had been obtained. In view of this only the run at low pH was carried out in the present work, although the electrophoretic conditions were altered with a view to achieving improved separation. Fraction 2Bl was separated into sub-fractions by high-voltage electrophoresis on
STUDIES
ON
CERTAIN
PEPTIDE
FRACTIONS
ISOLATED
FROM
HUMAN
DENTINE
505
Total ASN 1 Amberliie
in 0.5N
HCI
IR-120
I
re-applied in aqueous solution
AKH‘$
I
/Tji[;“.ctio”
Aqueous eluate “Ammonia eluate (discarded) 2” combined with “Ammonia eluate I”
(discarded) I Sephadex G-IO /l Excluded Retarded fraction 281 fraction 282 (discarded) I H.KE.
FIG.
1.
Fractionation
of dentine ASN.
Whatman 3MM paper (78 x 10 cm) using a Locarte apparatus. A pH 1.98 buffer (O-58 M formic acid/O*21 M acetic acid) was used and 4 kV applied to the paper for 2 hr. In each run two strips loaded with 0.1 ml of fraction 2Bl were separated by a third unloaded strip. The three strips were placed on top of each other so that the central unloaded strip adsorbed peptide material during the run and acted as a marker. This was later stained with ninhydrin after drying and the areas of the loaded strips corresponding to the ninhydrin-positive bands cut out and eluted with distilled water overnight. Eight such runs were required to deal with the 1.6 ml of solution. In each run 10 sub-fractions were obtained and combined according to the distances run, which under the electrophoretic conditions used, varied from 18 * 1 to 18 -9 cm for fraction A to 48 a5-49 a9 cm for fraction J. Fraction H was rejected and the nine remaining fractions subjected to amino acid analysis. Amino acid analysis of hydrolysed and unhydrolysed fractions In the previous work on bone, peptide fractions retarded on Sephadex G-10 were found to contain both free and peptide bound amino acids, the former comprising 15.91 per cent and the latter 84.09 per cent of the total retarded material. The possibility that the excluded fraction might contain some free amino acids was not considered. In the present study, the excluded fraction 2Bl was somewhat disperse; hence each fraction was examined for free and peptide bound amino acids. One half of each fraction was hydrolysed in 6~ HCl in a sealed evacuated tube at 105°C for 24 hr, taken to dryness in a vacuum desiccator and finally dissolved in 1 ml of nor leucine solution. The remaining half of each fraction was evaporated without further treatment and re-dissolved in 1 ml of nor leucine solution. Each hydrolysed and unhydrolysed fraction was then individually analysed on the Technicon autoanalyser. One ml of the
A. G. LEAVER
506
final solution in nor leucine was applied to the resin column, carried out using the normal microbead
ion-exchange
Technicon
automatic
analysis being
20 hr run on a 150 cm column
resin. In all hydrolysate
chromatograms
of Type A
the amino
acid
levels were calculated on the basis of a standard amino acid mixture separated on the autoanalyser under identical conditions.
Analysis of the total fraction 2B1 One fifth of the total material in fraction 2Bl was set aside for analysis of nitrogen and phosphorus and for the determination of the overall amino acid composition. Nitrogen was determined by the ninhydrin method of JACOBS(1962) and phosphorus by the method of BERENBLUMand CHAIN (1938). The amino acid composition of the fraction
was determined
before
and after hydrolysis
as described
for the individual
separated fractions. RESULTS
Composition of the total fraction 281 Fraction
2Bl contained
9.24 mg of nitrogen and 0.67
mg of phosphorus.
related to the initial weight of dentine (66 g) the nitrogen content is 0.014 which is rather less than that previously
When per cent
isolated from dentine under similar conditions
(LEAVER and SHUTTLEWORTH,1967). It suggests a peptide content of about O-08 per cent of total tissue or 0.4 per cent of matrix. The ratio of nitrogen to phosphorus is approximately 14:l and is very closely similar to that found in the comparable tions from bone (LEAVER and SHUTTLEWORTH,1968). TABLE1. AMINOACID
COMPOSITION
OF TOTAL
FRACTION
2Bl
(EXPRESSED AS
frac-
MOLES PERCENT) -
Amino acid Hydroxyproline Aspartic Acid Serine Threonine Glutamic Acid Proline Glycine Alanine Cystine Valine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Ornithine Lysine Histidine Arginine
2Bl (peptide bound) 15.35 11.01 3.19 10.22 2.06 20.41 9.56 1.08 2.51 trace 1.06 5.59 2.32 trace 3.94 4.87 2.55 4.29
2Bl (free)
23.36 20.42 trace 18.42 20.02 8.86 trace -
4.88 4.04 -
STUDIES
ON
CERTAIN
PEPTIDE
FRACTIONS
ISOLATED
FROM HUMAN DENTINE
507
The fraction was found to consist of 75 *35 per cent peptide-bound and 24.65 per cent free amino acids. The percentage composition of the peptide-bound and free amino acids is given in Table 1. The overall composition of the bound amino acids in the fraction is quite comparable to that of the corresponding fraction found in ox bone (LEAVER and SHUTTLEWORTH, 1968). The chief difference is an increase in the level of aspartic acid at the expense of glycine. The amount of dicarboxylic acids is therefore correspondingly greater in the dentine peptide fraction whereas the level of basic amino acids is almost identical. A small amount of cystine was present while only a trace of phenylalanine was found. This is in contrast to the bone peptides which contained no cystine and in which the level of phenylalanine exceeded that of tyrosine. The presence of about 25 per cent of free amino acids was clearly due to the extended elution volume of this fraction 2B1. As the present study was confined to the peptide fraction no attempt was made to study the retarded fraction 2B2 which was, in any case, minimal. The determination of free amino acids, both in this total unseparated fraction and in the fractions resolved by electrophoresis, was carried out merely to ensure the precise determination of the composition of the true peptide fractions which was obtained by difference of the total and free amino acids. Amino acid composition of the fractions obtained by high-voltage electrophoresis of fraction 2Bl All of the nine fractions analysed were found to contain a proportion of free amino acids which varied from approximately 2-40 per cent. In each case a single amino acid accounted for at least two-thirds of those present in the free state. The percentage composition of peptide-bound amino acids occurring in the nine fractions is shown in Table 2. Fractions A, B, C and D fall into an obvious group. All are predominantly acidic and are also rich in the hydroxy amino acids. The glycine level is almost constant as is the total of alanine and leucine. A and B are unusual in containing a substantial proportion of tyrosine. Fraction E is again predominantly acidic but does TABLE 2. AMINO ACID COMPOSITIONOF SEPARATEDPEPTIDEFRACTIONS(EXPRESSEDAS MOLES PER CENT) Amino
acid
Asparatic Acid Threonine Serine Glutamatic Acid Glycine Alanine Valine Iso-leucine Leucine Tyrosine Ornithine Lysine Histidine Arginine
A
B
C
D
E
F
18.55 5.50 11.93 15.90 20.80 14.07
12.50 5.36 14.92 16.20 21.43 10.71 -
18.05 5.66 23.89 13.36 20.09 12.92 -
13.85 14.98 16.35 11.24 21.00 9.65 -
13.80 4.37 15.22 10.82 19*77 11.11 10.21 -
8.40 4.98 10.19 9.80 16.02 21.70 -
6.12 7.14 -
10.97 7.91
6.02 -
12.94 -
6.62 4.11 3.93 -
4.20 2.33 7.15 7.15 8.09
-
G
I
J
4.55 2.87 11.00 5.98 14.11 9.33 -
-
6.50 3.38 9.75 15.50 10.63 7.63 3.38 2.13 5.13 -
4.80 6.24 4.68 8.64
15.00 8.38 6.00 6.63
17.22 24.12 3.83 6.98
5.34 4.20 8.16 8.28 42.24 7.48 -
-
-
508
A. G. LEAVER
contain some basic amino acids. It is also notable for its high valine content. Fractions F and G fall into the category of “amphoteric” peptides in which both dicarboxylic and basic amino acids are present, although the latter are present in greater amounts. Peptide fraction J contains 52 per cent of basic amino acids and is highly basic whereas I, though including 36 per cent of these, contains 22 per cent of dicarboxylic acids and is, therefore, more comparable to F and G. It is also unusual in possessing the full range of aliphatic amino acids. All the fractions containing basic amino acids included ornithine. A final point is that all nine fractions contained an excess of ammonia far greater than could have been derived from the breakdown of amide groups in glutamine and asparagine or from the loss of small proportions of arginine and hydroxyacids during hydrolysis. It appears likely that this was due to incomplete removal of the ammonia used in the elution of the IR-120 column. There is little information available concerning the molecular weight of the peptides although, as they were obtained in association with free amino acids, quite small peptides might be expected to be present. These dentine peptide fractions, although obtained from only a single electrophoretic run, were obtained under better and more stringent conditions than were those isolated from bone and are likely to be no more heterogeneous. They are likely to represent either mixtures of closely related peptides or fractions in which one peptide predominates but is contaminated by traces of others within the group.
DISCUSSION
A major purpose of the study was to compare the nature of the peptide fractions in the molecular weight range 750-5000 with those isolated from bone. As suggested earlier, the presence of free amino acids in the fractions did indicate the possibility that peptides of molecular weight < 750 might also be present. Although only nine peptide fractions were obtained from dentine compared with twenty-one from bone, several overall similarities may be noted. Glycine is consistently the most abundant amino acid, proline and hydroxyproline are totally absent and ornithine is invariably present in fractions containing basic amino acids. The majority of peptides from both tissues are rich in hydroxyacids (serine and threonine). On the other hand the acidic dentine peptides tend to have rather more aspartic acid than glutamatic and two such peptides contain tyrosine which only appeared in highly basic fractions isolated from bone. The composition of the dentine peptides, although showing considerable individual variations, does not reveal the very wide variation between fractions shown in the case of bone. Thus, except for the very high glycine content of G (42.24 per cent), no individual fraction contained more than 25 per cent of any single amino acid, whereas three of the bone fractions contained over 50 per cent of a single amino acid. In the light of the results of the present study it seems possible, though by no means certain, that those bone fractions containing very high proportions of a single amino acid might have included a substantial amount of that amino acid in the free form or in the form of a simple dipeptide. It also seems possible that one fraction containing 43 per cent glycine and 41 per cent alanine may have consisted predominantly of glycylalanine.
STUDIES
ON
CERTAIN
PEPTIDE
FRACTIONS
ISOLATED
FROM
HUMAN
DENTINE
509
With these various reservations in mind, it seems reasonable to conclude that the dentine peptide fractions are essentially similar to those isolated from bone. If this is so, then it is appropriate to consider the peptides in the light of the differences between the two tissues. In the first place, dentine, once laid down, is not subject to the processes of resorption and remodelling, which would tend to support the view that they are unlikely to be involved in calcium binding or transport. As reported recently (LEAVER, 1968), preliminary studies using various bone peptide fractions have revealed little calcium binding ability. Secondly, as the odontoblast layer of the dentine was removed prior to demineralization, it would seem unlikely that the dentine peptides (and, by implication, those of bone) could have been derived from the cellular fraction. A possible association between the mucopolysaccharide or glycoprotein fractions of bone and the peptides was previously suggested. Very little is known of the non-collagenous nitrogen of dentine (EASTOE,1967) although it seems unlikely that any single entity is present at anything like the level of the “mucoprotein II” described by HERRING(1964) which is probably similar to the “mucopolysaccharideprotein complex” described by EASTOEand EASTOE(1954). Any attempt to gain further information concerning a possible relationship between the peptide fractions and the mucopolysaccharide and glycoproteins will have to be confined to bone (and in particular to very young or embryonic bone) until more information is available concerning the minor organic fractions of dentine. The most positive suggestion made by LEAVERand SHUTTLEWORTH (1968) was that the bone peptides might be closely related to the fractions isolated from acid soluble calf-skin collagen by STEVENand TRISTRAM(1962). These workers considered that the peptides were associated firmly with collagen at physiological pH and released at pH extremes. LEAVERand SHUTTLEWORTH (1968) suggested that a regular arrangement of peptides, bound to collagen at physiological pH and labile to pH changes, might have a function in the mineralization process. Dentine collagen, however, is unusual in its very high content of organic phosphate which has been shown to be due in the main to the presence of a phosphoprotein linked to collagen through an oligosaccharide and hydroxylysine (VEISand PERRY,1967). As both bone and dentine have been shown to contain phosphate-containing peptides in similar amounts it is unlikely that those from dentine are in any way related to this phosphoprotein. GL~MCHERand LEVINE (1966) described soluble protein and peptide fractions isolated from bovine dental enamel. These fractions were obtained by gel-filtration only and were clearly much more heterogeneous than either the peptide fractions from bone or those dentine fractions described in this present study. It is, therefore, not strictly valid to attempt any precise comparison between our bone and dentine fractions and those from bovine enamel. Only three of the twenty-one bone peptide fractions were considered to resemble the enamel fractions of GLIMCHERand LEVINE(1966) whereas four of the dentine fractions (A, B, C and D) could be placed in this category. Two fractions (A and B) were characterized by the presence of tyrosine, which was absent from all the bone fractions except two highly basic ones. Recently GLIMCHER(1968) has described the isolation of two tripeptides after partial acid hydrolysis of soluble proteins from embryonic bovine enamel. These were identified as yglutamyl-o-
510
A. G.
LEAVER
phosphoseryl-tyrosine and yglutamyl-o-phosphoseryl-leucine. No fraction approximating to these peptides has been found in either bone or dentine, although degradation of fractions such as A might conceivably give rise to such compounds. At the present state of our knowledge of the peptides of adult ox bone and human dentine, we can conclude that they are probably extra-cellular, that they are unlikely to play a part in calcium transport or mobilization and that they are more likely to be associated with collagen than with any non-collagenous fraction. The choice would appear to be between a continuing association with collagen or a previous relationship with mucopolysaccharide or glycoprotein, which seems more feasible in bone than in dentine. A final possibility, of course, is that they represent vestigial material trapped during the mineralization process and possessing no continuing function. We have recently isolated a number of peptide fractions from very young (veal) bone and also from articular cartilage. It is hoped later to investigate any similar fractions which might occur in ossifying epiphyseal cartilage. Analysis of all such fractions may well shed further light on the significance and function of peptides occurring in mineralized tissues. Acknowledgement-I assistance.
should like to thank Miss G. RIXOMfor skilled technical
R&urn&Les composes azotes acido-solubles de la dentine humaine sont separes par filtration sur gel et par chromatographie avec echangeurs d’ions. La fraction, de poids mol6culaire suppose de 750-5000, qui s’est aver&e contenir ulterieurement des acides amines libres, comprend environ 0,OS pour cent de dentine et contient 20 pour cent de glycine, 25 pour cent d’acides amines dicarboxyliques et 15 pour cent d’acides amines basiques, comprenant de l’ornithine. L’electrophorese sur papier, effectuee sous tension Blev&+ a permis d’isoler neuf fractions, qui ont et6 analysees. Cinq des fractions peptidiques sont surtout acides et riches en acides amines hydroxyles, alors que trois sont plus riches en acides amines basiques, bien que contenant des acides amines dicarboxyliques. Une fraction hautement basique a aussi Cte isolde. Les fractions peptidiques semblent identiques a des fractions isolees anterieurement &partir d’os de boeufs. Bien que ne faisant pas partie des chaines principales de tropocollagene, ces fractions semblent associees soit au collagene a des pH physiologiques, soit aux glycoproteines ou aux muco-polysaccharides. Zusammenfassung-Die siureloslichen Stickstoffanteile des menschlichen Dentins wurden durch Gelfiltration und Ionenaustauschchromatographie fraktioniert. Die Fraktion schien sich im Bereich eines Molekulargewichts von 750-5000 zu halten, enthielt splter aber einige freie Aminosauren, umfabte etwa 0,08 Prozent des Dentins und enthielt 20 Prozent Glycin, 25 Prozent Aminodikarbonsauren und 15 Prozent basische Aminosauren einschlieblich Ornithin. Mit Hilfe der Hochspannungspapierelektrophorese wurden 9 Fraktionen analysiert. Fiinf von den Peptidfraktionen waren vorwiegend sauer und reich an Hydroxyaminsauren, wlhrend 3 trotz ihres Gehaltes an Aminodikarbons%uren reicher an basischen Aminosluren waren. Auch eine hochgradig basische Fraktion wurde gefunden. Die Peptidfraktionen 1ieRen sich mit frtiher isolierten Fraktionen vom Rinderknochen vergleichen. Obwohl keinerlei Fragmente der Tropokollagenketten vorhanden waren, schienen sie entweder bei physiologischem pH mit Kollagen zusammenzuh&igen oder mbglicherweise ursprtinglich mit Glykoprotein oder Mucopolysacchariden verbunden gewesen zu sein.
STUDIES ON CERTAIN PEPTIDE FRACTIONSISOLATEDFROM HUMANDENTINE
511
REFERENCES BERENBLUM, I and CHAIN, E. 1938. An improved method for the calorimetric determination of phosphate. Biochem. J. 32,295-298. EASTOE, J. E. 1967. In: StructuraI and Chemical Organisation of Teeth. (Edited by MILES, A. E. W.) Vol. 2, pp. 279-315, Academic, New York. EASTOE, J. E. and EASTOE, B. 1954. The organic constituents of mammalian compact bone. Biochem. J.
57,453-459. GLIMCHER, M. J. 1968. Studies of the early mineral deposits in bone and enamel, and of the organic matrix of enamel. Calc. Tiss. Rex 2, Suppl. 1. GLIMCHER, M. J. and LEVINE, P. T. 1966. Studies of the proteins, peptides and free amino acids of mature bovine enamel. Biochem. J. 98, 742-753. HERRING, G. M. 1964. Chemistry of the bone matrix, Clin. Orthopd. 36, 169-183. JACOBS, S. 1962. The quantitative determination of nitrogen by a modification of the indanetrione hydrate method. Analyst, Lond. 87, 53-57. LEAVER, A. G. 1968. The nature and possible significance of peptides isolated from bone and dentine. Calc. Tiss. Res. 2, Suppl. 85. LEAVER, A. G., EASTOE, J. E. and HARTLES, R. L. 1960. Citrate in mineralized tissues II. The isolation from human dentine of a complex containing citric acid and a peptide. Archs oral BioI. 2,12&126. LEAVER, A. G. and SHUTTLEWORTH, C. A. 1966. The isolation from human dentine and ox bone of phosphate-containing peptides. Archs oral Biol. 11, 1209-1211. LEAVER, A. G. and SHUTTLEWORTH,C. A. 1967. Fractionation of the acid-soluble nitrogen of bone and dentine. Archs oral Biol. 12,947-958. LEAVER, A. G. and SHUTTLEWORTH, C. A. 1968. Studies on the peptides, free amino acids and certain related compounds isolated from ox bone. Archs oral Biol. 13, 509-525. LEAVER, A. G., SHUTTLEWORTH,C. A. and TRIFFIT~, J. T. 1965. The separation of citric acid from other bone constituents by a series of chromatographic procedures. J. dent. Res. 44, 1177-l 178. STEVEN, F. S. and TRISTRAM, G. R. 1962. The presence of non-protein nitrogen in acetic-acid soluble calf-skin collagen. Biochem. J. 83,240-244. VEIS, A. and PERRY, A. 1967. The phosphoprotein of the dentine matrix. Biochemistry 6, 2409-2415.