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Studies on the peptides, free amino acids and certain related compounds isolated from ox bone
Archs oral Biol.
Vol. 13, pp. 509425,
1968.
Pergamon Press.
Printed in Gt. Britain.
STUDIES ON THE PEPTIDES, FREE AMINO ACIDS AND CERTAIN RELATED COMPOUNDS ISOLATED FROM OX BONE A. G. LEAVER and C. A. SHUTTLEWORTH* Department of Dental Sciences, School of Dental Surgery, University of Liverpool, Liverpool, England Summary--The acid-soluble nitrogenous components of ox bone were fractionated by gel filtration and ion-exchange chromatography. The overall amino acid composition and the relative proportions of free and peptidsbound amino acids in the molecular weight ranges < 750 and 750-5000, were determined by automatic amino acid analysis. The material of mol.wt. < 750 was shown to include 84 per cent bound and 16 per cent free, amino acids. Glycine was predominant (over 50 per cent) with substantial amounts of both dicarboxylic and basic amino acids. Ornithine existed only in the free form while lysine and serine occurred more plentifully in the free than in the peptide bound state. The overall composition of the peptides in the 750-5000 mol.wt. range showed 28 per cent glycine, 13 per cent alanine, 15 per cent basic and 13 per cent dicarboxylic amino acids. High voltage electrophoresis was used to obtain thirteen fractions in each of which one peptide predominated and eight fractions which contained closely related peptides. Arniio acid analysis showed these to include fractions containing 80 per cent dicarboxylic acids, others containing 60 per cent basic amino acids, together with “neutral” fractions in which both basic and dicarboxylic acids accounted for over 20 per cent of the total. “Non-polar” peptides with 80 per cent of glycine and alanine were also found together with other fractions rich in serine. No fractions included hydroxyproline and proline was present in only a few. Preliminary investigations of the fractions of mol. wt. > 5000 suggested the possible occurrence of free peptides and glycopeptides, relatively low in carbohydrate content. These fractions contained both
hydroxyproline and hydroxylysine but were clearly not collagen fragments.
THE PRESENCEof acid-soluble peptides in human dentine and ox bone was first reported by LEAVER, EASTOE and HARTLEY(1960). It was believed at that time that these peptides were associated with the citric acid of the tissue. That such an association was probably fortuitous was shown by LEAVER, SHUTTLEWORTHand TRIFFITT (1965) who separated bone citric acid from what appeared to be a mixture of soluble collagen, glycoproteins and various peptides, by a series of chromatographic procedures, which were the basis for the extensive fractionation of the acid-soluble nitrogenous compounds of bone described recently (LEAVER and SHUTTLEWORTH, 1967b). A series of prelimary studies on the minor nitrogenous fractions of bone and *Present address: Massachusetts General Hospital, Boston, Mass., U.S.A. 509
510
A. G. LEAVER ANDC. A. SHUTTLEWORTH
dentine (LEAVERand SHUTTLEWORTH, 1966a, b, 1967a.) demonstrated that peptides, rich in glycine, alanine, serine and in either dicarboxylic or basic amino acids (occasionally both) were released in similar quantity when the tissues were demineralized either with dilute acid or with EDTA at neutral pH. The observations that none of the peptides contained hydroxyproline, that several contained phosphate and that the different peptides showed wide variations in amino acid content suggested that they were worthy of further study. Peptides have been shown to comprise between 11 and 22 per cent of the acidsoluble nitrogen of bone (LEAVERand SHUTTLEWORTH, 1967b; SHUTTLEWORTH, 1967). The major fractions are in the molecular weight range 750-5000, the remaining material representing small peptides and free amino acids. The purpose of the investigations described in this present study was to determine the overall amino acid composition of the peptides in the different molecular weight ranges, to determine the proportions of free and peptide-bound amino acids in the fractions of mol. wt. < 750 and to isolate and determine the composition of individual peptides in the main group (mol. wt. 750-5000). The results of the previous study (LEAVERand SHUTTLEWORTH, 1967b) showed that only a small proportion of the mucoids and glycoproteins, known to be present in bone (HERRING,1964), was released by acid demineralization of the tissues. It was not, therefore, originally intended to investigate further the fraction comprising material of mol. wt. > 5000. In view, however, of the possibility that this fraction might include traces of compounds previously unrecognized in bone (which could, of course, include larger peptides), some preliminary analyses were carried out. EXPERIMENTAL
AND RESULTS
The acid-soluble nitrogenous material (ASN) was obtained from ox bone as described by LEAVERand SHUTTLEWORTH (1967b). The results presented here refer to work carried out over an extended period during which certain aspects of the fractionation techniques previously described were modified as shown in Figs. 1, 2, 3(a) and 3(b). The actual use of ion-exchange resin and dextran gels and the collection and monitoring of eluates were, however, carried out in precisely the same manner as before. Figure 1 indicates the modified fractionation scheme used in the separation of the ASN into groups of compounds of comparable molecular weight. The use of ion-exchange resin resulted in the complete de-salting of the material whilst also achieving a further degree of fractionation. Table 1 shows the distribution of nitrogen and phosphate between the various fractions. Nitrogen was determined by the ninhydrin method of JACOBS (1962) and phosphate by the method of BERENBLUM and CHAIN(1938). The distribution of nitrogen between the fractions is similar to that reported in a previous study (LEAVERand SHUTTLEWORTH, 1967b) except that rather more has been left unaccounted for. This may be ascribed to the omission of Fraction 0 from the analysis and from losses accruing during the present more extensive fractionation. As
PEPTIDESAND FREEAMINOACIDSOF OX BONE
511
ASN
I
in 0.5 N HCl
Amberlite IR-120 \
/ “Acid, eluok I’ re-applied to IR-I20 in aqueous solution ;utee wih 4
/
“Aqueous eluote” (Organic acids traces of nitrogen)
eluted with N NH,OH
\
I Sephadex
I
/ Fraction 10 nKll wt. >5000 I Sephodex G-0
G-25 Fraction ‘I’ 28 mol. wt. 750-5000
/ Fraction IA mol. wt. > 5000
Fraction 48 mol. wt. <750
I Fractwxl 38 mol. wt. <750
?
Fmctii 2A~s~~~ mol. wt. 750-5000
elwte”
Sephadex G-25
“Ammonia eluote”
Fraction 0 (Disuxded)
“Ammonia
~?kctica 4A mol. wt.<750 mol. wt.<750
FIG. 1. Fractionation of bone ASN after precipitation of calcium as oxalate. (Use of Amberlite IR-120, Sephadex G-25 and G-10 as described by LEAVER and SHUT-~LEWORTII (1967b).
TABLE1. NrrnooE~ AND PHOSPHATE (P) CONTENTOF FRACTIONS SHOWNIN FIG. 1
Fraction
(Toti ASN) 1B
fi
2A 2B 3A 3B 4A 4B *Unaccounted
for
*Lost in fractionation;
Mol. wt. range
r5000 >5000 750-5000 750-5000 < 750 < 750 < 750 < 750
Nitrogen (%of total bone)
% of ASN
0.138 0.030 0.014 o-012 O-008 0.006 o-005 o-001 0.001
loo*0 21.8 10.1 8.7 5.6 4.3 3.6 o-7 0.7
0.061
44.2
Organic Phosphate (P) (%of . total bone) 0~0017 0~00095 0~00045 o*ooo2 O*OOOl -
includes some nitrogen in fraction 0 and may include volatile material.
512
A. G. LEAVER ANDC. A. SHU~LEWORTH
previously observed in a qualitative study (LEAVERand SHUTTLEWORTH 1966b), phosphate was absent from the large molecular fractions and predominated in the peptide fractions in the 7.50-5000 mol. wt. range. In the material of mol. wt. < 750, phosphate was confined to those fractions (3A, 4A) initially eluted with HCl. Prelimary fractionation and analysis of the large molecular fractions
The fractions excluded from Sephadex G-25 (IA and IB) were each further fractionated by applying in 0.1 M NaCl to a 200 g column of Sephadex G-50. The U.V.absorption of the eluate at 280 rnp was, as usual, used to indicate the appearance of sub-fractions. Both 1A and 1B gave very similar elution patterns, each showing an “excluded” fraction (lA1, 1Bl) and two retarded fractions (lA2, lA3, lB2, lB3). The excluded fractions presumably represented material of mol. wt. > 30,000 and the retarded fractions material in the mol. wt. range 5000-30,000. All six fractions were examined for hexose, hexosamine and amino acid content. Aliquots were hydrolysed with 2 N HCl in sealed tubes for 3 hr at 100°C and the hydrolysates investigated by thin-layer chromatography on Kieselgel G using a solvent mixture containing 65 ml ethyl acetate and 35 ml of 66 per cent aqueous iso-propanol. Sugars were located by the anisaldehydejsulphuric acid reagent as described by STAHL and KALTENBACH (1965). Further samples of the six fractions were examined semi-quantitatively for sugar content by an anthrone method described by EASTOEand COURTS(1963). Four fractions (1Al, lA2, 1Bl, 1B2) were found to contain sugar, chiefly glucose, together with small amounts of hexosamine. During the course of the amino acid analyses, described later, it was clear that galactosamine was the predominant sugar amine. The amounts of sugar found were considerably less than had been expected (on the assumption that the four fractions were likely to be representative of known bone mucoids or glycoproteins) and it was not possible to repeat the analyses on larger samples. The two retarded fractions (lA3, 1B3) did not appear to contain any sugar or hexosamine. Each of the six fractions was hydrolysed with 6 N HCl in a sealed tube for 24 hr and the amino acid content of the hydrolysate determined using the Technicon autoanalyser as described later. The amino acid composition of the six fractions is shown in Table 2. Four of the fractions contain both hydroxyproline and hydroxylysine, amino acids typical of, though not peculiar to, collagen. It is clear, however, that not only is the level of glycine in all fractions too low to account for all the hydroxyproline and hydroxylysine being present as part of a soluble collagen fraction, but the general amino acid content of these four fractions is quite unlike that of collagen. Overall amino acid composition of fractions 2A and 2B
Fractions 2A and 2B (representing material of mol. wt. 750-5000 and together comprising 14.3 per cent of total ASN) are of particular interest in that previous work has shown them to consist entirely of a mixture of peptides, several of which have been separated and qualitatively investigated (LEAVERand SHUTTLEWORTH
PEPTLDESAND
TABLE 2. AMINO
513
FREEAMINOACIDSOFOXBONE
ACID COMPOSITION OF FRACTIONS
lA1, 2, 3, lB1, 2, 3 (expressed
as tLmoles per cent) Amino acid AQ* Hydroxyproline Aspartic acid Serine Threonine Glutamic acid Proline Glycine Alanine Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Hydroxylysine Lysine Histidine Arginine
1Al 0.31 2.67 11.22 5.68 5.53 13.89 9.17 9.81 7.14 6.29 3.46 6.61 2.43 2.86 0.88 0.25 4.99 2.31 4.41
lA2 1.12 6.08 9.14 3.51 2.45 12.88 11.89 12.64 7.29 4.18 2.66 6.38 3.39 3.31 0.40 0.37 3.76 2.28 5.56
lA3
1Bl
lB2
lB3
0.48 8.74 7.46 3.84 2.05 10.98 9.93 23.66 8.05 2.88 1.64 3.84 1.28 1.73 0.54 0.68 4.39 1.32 6.40
9.46 9.07 4.94 3.42 12.88 8.85 18.05 7.90 3.64 2.12 4.64 l-69 2.12 0.47 0.52 4.20 1.30 4.64
12.95 1.81 0.90 15.20 13.66 9.13 9.45 4.27 2.33 9.39 4.66 3.82 3.23 3.10 6.02
13.00 5.24 3.10 17.28 8.73 15.33 8.34 3.68 2.11 6.60 2.71 5.63 2.91 4.66
*AQ represents an unidentified peak preceding aspartic acid.
1966b). In the present studies material in this molecular weight range was selected for extensive investigation and for the separation of individual peptides for analysis. In the interest of continuity, however, the overall composition of the peptide mixtures 2A and 2B are presented at this stage. (Table 3). Samples of fractions 2A and 2B were hydrolysed with 6 N HCI and the hydrolysates analysed on the Technicon autoanalyser. Fractions 3A, 3B, 4A and 4B
These fractions represent material of mol. wt. < 750 and are composed of small peptides and free amino acids. Fractions 3A and 3B which contain relatively small proportions of free amino acids together comprised 7 -9 per cent of total bone ASN. Fractions 4A and 4B are composed of approximately equal proportions of small peptides and free amino acids but together make up only l-4 per cent of total bone ASN. Fractions 3A, 3B, 4A and 4B were each divided into two parts so that they could be applied to the auto-analyser unhydrolysed and also after the usual hydrolysis with 6 N HCI. In this way it was possible to estimate free and peptide-bound amino acids and also to determine the proportions of amino acids occurring in the bound and free states.
A.G.
514
LEAVERAND
C.A. SHUTTLEWORTH
TABLE 3. AMINO ACID COMPOSITION OF FRACJTIONS2A, 2B AND OF 2A AND 2B COMBINED (expressed as pmoles per cent) Amino acid
AQ* Aspartic acid Serine Threonine Glutamic acid Proline Glycine Alanine Valine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Omithine Lysine Histidine Arginine
*Unidentified
2A 0.95 4.57 7.30 5.31 11.12 3.90 28.67 14.10 3.20 1.40 3.17 0.90 l-23 0.26 4.37 3.66 3.00 2.79
2B 0.99 3.06 6.51 2.45 5.77 2.23 29.60 13.13 2.48 1.90 4.60 1.59 4.59 0.16 5.19 2.55 5.99 7.10
2A and 2B 0.97 4.13 7.04 4.50 9.56 3.43 28.81 13.83 3.00 I.55 3.58 1.10 2.21 0.23 4.63 3.31 3.89 4.06
peak preceding aspartic acid.
Table 4 shows the proportions of free and peptide-bound amino acids present in the four fractions and in the total material of mol. wt. < 750. Table 5 shows the percentage composition of total amino acids (i.e. peptidebound plus free) and of peptide-bound amino acids in the four fractions. Separation of individual peptides from the fractions of mol. wt. 750-5000 Throughout this work it has been clear that these fractions are of particular interest. They consist entirely of a mixture of peptides free both from carbohydrate and free amino acids and comprise 14.3 per cent of the total ASN. Preliminary studies have shown them to include peptides of widely varying composition and to appear in comparable quantity and form under different conditions of demineralization. It was, therefore, decided to study these fractions in detail and to attempt to separate individual peptides by high voltage electrophoresis at different pH levels. As stated at the beginning of this section, the work described in this paper was carried out over a long period during which the fractionation techniques were modified. A preliminary stage in the fractionation (LEAVER and SHU-ITLEWORTH, 1967b) involved the reprecipitation of bone minerals by making the demineralizing solution alkaline (immediately after filtering off the collagen). When this was done it was found that the large molecular fraction (mol. wt. > 5000) was co-precipitated. During the
515
PEPTIDES ANLI FREEAMINO ACIDS OF OX BONE TABLE 4. PERCENTAGE OF PEPTIDE-BOUND AND FREE AMINO ACIDS IN FRACTIONS OF MOL. WT. < 750
Total ASN (% of)
Fraction
3A 3B 4A 4B Total material < 750
Peptidebound amino acid (%)
(4.3)
Free amino acid (%)
78.87 91.39 51.09 56.91 84.09
ii:; (0.7) (9.3)
21.13 8.61 48.91 43.09 15.91
TABLE 5. AMINO ACID COMPOSITIONOF FRACTIONS 3A, 3B, 4A, 4B (columns Prefer to peptidebound amino acids and T to total free a.nd peptide-bound amino acids)
Amino acid
3A(P)
3A(I)
AC!* Aspartic acid Serine Threonine Glutamic acid Glycine Alanine Valine
0.85 7.09 3.39 1.46 8.60 53.80 4.63 2.03
0.75 6.97 4.17 1.30 9.41 50.33 5.35 1.81
4<4 1.82 0.98 2.82 65.94 2.33 0.98
Iso-leucine Leucine Tyrosine Phenylalanme Omithine Lysine Histidine Arginine
4.43 0.61 2.22 2.45 1.45 2.80 4.13
0.54 3.95 1.98 2.19 3.08 1.91 2.50 3.68
7.26 2.53 1.60 0.88 0.74 1.60 5.51
3B(P)
3B(T)
4A(P)
4A(T)
4.52 3.78 0.89 2.58 61.21 2.13 0.89
2.60 8.79 3.52 2.89 11.84 45.81 4.59 0.09
1.33 7.31 7.38 3.63 12.25 36.48 8.44 1.58
6;3 2.31 3.03 4.21 1.25 1.47 5.04
3.81 0.77 4.49 0.82 6.04 3.86
3-18 1.35 2.29 6.32 2.56 3.08 2.74
4B(P)
4B(T)
-
-
15.21 1.26 5.97 12.50 38.94 IO.86 -
8.65 9.79 3.40 7.11 30.16 6.18 -
5.07 9.23 -
2.88 5.25
1.26 -
20;1 6.28 -
*Represents an unidentified peak preceding aspartic acid.
course of further investigation of the various peptide fractions it was found that the smaller molecules distributed themselves between the “alkaline precipitate” and “titrate” so that an identical peptide would appear in each of these two main subfractions. Thus, the fractionation scheme described below is unnecessarily complex and the alkaline precipitation stage will not be repeated in future work. Figure 2 shows the preliminary fractionation of bone ASN while Figs. 3a and 3b show the procedures used to isolate the sub-fractions from which peptides were subsequently isolated by high voltage electrophoresis. Fractions A, B, C and D (Figs. 3a and 3b) represent material of mol. wt. 750-5000 and correspond to 2A and 2B in the fractionation scheme described in the earlier part of the paper (Fig. 1). h.0.B.13/5--c
516
A. G.
LEAVER AND C. A. SHUTTLEWORTH 2OOg bone 2.51 N HCL ASN
I bone salts
I
NaOH pH II
1 Precipitate
/
Filtrote A
(Fig.301
t Redissolved in N HCl Potossium oxalate solution
Precipitate ( No meosuroble nitrogen)
FIG.
2. Preliminary fractionation
Filtrate
B
(Fig. 3b) of bone ASN.
Separation oj’peptideJLactions by high aoltage paper electrophoresis Fractions A, B, C and D were further fractionated by high voltage electrophoresis on Whatman No. 3 paper (59 x 36 cm), using a Phaerograph high voltage apparatus. Pyridine/pyridinium acetate buffer (0.2 M, pH 5.4) was used and 1000 V applied to the paper for 90 min. Peptides were located by spraying a portion of the sheet with ninhydrin and the corresponding areas were eluted with water. In this way sixteen peptide sub-fractions were obtained. Only one band was common to A, B, C and D, two were common to A and D, and two common to C and D. These apparently identical sub-fractions were combined as suggested by their electrophoretic behaviour. The three combined fractions (referred to as X, Y, Z) were further separated by electrophoresis at pH 2-O (formic acid/acetic acid, O-2 M) using the same electrophoretic conditions. The remaining eight fractions from the initial run at pH 5 *4 were not subjected to this further run at pH 2.0; they represented fast running (i.e. highly charged) material and are referred to as fractions W 1-8. Electrophoresis at pH 2 *O separated a total of thirteen peptides from the three combined fractions. These are designated X l-4, Y 1-4 and Z l-5. The electrophoretic behaviour suggested that the peptides in the X, Y and Z groups represented single homogeneous entities while the W group represented eight sub-fractions in each of which either one peptide predominated or the mixture consisted of peptides of closely similar composition. The results of the amino acid analyses, described below, confirmed this view in respect of the W group but made it clear that each of the thirteen peptides of the X, Y and Z groups, though apparently electrophoretically homogeneous, was, in fact, contaminated with traces of other peptides.
PEITIDES
AND
FREE AMINO
ACIDS
517
OF OX BONE
FlItrote A I Amberlite
in 0.5 N HCL IR-120
/ “Acid eluote”
1 IR-120
/
re-opplied I” 0q”eous solution
+
“\et
T”“‘” No excluded fraction
eluted with N NH,OH I\ Aqueous eluate (Discorded)
eluted with N NH40H “Ammonia &ate”
or ded
fraction
I Sephadex G-IO
“Ammonia eluote”
/\ Fraction B (mol. wt. 750-5000)
Retarded froctton (DIscorded)
Sephadefc G-25
No excld fraction
Re!arded
fraction
1 SeDhadex G-IO
Fraction /
barded fraction (D Iscorded 1
(mol.wt. 750-5000)
FIG. 3(a). Fractionation
of ASN in filtrate A.
FlItrote B 1” 0.5 N HCI i Amberlite IR-120
“Acid eluote”
/
\
re-opplied in oqueous I solution IR-120
/\ Aqueous eluafe
&ted with N NH,OH
(Discorded 1
“Ammonia
eluded with N NH,OH “Ammonia
eluote”
+
Excluded Saphodex fraction (Discarded)
eluote”
?%orded
Sephadex
/ Fraction D (mol. wt 750-5000)
fraction
I G-IO
Rbd fraction (Discarded)
Sephode? G-25 / Excluded fraction (Discorded)
\ Retorded
fraction
1
(mol. wt. 750-5000)
FIG. 3(b). Fractionation
(Discorded)
of ASN in filtrate B.
518
A. G. LEAVERAND C.
A. SH~T~EWORTH
The separation of the 21 peptide fractions, derived by preparative electrophoresis from fractions A, B, C and D (Figs. 3a and 3b) is summarized as follows: Fractions X1-X4. These fractions represent material derived from fractions A and D, which moved approximately 5 cm towards the anode at pH 5 -4 and was separated into four components by further electrophoresis at pH 2-O (at which pH all peptide fractions moved towards the cathode). Fractions Yl- Y4. These fractions represent material, derived from fractions C and D, which moved approximately 2 *5 cm towards the anode at pH 5.4 andwas separated into four components by further electrophoresis at pH 2.0. Fractions 21-25. These fractions represent material, derived from fractions A, B, C and D, which remained close to the origin at pH 5 -4 and was separated into five components by further electrophoresis at pH 2.0. Fractions Wl-W8. These eight fractions were obtained by electrophoresis at pH 5.4 only. Wl-W4 were derived from fraction B, W5 from fraction A, W6 from fraction C and W7 and W8 from fraction D. They represent individual bands, all of which moved towards the cathode for distances varying from 4.5 cm to 17 -0 cm. Amino acid analysis of individual peptide fractions
The peptide fractions obtained after preparative high voltage paper electrophoresis were hydrolysed with 6 N HCl in sealed evacuated tubes for 24 hr, taken to dryness in a vacuum desiccator and dissolved in a specific volume of nor-leucine solution. One ml of the final solution was applied to the resin column of the Technicon autoanalyser. Automatic analysis was carried out using the normal Technicon 20 hr run on a 150 cm column of Type A microbead ion-exchange resin. In all hydrolysate chromatograms the amino acid levels were calculated on the basis of a standard amino acid mixture separated on the autoanalyser under identical conditions. Amino acid composition of peptides and peptide fractions
Table 6 shows the amino acid composition of the peptide fractions. Amino acid content is expressed as pmoles per cent. Most fractions contained a large excess of ammonia which has not been included in the figures. In attempting to interpret the results several considerations must be borne in mind. In the first place the peptides are within the molecular weight range 750-5000 and presumably contain between 6 and 40 amino acid residues, though the upper limit would be higher for those rich in glycine and lower for those rich in basic amino acids. It is clear, therefore, that an amino acid occurring at a level of below 2.5 pmole per cent, and even in some cases below 4 pmole per cent, is unlikely to represent a true component of the major peptide in that fraction. Although fractions X l-4, Y 14 and Z l-5 appeared to be electrophoretically homogeneous they clearly comprise a
-
E.
0.59
0%
E 1.42 -
1% 1.30 0.51 0.51 0.82
:::: 17.18 22.68
0<4 0.10 -
4% -
‘E
16’49
54’52
“:::: 1*88 6 *21 1.78 0 -70 0.44 1.43 1.72
66-24 15ai 0.71 3 -27
f:E
X2
Yl
Xl
W5
0% -
0%
076
2k.E 3.68
1.50
j.;
1.46 .
.
2 -43
f ‘%
‘8’ .‘R
4.30 15.66
. ‘Ei . ‘i’::
YZ
:: 2-17
‘:x 1:60
12 ‘42 13.98 5.17 14.50
X4
171 3 -43 2.21 1.71 2.70 -
Y3
.
::g 4’50 -
;.5:
. 345:
3.58 -
19.82 ‘:‘;: . 2.42 4.83 1.51
-
272 2.80 2.44
-
2.37 1.25 2.08
. “i’E
4.21 9.02
X3
‘Et
co -ON
W3
‘g7-E .. 1%
W4
TABLE 6. AMINO ACID
2 -98
::g
175 2.16
::g 7 -20
;:g
8 ‘85 4.63 11.01 24.51 9.47
Zl
1.21 -
O-84
;T;:
-
d: .‘E! -
. :‘:i
22
23
pRAmoNs
0.87 3.86
OP *aPnoB
Y4
4.57 17.14 24.91 9.95 -
‘22:
W7
-
0.75 0.72 10.76 12.18 5.55 5.81
Wl
W2
173 -
“% 2 -28
:::: lO*OO
7 -53
24
3.34 18.16 15.06 8.56 0 -92 -
:k’s. 6.83 1.43 0.47 1.40 -
;*s.
8 ‘47
Z5
570 0.81 15.51 10.10 13.71 24.12 1 -83
Et
:::
0 -70 2 ~80 13ao 3.81 0 -79
W6
W8
e:
8
4
520
A. G. LEAVER AND C. A. SHUT~LEWORTH
major peptide contaminated with traces of others. Similarly fractions W l-8, believed to be mixtures of closely similar peptides, must include traces of material unrepresentative of that group. Nearly all the fractions contained an excess of ammonia far greater than could be accounted for by the breakdown of amide groups in asparagine and glutamine or by the loss of small quantities of serine, threonine and arginine. Although many of the peptides have been shown to contain phosphate (LEAVERand
SHUTTLEWORTH,1966b) which appeared to correspond closely to the serine and threonine content of the total peptide fractions (LEAVERand SHUTTLEWORTH, 1967a), no attempt was made to isolate serine phosphate by selective mild hydrolysis. It is, however, very likely that much of the serine (and possibly threonine) exists as the phosphate. The presence of this acid phosphate and of excess amonia may account for the fact that some of the fractions did not behave in the electrophoretic separations as would have been expected from their amino acid composition.
DISCUSSION Although the fractionation methods used in the present work differ slightly from those described recently, the general considerations behind the method of preparation of the tissue, the choice of acid demineralization and the use of gel filtration and ionexchange chromatography remain the same and have been extensively discussed previously (LEAVERand SHUTTLEWORTH, 1967b). One of the main conclusions of the previous study was that acid demineralization released only a small proportion of the large-molecular non-collagenous components and that further investigation of these fractions was not intended. It was, however, decided to carry out certain investigations of this fraction (i.e. material of mol. wt. > 5000) in order to see whether larger peptides, free from carbohydrate components, could be obtained and to get some idea of whether the various fractions did, in fact, correspond to known bone constituents (HERRING,1964).
The two fractions excluded by Sephadex G-50 (i.e. mol. wt. > 30,000) together with two of the retarded fractions did, in fact, contain sugars (chiefly glucose) and galactosamine but it appeared that the total carbohydrate proportion of the fractions was comparatively low. The two remaining fractions (IA3 and lB3) contained only minimal traces of carbohydrate and may be regarded as mixtures of peptides of mol. wt. somewhat in excess of 5000. The amino acid analyses showed that four of the six fractions contained both hydroxyproline and hydroxylysine. At first sight this would suggest the presence of traces of soluble collagen but in no case was the glycine content high enough to account for this. This observation, together with the fact that the general amino acid composition of the fractions is quite unlike that of collagen, would suggest that even if the fractions do contain traces of soluble collagen there may well becompounds present, possibly as glycopeptides, which are separate from collagen but contain both hydroxyproline and hydroxylysine. It does, therefore, appear that the large molecular components of bone ASN might be worth further study, particularly in view of the fact that it is in the molecular weight range 5000-30,000 that
PEPTIDES
AND
FREE AMINO
ACIDS
OF OX BONE
521
calcium-binding peptides, corresponding to that isolated from intestinal mucosre by WASSERMANN(1967), might be expected to occur. The fractions of mol. wt. -C 750 represented 9.3 per cent of the total ASN. The chief interest here was in the relative proportions of free and peptide-bound amino acids. Although free amino acids constituted nearly half of the minor fractions 4A and 4B, they represented only l-6 per cent of the total ASN of bone and approximately 16 per cent of the total material of mol. wt. < 750. Glycine was the predominant peptide-bound amino acid and also occurred free in substantial proportions. Ornithine existed almost exclusively as the free amino acid while serine and lysine were more plentiful in the free than the bound state. An unidentified peak, apparently acidic in nature, but not serine phosphate, occurred at a level of approximately 1 pmole per cent. Whether amino acids exist in the free state in the intact tissue is not clear but their isolation in such small quantities would suggest that very little breakdown of peptides during demineralization has occurred. This observation, confirming our previous work, contrasts with the results of GLIMCHERand LEVINE(1966) who found that acid demineralization of bovine enamel greatly increased the proportion of free amino acids when compared with demineralization with EDTA which resulted in most of the amino acids being recovered as components of peptides. Apart from the very high glycine content the overall composition of the fractions of mol. wt. < 750 is similar to that of the major peptide fractions (i.e. those of mol. wt. 7% 5000). These fractions, comprising 14.3 per cent of the total ASN, were considered to be of particular interest because of the considerable variation in composition shown qualitatively in an earlier study (LEAVERand SHUTTLEWORTH, 1966b). High voltage electrophoresis was used to isolate thirteen peptides which appeared electrophoretically pure but which were shown by analysis to represent fractions in which one predominant peptide was contaminated by traces of others. Eight partially purified fractions, representing either single peptides with some contamination from others within the group or groups of closely similar peptides were also analysed. In attempting to interpret the results the limitation placed on the possible number of residues in a peptide by the known molecular weight range (750-5000) must be kept in mind. Although it is clearly impossible to set any specific value (in pmoles per cent) as representing a single amino acid residue, the limitations previously mentioned indicate that several of the peptides must consist of no more than three or four individual amino acids. The twenty-one peptide fractions exhibited remarkable differences in amino acid composition varying from highly acidic (Glu. and Asp. 80 per cent) to very basic (Orn., Lys., His., Arg. 65-75 per cent). Between these limits are fractions (22, 23) low in polar amino acids and consisting largely of glycine and alanine, while others (Zl, X2, W5) are particularly rich in serine and threonine. Other obvious features include the presence of tyrosine in the highly basic fractions W6 and W8 and the presence of traces of hydroxylysine in these two fractions. The high level of leucine in Y2 is also noteworthy. Possibly the most striking observation was the fact that ornithine was the most abundant basic amino acid in the majority of fractions
522
A.G.
LEAVERAND
C. A.
SHUTTLEWORTH
containing reasonable proportions of basic amino acids. The ornithine levels are far too high to have been produced by the breakdown of arginine and the absence of ornithine from several fractions, together with the isolation of free ornithine in the small molecular fractions (which were not subjected to electrophoresis) precludes the possibility of its having been acquired adventitiously from the paper. We must, therefore, accept ornithine as a true and, in fact, major component of several of the fractions. JACKSON,LEACHand JACOBS(1958) reported ornithine as a component of neutral salt soluble collagen from skin, while HAMILTONand ANDERSON(1954) had previously reported its presence in hydrolysates of lime-treated gelatin but ascribed its appearance to arginine breakdown at alkaline pH. Hydroxyproline was absent from all twenty-one peptide fractions, hydroxylysine present in only two and proline in seven, reaching a value of 7 -2 per cent in fraction Zl. This clearly indicates that they cannot be derived from the main chains of the tropocollagen molecule. Although glycine is a predominant amino acid, comprising over 70 per cent in one fraction and over 30 per cent in four others, it would be expected to occur at a consistent level of about 33 per cent in peptides derived from collagen (HANNIGand NORDWIG,1964). The presence of phosphate, very probably as serine phosphate (LEAVERand SHUTTLEWORTH, 1966b, 1967a), invites comparison with the peptides isolated from bovine dental enamel by GLIMMER and LEVINE(1966). The main difference is in the IeveIs of basic amino acids which were present in only very small amounts in the fractions from bovine enamel. Of the twenty-one fractions described in the present study only those such as X2, X4 and W5, in which glycine, glutamic acid, aspartic acid and serine were the main constituents, are comparable to the various fractions described by GLIMCHERand LEVINE.None of the fractions isolated shows any resemblance to the papain-labile “telopeptides” described by RUBIN, et al. (1963), which contained far higher levels of aromatic amino acids. The “telopeptides” were considered to represent extra-helical components of the tropocollagen molecule. The size of the present series of peptides is such that they could not function as intrahelical cross-linkages and, as they clearly cannot be derived from the main chains, the only remaining possibility is that they represent material bound extra-helically and possibly as a series of repeating units. Any regular pattern of attachment would depend on the binding sites and the nature of the linkages. Their acid lability precludes the possibility of covalent linkages and, if binding does occur, it is most likely to be salt-like, particularly in view of the nature of the peptides, many of which are either very basic or very acidic. STEVENand TRISTRAM(1962) isolated peptides and free amino acids from acetic acid soluble calf skin collagen and suggested that such peptides, classi6ed as “non-protein nitrogen associated with collagen”, may be strongly bound to collagen at neutral pH and released at extremes of pH. As some of the peptides described here do, in fact, resemble those reported by STEVENand TRJ~TRAMit is possible that they are bound to collagen at physiological pH and released during acid demineralization, although we have found similar peptides to be released by demineralization with EDTA at pH 7 *4 (LEAVERand SHUTTLEWORTH, 1967a). Whether such binding does occur and, if so, whether it has any biological
PEPTIDES
AND
FBEE
AMINO
ACIDS
523
OF OX BONE
significance is not yet clear, though it is tempting to suggest that a regular arrangement of highly polar peptides, bound to collagen at physiological pH and labile to pH changes, might have a function in nucleation and thus in the mineralization process. If the peptides are unassociated with collagen then they must be free and separate entities, which may either have fulfilled a biological function and thus represent vestiges of previous material, or possess a continuing function in adult bone. It is convenient, therefore, to consider the possible derivation of the peptides together with function. Apart from two neutral peptides most are highly polar, including not only acidic and basic fractions but also others containing both dicarboxylic and basic amino acids in similar proportions and thus potentially amphoteric in character. There has been considerable interest recently in histochemical observations which suggest that the acidic groups of the mucopolysaccharides of ossifying cartilage are initially masked and that the masking agent disappears during ossification. CAMPO and DZIEWIATKOWSKI (1963) described this effect and implicated basic proteins as the masking agents while WILLIAMSON (1968) refers to the possibility of peptide blocking. It might be that the basic peptides isolated from bone represent vestiges of material originally present in cartilage, in which case it would be worthwhile attempting to gain more precise chemical information about the nature of the blocking agents and, in particular, to see whether similar peptides to those obtained from bone can, in fact, be isolated from cartilage. On the other hand it seems unlikely that such cartilage remnants would persist in adult cortical bone which is constantly undergoing resorption and remodelling under the control of osteoblasts and osteoclasts. If the peptides are separate entities, it is likely that they are derived from the bone cells and may be involved in the mineralization process, possibly in association with mucopolysaccharides or glycoproteins. The very high content of dicarboxylic acids in some of the peptides, and the presence of substantial proportions of dicarboxylic acids in the “amphoteric” peptides, suggests a possible role in calcium binding and transport. Although calcium binding is usually associated with larger molecules, such as that described by WASSERMANN (1967), it is intended to investigate the possible calcium-binding ability of all the fractions described in the earlier part of the present work (i.e. mixtures of ASN components in the specific molecular weight ranges). These considerations imply that the bone peptides might be bound to collagen and possess a role in nucleation or that they may be free and represent vestiges of material with an earlier function in cartilage or in the laying down of new bone by osteoblasts. A final possibility is a continuing role in calcium binding and transport. In view of the widely diverse nature of the peptides further research may lend support to one or more of these viewpoints. Acknowledgement-The
assistance.
authors wish to thank Miss G. A.
RIXOM
for technical
524
A.G.
LEAVERAND
C. A. SHUTTLEWORTH
R&n&--Les composes azotb acido-solubles de 1’0s de boeuf sont &pares par filtration sur gel et chromatographie par echanges d’ions. La composition totale en acides amines et les proportions relatives en acides amines libres et lies aux peptides dans le domaine des poids mol&culaires < 750 et 750-5000 sont determintes par analyse automatique d’acides amines. Le materiel de poids moEculaire < 750 comporte 84 pour cent d’acides amines lies et 16 pour cent d’acides amines libres. La glycine est prtdominante (audessus de 50 pour cent) avec des quantites importantes d’acides amines dicarboxyliques et basiques. L’ornithine n’existe que sous forme libre, alors que la lysine et la serine sont plus frequentes dans la fraction libre que dans la fraction IiCe aux peptides. La composition des peptides, dans la fraction depoidsmol&culaireallant de 750-5000,est la suivante: 28 pour cent de glycine, 13 pour cent d’alanine, 15 pour cent d’acides amines basiques et 13 pour cent d’acides amines dicarboxyliques. L’electrophorbse, sous haute tension, permet d’obtenir 13 fractions, dans lesquelles un peptide predomine et huit fractions qui contiennent des composts voisins des peptides. L’analyse d’acides amines montre qu’ils contiennent 80 pour cent d’acides dicarboxyliques; d’autres renferment 60 pour cent d’acides amines basiques ainsi que des fractions neutres dans lesquelles les acides basiques et dicarboxyliques constituent plus de 20 pour cent du total. Des peptides “non polarises”, renfermant 80 pour cent de glycine et alanine, sont retrouves avec d’autres fractions dans la serine. Aucune fraction ne comporte de l’hydroxyproline et la proline n’est pr&.sente que dans certaines portions. Des essais preliminaires des fractions de poids mol&culaire > 5000 suggerent la presence possible de peptides et glycopeptides libres, relativement faibles en hydrate de carbone. Ces fractions contiennent a la fois de l’hydroxyproline et l’hydroxylysine, mais il ne s’agit pas de collagene. Zusammenfassung-Durch Gel-Filtration und Ionenaustauscher-Chromatographie wurden die slureliislichen Stickstoffanteile von Rinderknochen fraktioniert. Der gesamte Aminosluregehalt und die relativen Anteile freier und Peptid-gebundener Aminosauren in Molekulargewichtsbereichen < 750 und 75&5000 wurden durch automatische Aminoslurea&yse bestimmt. Das Material mit einem Molekulargewicht < 750 enthielt 84% gebundene und 16 ‘A freie Aminosluren. Glycin stand mit ilber 50% im Vordergrund neben hemerkenswerten Mengen von Dicarbon- und basischen Aminosluren. Omithin kam nur frei vor, wahrend Lysin und Serin griil3erenteils frei als im Peptid-gebundenen Zustand auftraten. Die gesamten Peptide mit einem Molekularaewicht zwischen 750 und 5000 enthielten 28 “/, Glvcin, 13 “/, Alanin. 15 % basische und 13 ‘A Dicarbon-Aminosluren. Mit Hilfe der I-Iochspanmmgselektrophorese wurden 13 Fraktionen, in denen ein bestimmtes Peptid vorherrschte, weiterhin 8 Fraktionen mit nahe verwandten Peptiden erhalten. Die Aminosaureanalyse ergab, daR diese Fraktionen 80% Dicarbonsluren und andere 60% basische Aminoduren zusammen mit “neutralen” Fraktionen enthielten, bei welchen sowohl basische als such Dicarbonsauren im Anteil von je 20% vorhanden waren. Weiterhin wurden “nicht-polare” Peptide mit 80 % Glycin und Alanin zusammen mit anderen Serin-reichen Fraktionen gefunden. Keine der Fraktionen enthielten Hydroxyprolin, und Prolin war nur in wenigen vorhanden. Vorllufige Untersuchungen tiber die Fraktionen mit einem Molekulargewicht > 5000 deuten auf das miigliche Vorkommen freier Peptide und Glycopeptide mit relativ niedrigem Kohlenhydratgehalt hin. Diese Fraktionen enthielten sowohl Hydroxyprolin als such Hydroxylysin, es waren jedoch sicherlich nicht Kollagenfragmente.
PEPTIDESAND FREEAMINOACIDSOF OX BONE
525
REFERENCES BERENBLIJM,I. and CHAIN, E. 1938. An improved method for the calorimetric determination of phosphate. Biochem. J. 32,295-298. CAMPO, R. D. and DZIE~IATKOWSKI, D. D. 1963. Turnover of the organic matrix of cartilage and bone as visualized by autoradiography. J. biophys. biochem. Cytol. supp J. Cell. bioll8, 19-29. EASTOE, J. E. and COURTS, A. 1963. Practical Analytical Methods for Connective Tissue Proteins. Spon, London. 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. HAMILTON,P. B. and ANDERSON,R. A. 1954. Demonstration of ornithine in gelatin by ion-exchange chromatography. J. biol. Chem. 211, 95-102. HANNIG, K. and NORDWIG, A. 1964. Structure and Function of Connective and Skeletal Tissue. (Edited by FITTON JACKSON, S., HARKNESS, R. D., PARTRIDGE, S. M. and TRISTRAM, G. R.) p. 7. Butterworth, London. HERRING, G. M. 1964. Chemistry of the bone matrix. Clin. Orthopaed. 36, 169-183. JACKSON;D. S., LEACH, A. A. and JACOBS,S. 1958. The amino acid composition of the collagen fractions of rabbit skin. Biochim. bioohvs. Acta 27.418-420. JACOBS, S. 1962. The quantitative determination of nitrogen by a modification of the indanetrione hydrate method. AnaZyst, Lond. 87, 53-57. 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 Biol. 2,120-l 26. LEAVER, A. G. and SHUTTLEWOR~, C. A. 1966a. Peptides of bone and dentine. J. Bone Jt Surg. 48-B 851. LEAVER,A. G. and SHUTIZEWORTH,C. A. 1966b. The isolation from human dentine and ox bone of phosphate-containing peptides. Archs oral Biol. 11, 1209-1211. LEAVER, A. G. and SHUTIZEWORTH, C. A. 1967a. Observations on the composition of peptides isolated from bone and dentine. J. Bone Jt Surg. 49-B, 38 1. LEAVER,A. G. and SHUITLEWORTH,C. A. 1967b. Fractionation of the acid-soluble nitrogen of bone and dentine. Archs oral Biol. 12,947-958. LEAVER,A. G., SHUITLEWORTH,C. A. and TRIFFIIY~,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. RUBIN, A. L., PFAHL, D., SPEAKMAN,P. T., DAVISON,P. F. and SCHMITT,F. 0. 1963. Tropocollagen: significance of protease induced alterations. Science, M. Y. 139, 37-39. SHUT~LEWORTH,C. A., 1967. Studies on non-collagenous peptides isolated from ox bone. Ph.D. Thesis. University of Liverpool. STAHL, G. and KALTENBACH,U. 1965. In: Thin Layer Chromatography (Edited by STAHL, G.) p. 461. Springer, Berlin. 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. WASSERMANN,R. H. 1967. Fifth European Symposium on Calcified Tissues, Societe d’edition de l’eiseignment superieur, Bordeaux. In press. WILLIAMSON,M. 1968. The distribution of mucosubstances in the cartilage plate of young rabbits. J. Bone Jt Surg. In press.
Vol. 13, pp. 509425,
1968.
Pergamon Press.
Printed in Gt. Britain.
STUDIES ON THE PEPTIDES, FREE AMINO ACIDS AND CERTAIN RELATED COMPOUNDS ISOLATED FROM OX BONE A. G. LEAVER and C. A. SHUTTLEWORTH* Department of Dental Sciences, School of Dental Surgery, University of Liverpool, Liverpool, England Summary--The acid-soluble nitrogenous components of ox bone were fractionated by gel filtration and ion-exchange chromatography. The overall amino acid composition and the relative proportions of free and peptidsbound amino acids in the molecular weight ranges < 750 and 750-5000, were determined by automatic amino acid analysis. The material of mol.wt. < 750 was shown to include 84 per cent bound and 16 per cent free, amino acids. Glycine was predominant (over 50 per cent) with substantial amounts of both dicarboxylic and basic amino acids. Ornithine existed only in the free form while lysine and serine occurred more plentifully in the free than in the peptide bound state. The overall composition of the peptides in the 750-5000 mol.wt. range showed 28 per cent glycine, 13 per cent alanine, 15 per cent basic and 13 per cent dicarboxylic amino acids. High voltage electrophoresis was used to obtain thirteen fractions in each of which one peptide predominated and eight fractions which contained closely related peptides. Arniio acid analysis showed these to include fractions containing 80 per cent dicarboxylic acids, others containing 60 per cent basic amino acids, together with “neutral” fractions in which both basic and dicarboxylic acids accounted for over 20 per cent of the total. “Non-polar” peptides with 80 per cent of glycine and alanine were also found together with other fractions rich in serine. No fractions included hydroxyproline and proline was present in only a few. Preliminary investigations of the fractions of mol. wt. > 5000 suggested the possible occurrence of free peptides and glycopeptides, relatively low in carbohydrate content. These fractions contained both
hydroxyproline and hydroxylysine but were clearly not collagen fragments.
THE PRESENCEof acid-soluble peptides in human dentine and ox bone was first reported by LEAVER, EASTOE and HARTLEY(1960). It was believed at that time that these peptides were associated with the citric acid of the tissue. That such an association was probably fortuitous was shown by LEAVER, SHUTTLEWORTHand TRIFFITT (1965) who separated bone citric acid from what appeared to be a mixture of soluble collagen, glycoproteins and various peptides, by a series of chromatographic procedures, which were the basis for the extensive fractionation of the acid-soluble nitrogenous compounds of bone described recently (LEAVER and SHUTTLEWORTH, 1967b). A series of prelimary studies on the minor nitrogenous fractions of bone and *Present address: Massachusetts General Hospital, Boston, Mass., U.S.A. 509
510
A. G. LEAVER ANDC. A. SHUTTLEWORTH
dentine (LEAVERand SHUTTLEWORTH, 1966a, b, 1967a.) demonstrated that peptides, rich in glycine, alanine, serine and in either dicarboxylic or basic amino acids (occasionally both) were released in similar quantity when the tissues were demineralized either with dilute acid or with EDTA at neutral pH. The observations that none of the peptides contained hydroxyproline, that several contained phosphate and that the different peptides showed wide variations in amino acid content suggested that they were worthy of further study. Peptides have been shown to comprise between 11 and 22 per cent of the acidsoluble nitrogen of bone (LEAVERand SHUTTLEWORTH, 1967b; SHUTTLEWORTH, 1967). The major fractions are in the molecular weight range 750-5000, the remaining material representing small peptides and free amino acids. The purpose of the investigations described in this present study was to determine the overall amino acid composition of the peptides in the different molecular weight ranges, to determine the proportions of free and peptide-bound amino acids in the fractions of mol. wt. < 750 and to isolate and determine the composition of individual peptides in the main group (mol. wt. 750-5000). The results of the previous study (LEAVERand SHUTTLEWORTH, 1967b) showed that only a small proportion of the mucoids and glycoproteins, known to be present in bone (HERRING,1964), was released by acid demineralization of the tissues. It was not, therefore, originally intended to investigate further the fraction comprising material of mol. wt. > 5000. In view, however, of the possibility that this fraction might include traces of compounds previously unrecognized in bone (which could, of course, include larger peptides), some preliminary analyses were carried out. EXPERIMENTAL
AND RESULTS
The acid-soluble nitrogenous material (ASN) was obtained from ox bone as described by LEAVERand SHUTTLEWORTH (1967b). The results presented here refer to work carried out over an extended period during which certain aspects of the fractionation techniques previously described were modified as shown in Figs. 1, 2, 3(a) and 3(b). The actual use of ion-exchange resin and dextran gels and the collection and monitoring of eluates were, however, carried out in precisely the same manner as before. Figure 1 indicates the modified fractionation scheme used in the separation of the ASN into groups of compounds of comparable molecular weight. The use of ion-exchange resin resulted in the complete de-salting of the material whilst also achieving a further degree of fractionation. Table 1 shows the distribution of nitrogen and phosphate between the various fractions. Nitrogen was determined by the ninhydrin method of JACOBS (1962) and phosphate by the method of BERENBLUM and CHAIN(1938). The distribution of nitrogen between the fractions is similar to that reported in a previous study (LEAVERand SHUTTLEWORTH, 1967b) except that rather more has been left unaccounted for. This may be ascribed to the omission of Fraction 0 from the analysis and from losses accruing during the present more extensive fractionation. As
PEPTIDESAND FREEAMINOACIDSOF OX BONE
511
ASN
I
in 0.5 N HCl
Amberlite IR-120 \
/ “Acid, eluok I’ re-applied to IR-I20 in aqueous solution ;utee wih 4
/
“Aqueous eluote” (Organic acids traces of nitrogen)
eluted with N NH,OH
\
I Sephadex
I
/ Fraction 10 nKll wt. >5000 I Sephodex G-0
G-25 Fraction ‘I’ 28 mol. wt. 750-5000
/ Fraction IA mol. wt. > 5000
Fraction 48 mol. wt. <750
I Fractwxl 38 mol. wt. <750
?
Fmctii 2A~s~~~ mol. wt. 750-5000
elwte”
Sephadex G-25
“Ammonia eluote”
Fraction 0 (Disuxded)
“Ammonia
~?kctica 4A mol. wt.<750 mol. wt.<750
FIG. 1. Fractionation of bone ASN after precipitation of calcium as oxalate. (Use of Amberlite IR-120, Sephadex G-25 and G-10 as described by LEAVER and SHUT-~LEWORTII (1967b).
TABLE1. NrrnooE~ AND PHOSPHATE (P) CONTENTOF FRACTIONS SHOWNIN FIG. 1
Fraction
(Toti ASN) 1B
fi
2A 2B 3A 3B 4A 4B *Unaccounted
for
*Lost in fractionation;
Mol. wt. range
r5000 >5000 750-5000 750-5000 < 750 < 750 < 750 < 750
Nitrogen (%of total bone)
% of ASN
0.138 0.030 0.014 o-012 O-008 0.006 o-005 o-001 0.001
loo*0 21.8 10.1 8.7 5.6 4.3 3.6 o-7 0.7
0.061
44.2
Organic Phosphate (P) (%of . total bone) 0~0017 0~00095 0~00045 o*ooo2 O*OOOl -
includes some nitrogen in fraction 0 and may include volatile material.
512
A. G. LEAVER ANDC. A. SHU~LEWORTH
previously observed in a qualitative study (LEAVERand SHUTTLEWORTH 1966b), phosphate was absent from the large molecular fractions and predominated in the peptide fractions in the 7.50-5000 mol. wt. range. In the material of mol. wt. < 750, phosphate was confined to those fractions (3A, 4A) initially eluted with HCl. Prelimary fractionation and analysis of the large molecular fractions
The fractions excluded from Sephadex G-25 (IA and IB) were each further fractionated by applying in 0.1 M NaCl to a 200 g column of Sephadex G-50. The U.V.absorption of the eluate at 280 rnp was, as usual, used to indicate the appearance of sub-fractions. Both 1A and 1B gave very similar elution patterns, each showing an “excluded” fraction (lA1, 1Bl) and two retarded fractions (lA2, lA3, lB2, lB3). The excluded fractions presumably represented material of mol. wt. > 30,000 and the retarded fractions material in the mol. wt. range 5000-30,000. All six fractions were examined for hexose, hexosamine and amino acid content. Aliquots were hydrolysed with 2 N HCl in sealed tubes for 3 hr at 100°C and the hydrolysates investigated by thin-layer chromatography on Kieselgel G using a solvent mixture containing 65 ml ethyl acetate and 35 ml of 66 per cent aqueous iso-propanol. Sugars were located by the anisaldehydejsulphuric acid reagent as described by STAHL and KALTENBACH (1965). Further samples of the six fractions were examined semi-quantitatively for sugar content by an anthrone method described by EASTOEand COURTS(1963). Four fractions (1Al, lA2, 1Bl, 1B2) were found to contain sugar, chiefly glucose, together with small amounts of hexosamine. During the course of the amino acid analyses, described later, it was clear that galactosamine was the predominant sugar amine. The amounts of sugar found were considerably less than had been expected (on the assumption that the four fractions were likely to be representative of known bone mucoids or glycoproteins) and it was not possible to repeat the analyses on larger samples. The two retarded fractions (lA3, 1B3) did not appear to contain any sugar or hexosamine. Each of the six fractions was hydrolysed with 6 N HCl in a sealed tube for 24 hr and the amino acid content of the hydrolysate determined using the Technicon autoanalyser as described later. The amino acid composition of the six fractions is shown in Table 2. Four of the fractions contain both hydroxyproline and hydroxylysine, amino acids typical of, though not peculiar to, collagen. It is clear, however, that not only is the level of glycine in all fractions too low to account for all the hydroxyproline and hydroxylysine being present as part of a soluble collagen fraction, but the general amino acid content of these four fractions is quite unlike that of collagen. Overall amino acid composition of fractions 2A and 2B
Fractions 2A and 2B (representing material of mol. wt. 750-5000 and together comprising 14.3 per cent of total ASN) are of particular interest in that previous work has shown them to consist entirely of a mixture of peptides, several of which have been separated and qualitatively investigated (LEAVERand SHUTTLEWORTH
PEPTLDESAND
TABLE 2. AMINO
513
FREEAMINOACIDSOFOXBONE
ACID COMPOSITION OF FRACTIONS
lA1, 2, 3, lB1, 2, 3 (expressed
as tLmoles per cent) Amino acid AQ* Hydroxyproline Aspartic acid Serine Threonine Glutamic acid Proline Glycine Alanine Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Hydroxylysine Lysine Histidine Arginine
1Al 0.31 2.67 11.22 5.68 5.53 13.89 9.17 9.81 7.14 6.29 3.46 6.61 2.43 2.86 0.88 0.25 4.99 2.31 4.41
lA2 1.12 6.08 9.14 3.51 2.45 12.88 11.89 12.64 7.29 4.18 2.66 6.38 3.39 3.31 0.40 0.37 3.76 2.28 5.56
lA3
1Bl
lB2
lB3
0.48 8.74 7.46 3.84 2.05 10.98 9.93 23.66 8.05 2.88 1.64 3.84 1.28 1.73 0.54 0.68 4.39 1.32 6.40
9.46 9.07 4.94 3.42 12.88 8.85 18.05 7.90 3.64 2.12 4.64 l-69 2.12 0.47 0.52 4.20 1.30 4.64
12.95 1.81 0.90 15.20 13.66 9.13 9.45 4.27 2.33 9.39 4.66 3.82 3.23 3.10 6.02
13.00 5.24 3.10 17.28 8.73 15.33 8.34 3.68 2.11 6.60 2.71 5.63 2.91 4.66
*AQ represents an unidentified peak preceding aspartic acid.
1966b). In the present studies material in this molecular weight range was selected for extensive investigation and for the separation of individual peptides for analysis. In the interest of continuity, however, the overall composition of the peptide mixtures 2A and 2B are presented at this stage. (Table 3). Samples of fractions 2A and 2B were hydrolysed with 6 N HCI and the hydrolysates analysed on the Technicon autoanalyser. Fractions 3A, 3B, 4A and 4B
These fractions represent material of mol. wt. < 750 and are composed of small peptides and free amino acids. Fractions 3A and 3B which contain relatively small proportions of free amino acids together comprised 7 -9 per cent of total bone ASN. Fractions 4A and 4B are composed of approximately equal proportions of small peptides and free amino acids but together make up only l-4 per cent of total bone ASN. Fractions 3A, 3B, 4A and 4B were each divided into two parts so that they could be applied to the auto-analyser unhydrolysed and also after the usual hydrolysis with 6 N HCI. In this way it was possible to estimate free and peptide-bound amino acids and also to determine the proportions of amino acids occurring in the bound and free states.
A.G.
514
LEAVERAND
C.A. SHUTTLEWORTH
TABLE 3. AMINO ACID COMPOSITION OF FRACJTIONS2A, 2B AND OF 2A AND 2B COMBINED (expressed as pmoles per cent) Amino acid
AQ* Aspartic acid Serine Threonine Glutamic acid Proline Glycine Alanine Valine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Omithine Lysine Histidine Arginine
*Unidentified
2A 0.95 4.57 7.30 5.31 11.12 3.90 28.67 14.10 3.20 1.40 3.17 0.90 l-23 0.26 4.37 3.66 3.00 2.79
2B 0.99 3.06 6.51 2.45 5.77 2.23 29.60 13.13 2.48 1.90 4.60 1.59 4.59 0.16 5.19 2.55 5.99 7.10
2A and 2B 0.97 4.13 7.04 4.50 9.56 3.43 28.81 13.83 3.00 I.55 3.58 1.10 2.21 0.23 4.63 3.31 3.89 4.06
peak preceding aspartic acid.
Table 4 shows the proportions of free and peptide-bound amino acids present in the four fractions and in the total material of mol. wt. < 750. Table 5 shows the percentage composition of total amino acids (i.e. peptidebound plus free) and of peptide-bound amino acids in the four fractions. Separation of individual peptides from the fractions of mol. wt. 750-5000 Throughout this work it has been clear that these fractions are of particular interest. They consist entirely of a mixture of peptides free both from carbohydrate and free amino acids and comprise 14.3 per cent of the total ASN. Preliminary studies have shown them to include peptides of widely varying composition and to appear in comparable quantity and form under different conditions of demineralization. It was, therefore, decided to study these fractions in detail and to attempt to separate individual peptides by high voltage electrophoresis at different pH levels. As stated at the beginning of this section, the work described in this paper was carried out over a long period during which the fractionation techniques were modified. A preliminary stage in the fractionation (LEAVER and SHU-ITLEWORTH, 1967b) involved the reprecipitation of bone minerals by making the demineralizing solution alkaline (immediately after filtering off the collagen). When this was done it was found that the large molecular fraction (mol. wt. > 5000) was co-precipitated. During the
515
PEPTIDES ANLI FREEAMINO ACIDS OF OX BONE TABLE 4. PERCENTAGE OF PEPTIDE-BOUND AND FREE AMINO ACIDS IN FRACTIONS OF MOL. WT. < 750
Total ASN (% of)
Fraction
3A 3B 4A 4B Total material < 750
Peptidebound amino acid (%)
(4.3)
Free amino acid (%)
78.87 91.39 51.09 56.91 84.09
ii:; (0.7) (9.3)
21.13 8.61 48.91 43.09 15.91
TABLE 5. AMINO ACID COMPOSITIONOF FRACTIONS 3A, 3B, 4A, 4B (columns Prefer to peptidebound amino acids and T to total free a.nd peptide-bound amino acids)
Amino acid
3A(P)
3A(I)
AC!* Aspartic acid Serine Threonine Glutamic acid Glycine Alanine Valine
0.85 7.09 3.39 1.46 8.60 53.80 4.63 2.03
0.75 6.97 4.17 1.30 9.41 50.33 5.35 1.81
4<4 1.82 0.98 2.82 65.94 2.33 0.98
Iso-leucine Leucine Tyrosine Phenylalanme Omithine Lysine Histidine Arginine
4.43 0.61 2.22 2.45 1.45 2.80 4.13
0.54 3.95 1.98 2.19 3.08 1.91 2.50 3.68
7.26 2.53 1.60 0.88 0.74 1.60 5.51
3B(P)
3B(T)
4A(P)
4A(T)
4.52 3.78 0.89 2.58 61.21 2.13 0.89
2.60 8.79 3.52 2.89 11.84 45.81 4.59 0.09
1.33 7.31 7.38 3.63 12.25 36.48 8.44 1.58
6;3 2.31 3.03 4.21 1.25 1.47 5.04
3.81 0.77 4.49 0.82 6.04 3.86
3-18 1.35 2.29 6.32 2.56 3.08 2.74
4B(P)
4B(T)
-
-
15.21 1.26 5.97 12.50 38.94 IO.86 -
8.65 9.79 3.40 7.11 30.16 6.18 -
5.07 9.23 -
2.88 5.25
1.26 -
20;1 6.28 -
*Represents an unidentified peak preceding aspartic acid.
course of further investigation of the various peptide fractions it was found that the smaller molecules distributed themselves between the “alkaline precipitate” and “titrate” so that an identical peptide would appear in each of these two main subfractions. Thus, the fractionation scheme described below is unnecessarily complex and the alkaline precipitation stage will not be repeated in future work. Figure 2 shows the preliminary fractionation of bone ASN while Figs. 3a and 3b show the procedures used to isolate the sub-fractions from which peptides were subsequently isolated by high voltage electrophoresis. Fractions A, B, C and D (Figs. 3a and 3b) represent material of mol. wt. 750-5000 and correspond to 2A and 2B in the fractionation scheme described in the earlier part of the paper (Fig. 1). h.0.B.13/5--c
516
A. G.
LEAVER AND C. A. SHUTTLEWORTH 2OOg bone 2.51 N HCL ASN
I bone salts
I
NaOH pH II
1 Precipitate
/
Filtrote A
(Fig.301
t Redissolved in N HCl Potossium oxalate solution
Precipitate ( No meosuroble nitrogen)
FIG.
2. Preliminary fractionation
Filtrate
B
(Fig. 3b) of bone ASN.
Separation oj’peptideJLactions by high aoltage paper electrophoresis Fractions A, B, C and D were further fractionated by high voltage electrophoresis on Whatman No. 3 paper (59 x 36 cm), using a Phaerograph high voltage apparatus. Pyridine/pyridinium acetate buffer (0.2 M, pH 5.4) was used and 1000 V applied to the paper for 90 min. Peptides were located by spraying a portion of the sheet with ninhydrin and the corresponding areas were eluted with water. In this way sixteen peptide sub-fractions were obtained. Only one band was common to A, B, C and D, two were common to A and D, and two common to C and D. These apparently identical sub-fractions were combined as suggested by their electrophoretic behaviour. The three combined fractions (referred to as X, Y, Z) were further separated by electrophoresis at pH 2-O (formic acid/acetic acid, O-2 M) using the same electrophoretic conditions. The remaining eight fractions from the initial run at pH 5 *4 were not subjected to this further run at pH 2.0; they represented fast running (i.e. highly charged) material and are referred to as fractions W 1-8. Electrophoresis at pH 2 *O separated a total of thirteen peptides from the three combined fractions. These are designated X l-4, Y 1-4 and Z l-5. The electrophoretic behaviour suggested that the peptides in the X, Y and Z groups represented single homogeneous entities while the W group represented eight sub-fractions in each of which either one peptide predominated or the mixture consisted of peptides of closely similar composition. The results of the amino acid analyses, described below, confirmed this view in respect of the W group but made it clear that each of the thirteen peptides of the X, Y and Z groups, though apparently electrophoretically homogeneous, was, in fact, contaminated with traces of other peptides.
PEITIDES
AND
FREE AMINO
ACIDS
517
OF OX BONE
FlItrote A I Amberlite
in 0.5 N HCL IR-120
/ “Acid eluote”
1 IR-120
/
re-opplied I” 0q”eous solution
+
“\et
T”“‘” No excluded fraction
eluted with N NH,OH I\ Aqueous eluate (Discorded)
eluted with N NH40H “Ammonia &ate”
or ded
fraction
I Sephadex G-IO
“Ammonia eluote”
/\ Fraction B (mol. wt. 750-5000)
Retarded froctton (DIscorded)
Sephadefc G-25
No excld fraction
Re!arded
fraction
1 SeDhadex G-IO
Fraction /
barded fraction (D Iscorded 1
(mol.wt. 750-5000)
FIG. 3(a). Fractionation
of ASN in filtrate A.
FlItrote B 1” 0.5 N HCI i Amberlite IR-120
“Acid eluote”
/
\
re-opplied in oqueous I solution IR-120
/\ Aqueous eluafe
&ted with N NH,OH
(Discorded 1
“Ammonia
eluded with N NH,OH “Ammonia
eluote”
+
Excluded Saphodex fraction (Discarded)
eluote”
?%orded
Sephadex
/ Fraction D (mol. wt 750-5000)
fraction
I G-IO
Rbd fraction (Discarded)
Sephode? G-25 / Excluded fraction (Discorded)
\ Retorded
fraction
1
(mol. wt. 750-5000)
FIG. 3(b). Fractionation
(Discorded)
of ASN in filtrate B.
518
A. G. LEAVERAND C.
A. SH~T~EWORTH
The separation of the 21 peptide fractions, derived by preparative electrophoresis from fractions A, B, C and D (Figs. 3a and 3b) is summarized as follows: Fractions X1-X4. These fractions represent material derived from fractions A and D, which moved approximately 5 cm towards the anode at pH 5 -4 and was separated into four components by further electrophoresis at pH 2-O (at which pH all peptide fractions moved towards the cathode). Fractions Yl- Y4. These fractions represent material, derived from fractions C and D, which moved approximately 2 *5 cm towards the anode at pH 5.4 andwas separated into four components by further electrophoresis at pH 2.0. Fractions 21-25. These fractions represent material, derived from fractions A, B, C and D, which remained close to the origin at pH 5 -4 and was separated into five components by further electrophoresis at pH 2.0. Fractions Wl-W8. These eight fractions were obtained by electrophoresis at pH 5.4 only. Wl-W4 were derived from fraction B, W5 from fraction A, W6 from fraction C and W7 and W8 from fraction D. They represent individual bands, all of which moved towards the cathode for distances varying from 4.5 cm to 17 -0 cm. Amino acid analysis of individual peptide fractions
The peptide fractions obtained after preparative high voltage paper electrophoresis were hydrolysed with 6 N HCl in sealed evacuated tubes for 24 hr, taken to dryness in a vacuum desiccator and dissolved in a specific volume of nor-leucine solution. One ml of the final solution was applied to the resin column of the Technicon autoanalyser. Automatic analysis was carried out using the normal Technicon 20 hr run on a 150 cm column of Type A microbead ion-exchange resin. In all hydrolysate chromatograms the amino acid levels were calculated on the basis of a standard amino acid mixture separated on the autoanalyser under identical conditions. Amino acid composition of peptides and peptide fractions
Table 6 shows the amino acid composition of the peptide fractions. Amino acid content is expressed as pmoles per cent. Most fractions contained a large excess of ammonia which has not been included in the figures. In attempting to interpret the results several considerations must be borne in mind. In the first place the peptides are within the molecular weight range 750-5000 and presumably contain between 6 and 40 amino acid residues, though the upper limit would be higher for those rich in glycine and lower for those rich in basic amino acids. It is clear, therefore, that an amino acid occurring at a level of below 2.5 pmole per cent, and even in some cases below 4 pmole per cent, is unlikely to represent a true component of the major peptide in that fraction. Although fractions X l-4, Y 14 and Z l-5 appeared to be electrophoretically homogeneous they clearly comprise a
-
E.
0.59
0%
E 1.42 -
1% 1.30 0.51 0.51 0.82
:::: 17.18 22.68
0<4 0.10 -
4% -
‘E
16’49
54’52
“:::: 1*88 6 *21 1.78 0 -70 0.44 1.43 1.72
66-24 15ai 0.71 3 -27
f:E
X2
Yl
Xl
W5
0% -
0%
076
2k.E 3.68
1.50
j.;
1.46 .
.
2 -43
f ‘%
‘8’ .‘R
4.30 15.66
. ‘Ei . ‘i’::
YZ
:: 2-17
‘:x 1:60
12 ‘42 13.98 5.17 14.50
X4
171 3 -43 2.21 1.71 2.70 -
Y3
.
::g 4’50 -
;.5:
. 345:
3.58 -
19.82 ‘:‘;: . 2.42 4.83 1.51
-
272 2.80 2.44
-
2.37 1.25 2.08
. “i’E
4.21 9.02
X3
‘Et
co -ON
W3
‘g7-E .. 1%
W4
TABLE 6. AMINO ACID
2 -98
::g
175 2.16
::g 7 -20
;:g
8 ‘85 4.63 11.01 24.51 9.47
Zl
1.21 -
O-84
;T;:
-
d: .‘E! -
. :‘:i
22
23
pRAmoNs
0.87 3.86
OP *aPnoB
Y4
4.57 17.14 24.91 9.95 -
‘22:
W7
-
0.75 0.72 10.76 12.18 5.55 5.81
Wl
W2
173 -
“% 2 -28
:::: lO*OO
7 -53
24
3.34 18.16 15.06 8.56 0 -92 -
:k’s. 6.83 1.43 0.47 1.40 -
;*s.
8 ‘47
Z5
570 0.81 15.51 10.10 13.71 24.12 1 -83
Et
:::
0 -70 2 ~80 13ao 3.81 0 -79
W6
W8
e:
8
4
520
A. G. LEAVER AND C. A. SHUT~LEWORTH
major peptide contaminated with traces of others. Similarly fractions W l-8, believed to be mixtures of closely similar peptides, must include traces of material unrepresentative of that group. Nearly all the fractions contained an excess of ammonia far greater than could be accounted for by the breakdown of amide groups in asparagine and glutamine or by the loss of small quantities of serine, threonine and arginine. Although many of the peptides have been shown to contain phosphate (LEAVERand
SHUTTLEWORTH,1966b) which appeared to correspond closely to the serine and threonine content of the total peptide fractions (LEAVERand SHUTTLEWORTH, 1967a), no attempt was made to isolate serine phosphate by selective mild hydrolysis. It is, however, very likely that much of the serine (and possibly threonine) exists as the phosphate. The presence of this acid phosphate and of excess amonia may account for the fact that some of the fractions did not behave in the electrophoretic separations as would have been expected from their amino acid composition.
DISCUSSION Although the fractionation methods used in the present work differ slightly from those described recently, the general considerations behind the method of preparation of the tissue, the choice of acid demineralization and the use of gel filtration and ionexchange chromatography remain the same and have been extensively discussed previously (LEAVERand SHUTTLEWORTH, 1967b). One of the main conclusions of the previous study was that acid demineralization released only a small proportion of the large-molecular non-collagenous components and that further investigation of these fractions was not intended. It was, however, decided to carry out certain investigations of this fraction (i.e. material of mol. wt. > 5000) in order to see whether larger peptides, free from carbohydrate components, could be obtained and to get some idea of whether the various fractions did, in fact, correspond to known bone constituents (HERRING,1964).
The two fractions excluded by Sephadex G-50 (i.e. mol. wt. > 30,000) together with two of the retarded fractions did, in fact, contain sugars (chiefly glucose) and galactosamine but it appeared that the total carbohydrate proportion of the fractions was comparatively low. The two remaining fractions (IA3 and lB3) contained only minimal traces of carbohydrate and may be regarded as mixtures of peptides of mol. wt. somewhat in excess of 5000. The amino acid analyses showed that four of the six fractions contained both hydroxyproline and hydroxylysine. At first sight this would suggest the presence of traces of soluble collagen but in no case was the glycine content high enough to account for this. This observation, together with the fact that the general amino acid composition of the fractions is quite unlike that of collagen, would suggest that even if the fractions do contain traces of soluble collagen there may well becompounds present, possibly as glycopeptides, which are separate from collagen but contain both hydroxyproline and hydroxylysine. It does, therefore, appear that the large molecular components of bone ASN might be worth further study, particularly in view of the fact that it is in the molecular weight range 5000-30,000 that
PEPTIDES
AND
FREE AMINO
ACIDS
OF OX BONE
521
calcium-binding peptides, corresponding to that isolated from intestinal mucosre by WASSERMANN(1967), might be expected to occur. The fractions of mol. wt. -C 750 represented 9.3 per cent of the total ASN. The chief interest here was in the relative proportions of free and peptide-bound amino acids. Although free amino acids constituted nearly half of the minor fractions 4A and 4B, they represented only l-6 per cent of the total ASN of bone and approximately 16 per cent of the total material of mol. wt. < 750. Glycine was the predominant peptide-bound amino acid and also occurred free in substantial proportions. Ornithine existed almost exclusively as the free amino acid while serine and lysine were more plentiful in the free than the bound state. An unidentified peak, apparently acidic in nature, but not serine phosphate, occurred at a level of approximately 1 pmole per cent. Whether amino acids exist in the free state in the intact tissue is not clear but their isolation in such small quantities would suggest that very little breakdown of peptides during demineralization has occurred. This observation, confirming our previous work, contrasts with the results of GLIMCHERand LEVINE(1966) who found that acid demineralization of bovine enamel greatly increased the proportion of free amino acids when compared with demineralization with EDTA which resulted in most of the amino acids being recovered as components of peptides. Apart from the very high glycine content the overall composition of the fractions of mol. wt. < 750 is similar to that of the major peptide fractions (i.e. those of mol. wt. 7% 5000). These fractions, comprising 14.3 per cent of the total ASN, were considered to be of particular interest because of the considerable variation in composition shown qualitatively in an earlier study (LEAVERand SHUTTLEWORTH, 1966b). High voltage electrophoresis was used to isolate thirteen peptides which appeared electrophoretically pure but which were shown by analysis to represent fractions in which one predominant peptide was contaminated by traces of others. Eight partially purified fractions, representing either single peptides with some contamination from others within the group or groups of closely similar peptides were also analysed. In attempting to interpret the results the limitation placed on the possible number of residues in a peptide by the known molecular weight range (750-5000) must be kept in mind. Although it is clearly impossible to set any specific value (in pmoles per cent) as representing a single amino acid residue, the limitations previously mentioned indicate that several of the peptides must consist of no more than three or four individual amino acids. The twenty-one peptide fractions exhibited remarkable differences in amino acid composition varying from highly acidic (Glu. and Asp. 80 per cent) to very basic (Orn., Lys., His., Arg. 65-75 per cent). Between these limits are fractions (22, 23) low in polar amino acids and consisting largely of glycine and alanine, while others (Zl, X2, W5) are particularly rich in serine and threonine. Other obvious features include the presence of tyrosine in the highly basic fractions W6 and W8 and the presence of traces of hydroxylysine in these two fractions. The high level of leucine in Y2 is also noteworthy. Possibly the most striking observation was the fact that ornithine was the most abundant basic amino acid in the majority of fractions
522
A.G.
LEAVERAND
C. A.
SHUTTLEWORTH
containing reasonable proportions of basic amino acids. The ornithine levels are far too high to have been produced by the breakdown of arginine and the absence of ornithine from several fractions, together with the isolation of free ornithine in the small molecular fractions (which were not subjected to electrophoresis) precludes the possibility of its having been acquired adventitiously from the paper. We must, therefore, accept ornithine as a true and, in fact, major component of several of the fractions. JACKSON,LEACHand JACOBS(1958) reported ornithine as a component of neutral salt soluble collagen from skin, while HAMILTONand ANDERSON(1954) had previously reported its presence in hydrolysates of lime-treated gelatin but ascribed its appearance to arginine breakdown at alkaline pH. Hydroxyproline was absent from all twenty-one peptide fractions, hydroxylysine present in only two and proline in seven, reaching a value of 7 -2 per cent in fraction Zl. This clearly indicates that they cannot be derived from the main chains of the tropocollagen molecule. Although glycine is a predominant amino acid, comprising over 70 per cent in one fraction and over 30 per cent in four others, it would be expected to occur at a consistent level of about 33 per cent in peptides derived from collagen (HANNIGand NORDWIG,1964). The presence of phosphate, very probably as serine phosphate (LEAVERand SHUTTLEWORTH, 1966b, 1967a), invites comparison with the peptides isolated from bovine dental enamel by GLIMMER and LEVINE(1966). The main difference is in the IeveIs of basic amino acids which were present in only very small amounts in the fractions from bovine enamel. Of the twenty-one fractions described in the present study only those such as X2, X4 and W5, in which glycine, glutamic acid, aspartic acid and serine were the main constituents, are comparable to the various fractions described by GLIMCHERand LEVINE.None of the fractions isolated shows any resemblance to the papain-labile “telopeptides” described by RUBIN, et al. (1963), which contained far higher levels of aromatic amino acids. The “telopeptides” were considered to represent extra-helical components of the tropocollagen molecule. The size of the present series of peptides is such that they could not function as intrahelical cross-linkages and, as they clearly cannot be derived from the main chains, the only remaining possibility is that they represent material bound extra-helically and possibly as a series of repeating units. Any regular pattern of attachment would depend on the binding sites and the nature of the linkages. Their acid lability precludes the possibility of covalent linkages and, if binding does occur, it is most likely to be salt-like, particularly in view of the nature of the peptides, many of which are either very basic or very acidic. STEVENand TRISTRAM(1962) isolated peptides and free amino acids from acetic acid soluble calf skin collagen and suggested that such peptides, classi6ed as “non-protein nitrogen associated with collagen”, may be strongly bound to collagen at neutral pH and released at extremes of pH. As some of the peptides described here do, in fact, resemble those reported by STEVENand TRJ~TRAMit is possible that they are bound to collagen at physiological pH and released during acid demineralization, although we have found similar peptides to be released by demineralization with EDTA at pH 7 *4 (LEAVERand SHUTTLEWORTH, 1967a). Whether such binding does occur and, if so, whether it has any biological
PEPTIDES
AND
FBEE
AMINO
ACIDS
523
OF OX BONE
significance is not yet clear, though it is tempting to suggest that a regular arrangement of highly polar peptides, bound to collagen at physiological pH and labile to pH changes, might have a function in nucleation and thus in the mineralization process. If the peptides are unassociated with collagen then they must be free and separate entities, which may either have fulfilled a biological function and thus represent vestiges of previous material, or possess a continuing function in adult bone. It is convenient, therefore, to consider the possible derivation of the peptides together with function. Apart from two neutral peptides most are highly polar, including not only acidic and basic fractions but also others containing both dicarboxylic and basic amino acids in similar proportions and thus potentially amphoteric in character. There has been considerable interest recently in histochemical observations which suggest that the acidic groups of the mucopolysaccharides of ossifying cartilage are initially masked and that the masking agent disappears during ossification. CAMPO and DZIEWIATKOWSKI (1963) described this effect and implicated basic proteins as the masking agents while WILLIAMSON (1968) refers to the possibility of peptide blocking. It might be that the basic peptides isolated from bone represent vestiges of material originally present in cartilage, in which case it would be worthwhile attempting to gain more precise chemical information about the nature of the blocking agents and, in particular, to see whether similar peptides to those obtained from bone can, in fact, be isolated from cartilage. On the other hand it seems unlikely that such cartilage remnants would persist in adult cortical bone which is constantly undergoing resorption and remodelling under the control of osteoblasts and osteoclasts. If the peptides are separate entities, it is likely that they are derived from the bone cells and may be involved in the mineralization process, possibly in association with mucopolysaccharides or glycoproteins. The very high content of dicarboxylic acids in some of the peptides, and the presence of substantial proportions of dicarboxylic acids in the “amphoteric” peptides, suggests a possible role in calcium binding and transport. Although calcium binding is usually associated with larger molecules, such as that described by WASSERMANN (1967), it is intended to investigate the possible calcium-binding ability of all the fractions described in the earlier part of the present work (i.e. mixtures of ASN components in the specific molecular weight ranges). These considerations imply that the bone peptides might be bound to collagen and possess a role in nucleation or that they may be free and represent vestiges of material with an earlier function in cartilage or in the laying down of new bone by osteoblasts. A final possibility is a continuing role in calcium binding and transport. In view of the widely diverse nature of the peptides further research may lend support to one or more of these viewpoints. Acknowledgement-The
assistance.
authors wish to thank Miss G. A.
RIXOM
for technical
524
A.G.
LEAVERAND
C. A. SHUTTLEWORTH
R&n&--Les composes azotb acido-solubles de 1’0s de boeuf sont &pares par filtration sur gel et chromatographie par echanges d’ions. La composition totale en acides amines et les proportions relatives en acides amines libres et lies aux peptides dans le domaine des poids mol&culaires < 750 et 750-5000 sont determintes par analyse automatique d’acides amines. Le materiel de poids moEculaire < 750 comporte 84 pour cent d’acides amines lies et 16 pour cent d’acides amines libres. La glycine est prtdominante (audessus de 50 pour cent) avec des quantites importantes d’acides amines dicarboxyliques et basiques. L’ornithine n’existe que sous forme libre, alors que la lysine et la serine sont plus frequentes dans la fraction libre que dans la fraction IiCe aux peptides. La composition des peptides, dans la fraction depoidsmol&culaireallant de 750-5000,est la suivante: 28 pour cent de glycine, 13 pour cent d’alanine, 15 pour cent d’acides amines basiques et 13 pour cent d’acides amines dicarboxyliques. L’electrophorbse, sous haute tension, permet d’obtenir 13 fractions, dans lesquelles un peptide predomine et huit fractions qui contiennent des composts voisins des peptides. L’analyse d’acides amines montre qu’ils contiennent 80 pour cent d’acides dicarboxyliques; d’autres renferment 60 pour cent d’acides amines basiques ainsi que des fractions neutres dans lesquelles les acides basiques et dicarboxyliques constituent plus de 20 pour cent du total. Des peptides “non polarises”, renfermant 80 pour cent de glycine et alanine, sont retrouves avec d’autres fractions dans la serine. Aucune fraction ne comporte de l’hydroxyproline et la proline n’est pr&.sente que dans certaines portions. Des essais preliminaires des fractions de poids mol&culaire > 5000 suggerent la presence possible de peptides et glycopeptides libres, relativement faibles en hydrate de carbone. Ces fractions contiennent a la fois de l’hydroxyproline et l’hydroxylysine, mais il ne s’agit pas de collagene. Zusammenfassung-Durch Gel-Filtration und Ionenaustauscher-Chromatographie wurden die slureliislichen Stickstoffanteile von Rinderknochen fraktioniert. Der gesamte Aminosluregehalt und die relativen Anteile freier und Peptid-gebundener Aminosauren in Molekulargewichtsbereichen < 750 und 75&5000 wurden durch automatische Aminoslurea&yse bestimmt. Das Material mit einem Molekulargewicht < 750 enthielt 84% gebundene und 16 ‘A freie Aminosluren. Glycin stand mit ilber 50% im Vordergrund neben hemerkenswerten Mengen von Dicarbon- und basischen Aminosluren. Omithin kam nur frei vor, wahrend Lysin und Serin griil3erenteils frei als im Peptid-gebundenen Zustand auftraten. Die gesamten Peptide mit einem Molekularaewicht zwischen 750 und 5000 enthielten 28 “/, Glvcin, 13 “/, Alanin. 15 % basische und 13 ‘A Dicarbon-Aminosluren. Mit Hilfe der I-Iochspanmmgselektrophorese wurden 13 Fraktionen, in denen ein bestimmtes Peptid vorherrschte, weiterhin 8 Fraktionen mit nahe verwandten Peptiden erhalten. Die Aminosaureanalyse ergab, daR diese Fraktionen 80% Dicarbonsluren und andere 60% basische Aminoduren zusammen mit “neutralen” Fraktionen enthielten, bei welchen sowohl basische als such Dicarbonsauren im Anteil von je 20% vorhanden waren. Weiterhin wurden “nicht-polare” Peptide mit 80 % Glycin und Alanin zusammen mit anderen Serin-reichen Fraktionen gefunden. Keine der Fraktionen enthielten Hydroxyprolin, und Prolin war nur in wenigen vorhanden. Vorllufige Untersuchungen tiber die Fraktionen mit einem Molekulargewicht > 5000 deuten auf das miigliche Vorkommen freier Peptide und Glycopeptide mit relativ niedrigem Kohlenhydratgehalt hin. Diese Fraktionen enthielten sowohl Hydroxyprolin als such Hydroxylysin, es waren jedoch sicherlich nicht Kollagenfragmente.
PEPTIDESAND FREEAMINOACIDSOF OX BONE
525
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