::55n
IY..I.SCBROEDER, L. Lf. KAY,J. LEGETTE,L. HONNEN AND F. C. GREEX
[CONTRIBUTIOY S O
1875 FROM
THE
GATESAND CRELLIN LABORATORIES O F CHEMISTRB, CALIFORVIA
1701.76
IVSTITVIC OF
TECMYOLOGY]
The Constitution of Gelatin.
Separation and Estimation of Peptides in Partial Hydrolysates
BY IY.A . SCHROEDER, LOISX I . K.zu, JOANN LEGETTE,LEWISHONNEN AND F. C I I ~ R L O TGREEN TE RECEIVED JATCART 25, 1953 The peptides in partial acidic and basic hydrolysates of gclatin have been separated initially by chromatographing oil thc ion exchanger Dowes-30, and then further resolved by chromatographing in the form of the DSP-peptides on silicic acidCclite. In this manner, 34 peptides (mainly dipeptides) have been isolated, identified and quantitatively estimated. Thesc peptides account for a minimum of 32% of the threonine, 27% of the serine, 22% of the proline, 13% of the hytlroxyproliiic and 20% of the glycine and alanine in gelatin. The nature and quantity of the peptides which have been isolated from gelatin in this study as well as those which other investigators have identified do not support the assumption that the structure of collagen and gelatin may be described by the repetitive sequence -P-G-K- in which P represents prolyl or hydroxyprolyl, G glycyl and R other amino acid residues. The analytical data provide some evidence that the sequence -gly-pro-hyl)ro-gly(or similar sequence) may be of frequcnt occurrence in gelatin and collagen.
I n preliminary reports,'a2 we have presented the initial results of an exploratory small-scale study of partial hydrolysates of gelatin. This study showed that chromatography on the ion exchanger Dowex50 was very effective in separating the complex mixture into several discrete peptide zones. Some of these peptide zones were then converted into the dinitrophenyl (DNP) derivatives and further fractionated by chromatography on silicic acid; the individual peptides so isolated were then identified. The further study of these hydrolysates 011 a larger scale has now resulted in the isolation of 45 peptides of which 34 have been identified with certainty or with reasonable certainty; in addition, inany peptides were present in amounts too small to warrant detailed investigation. Throughout this work, stress has been placed upon the acquisition of quantitative data with respect both to the amount of each peptide in the hydrolysate and to the molar proportions of each amino acid in each peptide; such quantitative information is of greater assistance in the unambiguous interpretation of the data than mere qualitative itemizing would be. In the present report, we present a detailed description of experimental techniques which have been used in this larger scale isolation and identification of the peptides and a discussion of the data obtained, with special reference t o their bearing upon the structure of gelatin and collagen.
Experimental Source and Partial Hydrolysis of Gelatin.-The gelatin which was used for the hydrolyses was Wilson U-COP-CO Special Son-Pyrogenic Gelatin No. 47033 from pigskin. For the acidic hydrolysis, a 2-g. sample of gelatin was dissolved in 2 1 in1. of 3.6 ,V hj-drochloric acid arid heated a t 37" for one week. The originally light tan-colored solution rapidly became a deeper orange-brown but underwent no apparent change in color aftcr about a day of heating. For the basic hydrolysis, 1 g. of gelatin was dissolved in 10 ml. of 0.5 N sodium hydroxide and maintained a t 37" for one week. The hydrolysate was pale yellow. Ion Exchange Chromatography of the Hydrolysates.-The initial separation of the components of the partial hydrolysates was macle on the ion exchanger Dowex-50 by a procedure which differed only in minor detail from the method of Moore and Stein3 for the separation of amino acids on 100(1) W.A . Schroeder, L. IIonnen and 17. C . Green, Proc. N a l . A c n d . S c i . , 39,23 (1!)53), (2) W. A. Schroeder, " T h e Chemical Striicture of Proteins," Ciha Foundation, 1953, p. 184. (3) S.hfnore nnd W. JJ. Stein, J . A i d , Chrm , 102, 663 (1051),
cm. columns. The chromatographic column, 3 . 5 cni. in diameter and 100 cm. long, was prepared exactly as they described. The buffers mere adjusted within the suggcstcd limits of pH and differed only in that the dctcrgent R R I J 35 was omitted. The major variation in procedurc lics iii the fact that the temperatnrc of the chro~natogramsW:LS not altered as recommended by Moore and Stcin but inste:irl was maintained a t 37.5" until the changc to 0.2 sotliuin hydroxide a t which time the column was allowed t o come to room temperature. The flow rate of tlcvc1o~)crthrough the columns iras adjusted t o about 50 nil. per hour :itit1 approsimately .5-ml. fractions were collected. The buffcrs ircri' progressively changed as shown in Fig. 2. The progi-cs? of the chromatogram was asscsscd by removing 0.5-nil. ])ortions of alternate fractions ant1 applying thc riinhytlriii procedure of Moore and Stein.4 One advantage of ion exchange chromatography for the separation of peptides from partial hytlrolysntcs lics in thc fact that the hydrolysate need not be evaporatctl ticfore chromatography. A sample of the acidic hydrolysate ~ v a ~ prepared for chromatography as follows: 4 mi. of h)-clrolysate was diluted to 105 ml. with p€I 4.25 buffer and 1 ml. of B N hydrochloric acid was added. The PH of the solution was 1.4; 100 ml. of this solution (containing the products from 262 mg. of gelatin corrected for moisture and a s h ) WYLY then placed on the ion exchange column. Likcwisc, 3 . 5 nil. of the basic hydrolysate and 2.3 ml. of G N hydrochloric acid were diluted to 106 ml. with PH 4.25 buffer to give :L />IT of 1.75; 100 ml. of this solution (equivalent t o 274 rng. of gelatin) was placed on the column. Although the voluinc, of the sample is large, there is no deleterious effect 011 the subsequent separations if the pH of the sample is lictirecii 1 and 2. After a zone had been detected by thc ninhydrin mcthotl, the appropriate fractions were combined, neutralized to />I1 6.5-7.5, and evaporated to dryness over concd. sulfuric acid in a vacuum desiccator. The residues were stored 31 room temperature. Dinitrophenylation of the Peptides.-As previously tliscussed,' the separation of the peptides from thc acconiganying buffer salts was accomplished by dinitrop1ieii)lntion which was carried out in the following way. All or part of the dried residue from the combined fractions of a zoiie w a q dissolved in an amount of water which varied from 3 to 15 ml. depending upon the amount of salt to be tlissolvetl. To this solution were added 0.1 g. of sodiurn 1)ic~l)on:ite and 0.1 nil. of 2,4-dinitrofluorobenzcne (DKFI3) \rhich 11x1 been dissolved in a volume of absolute akohol equal to twice the volume of water required to dissolve the salts. The mixture was then mechanically shaken for 3 hr. tluriiig which time the salts precipitated. The reaction misturr was next transferred to a separatory funnel with 20 ml. of water and extracted as shown in Fig. 1. The extraction of s o h . 1 with ether removes excess DNFB. Solution 7 is ycllow partly because of the presence of dinitrophenol and partly because the presence of alcohol increases the miscibility of the ether and aqueous phases and thus causes sonic DNPcompound to be carried into the ethcr. Acidification of soli1. 2 and extraction with ethyl acetate will remove :ill but long DNP-peptides and DNP-arginpl ~)rptides. IT solti. 4 is ( 4 ) S , Moore and W.
H. Stein. < b i d . 176, 307 ( 1 W I H )
July 5 , 1954
SEPARATION OF PEPTIDES IN PARTIAL HYDROLYSATES OF GELATIN
very yellow, it probably contains the latter and requires treatment by a procedure to be described below; it is often pale yellow as a consequence of the presence of unidentified materials. Solution 3 was washed to remove any trace of salt; acidification of the water was necessary to prevent extensive return of DNP-peptides to the water phase. The isolation of any DNP-peptides present in s o h . 7 was accomplished by re-extracting into water by the use of the washings (soln. 6). Acidification, extraction, washing and combination with s o h . 5 then followed. Solution 9 will USUally be yellow because of the presence of dinitrophenol which, of course, will turn colorless if acid is added. Solutions 11 and 13 were colorless. Chromatography of DNP-peptides on Silicic Acid-Celite. -The DNP-oeotides which had been mepared and isolated as described t h e preceding section were finally chromatographed on silicic acid-Celite for further separation of mixtures. The chromatographic apparatus, the adsorbent, the packing and prewashing of the column, the types of developer and the method of elution are identical with those used by Green and Kay6 for the separation of DNP-amino acids on silicic acid-Celite. The extension of the methods of Green and Kay to the separation of DNP-peptides has been described p r e v i ~ u s l y . ' ~The ~ ~ ~sequence of chromatograms and developers which is suggested in references 1 and 7 is often sufficient to separate a simple mixture of DNPpeptides into its components but it is sometimes helpful to chromatograph once more and to develop with 12AA6AL,8 16AA8AL or a similar developer. The separation of DNPpeptides in this way is generally satisfactory. Even if the interzones between the DNP-peptides are not wide, the mutual contamination is negligible. Sometimes, of course, complete separation cannot be achieved, but even then quantitative data often permit a conclusion as t o the nature of the peptides. When a DNP-peptide was not extracted by ethyl acetate but remained in soln. 4 (Fig. I ) , arginyl peptides were usually present and required a different procedure from that described above. The aqueous solution was evaporated to dryness over concd. sulfuric acid and then chromatographed on 1: 1 talc-Celite with N hydrochloric acid as the developer. In this procedure, which is similar to one of Sanger? the DNP-arginyl peptides remain fixed while the contaminating salts wash into the filtrate. Elution of the DNP-peptides required 4:1alcohol-6 N hydrochloric acid (by volume) and tended to be somewhat incomplete. The separation of the DNP-arginyl peptides was achieved by the use of the MM system of Sanger.9 Identification of the DNP-peptides.-After a DNP-peptide had been separated, an aliquot portion was removed, the ultraviolet spectrum was taken, and the quantity was determined. A known amount of peptide was then hydrolyzed in 10 ml. of refluxing doubly glass-distilled 6 N hydrochloric acid. The time of hydrolysis was 2 hr. for a DNP-prolyl or -hydroxyprolyl peptide and 4 hr. for all other peptides. As previously pointed out,' the spectrum of a DNP-prolyl or -hydroxyprolyl peptide is characteristic and permits ready distinction from other DNP-peptides. A 4hr. period of hydrolysis has been found to hydrolyze completely all peptides which have been encountered in this work with the exception of DNP-val-gly.lo Experiments on the hydrolysis of known DNP-glycyl, -prolyl and -hydroxypmlyl peptides" have shown that about two-thirds of the DNP-glycine survives a 4-hr. hydrolysis in refluxing 6 N hydrochloric acid, so that no difficulty is encountered in detecting peptides with N-terminal glycine. On the other hand, no DNP-proline survives the 2-hi. hydrolysis of a DNP-prolyl peptide under these conditions. However, about 20% of N-terminal DNP-proline is converted to free proline and unless care is taken in the interpretation of the results it may be concluded erroneously that proline occupied a position in the peptide other than N-terminal. When a DNP-hydroxyprolyl peptide is hydrolyzed for two
Dinttrophenylationmixture lSoln I) o n d 2Cmt woter Extract w i h 4x25ml ether
A c i d l y with 6 N HCI E x t r o c t with 2x25ml and 5110 mi EtOAc
Wosh w t h
h
( 5 ) F.
C.Green and L. M. Kay, A n d . Chem., Z4, 726 (1952). (6) W.A. Schroeder, THISJOURNAL, 74, 5118 (1952). (7) W.A. Schroeder and L. R . Honnen, ibid., 76, 4615 (1953). (8)Abbreviations as in Green and Kay.5 (9) F. Sanger, Biochem. J . , 46, 563 (1949). (10) The abbreviations and representation of amino acid sequences follow E. Brand (Ann. N . Y.Acod. Sci., 47,187 (1946)) and F.Sanger and H. Tuppy (Biochrm. J . , 49,463 and 481 (1951)). (11) F. C. Qreen, unpublished results.
3557
1sa1n 51 discard
Make bosic
W m h os Soln 3
I
(Soln 131 discord '
s
o
l
i
~
p
o
d
EtOAc e
Resjdue of ONP-compounds
Fig. 1,-Procedure for the extraction of DNP-peptides from the dinitrophenylation mixture. hours, about 40% of the DNP-hydroxyproline escapes destruction and another 25% is converted to free hydroxyproline. Accordingly, DNP-prolyl and -hydroxyprolyl peptides may be detected readily because of their characteristic spectra and may be distinguished easily on the basis of their hydrolytic behavior. After the DNP-peptide had been hydrolyzed, the extraction of the N-terminal DNP-amino acid and the subsequent dinitrophenylation of the amino acids which constituted the remainder of the peptide followed the described proceduree with the exception that during the latter half of the work the conditions of dinitrophenylation were altered to conform with the best procedure which had been found in studies of the dinitrophenylation of amino acids.l2 The DNP-amino acids so obtained from the peptide were identified by the method of Green and Kay.' After identification, the quantity of each amino acid was determined spectrophotometrically as described below, When DNP-arginyl peptides were being studied a different procedure was necessary. If arginine was N-terminal, the DNP-arginine did not extract from the hydrolysate and had to be separated from the free amino acids by means of talcCelite as described above. During this procedure the free amino acids passed into the filtrate and could subsequently be dinitrophenylated and studied as required while the arginine was eluted and identified on a Sanger MMc01umn.~ If arginine was not N-terminal, it was present in the extracted hydrolysate and could then be dinitrophenylated as usual. After extraction of the dinitrophenylation mixture first to remove ether-soluble DNP-amino acids, the aqueous solution of DNP-arginine was passed through talcCelite to remove salt and the DNP-arginine was then identified on the M Bcolumn. ~ A peptide usually could be identified unequivocally by comparing the molar proportions of the constituent amino acids with the amount of DNP-peptide which had been hydrolyzed. The recovery of the constituent amino acids usually ranged between 50 and 90% of the amount of DNPpeptide hydrolyzed. If the chromatography of the DNPpeptides on silicic acid did not result in complete scGaration of a mixture, the nature of the DNP-peptides in the mixture often could be deduced from the quantitative data. The more disparate the amounts of the peptides in the mixture, the simpler such an interpretation naturally was. As another criterion of identity, the chromatographic behavior of each DNP-peptide was compared either with that of the synthetic compound if it was available or with that of (12) W. A. Schroeder and J. LtGette, T H I J~O U R N ~ L , 7 6 , 4612 (lesa).
3558
76 I
I
I
I
I
ACIDIC HYDROLYSATE
19
-
-_
pH 3 4 2
~~
I
I
I
-
I
m.m.xxxI 8
10-
r k 3
I
I
I
I
I
pH 4 2 5 I
I
I'
23
1.0 -
24
/
28
0.5 -
-I
6
0 ta 0
BASIC HYDROLYSATE 1.0 -
0.5
-
100
r
.
200
300
pH 3.42
FRACTION NUMBER
400
-
500
-_
600
700
800
4
pH 4.25 -~
Fig. 2.--Separation of amino acids and peptides in partial acidic and basic hydrolysates of gelatin by means of chromatography on Dowex-50: column dimensions, 3.5 X 100 cm. ; temperature of chromatogram, 37.5'; developers, buffers of PH shown; fraction size, about 5 ml.; sample, equivalent to about 250 mg. of gelatin. similar compounds; the comparison was interpreted in the light of the generalizations between the structure of DNPpeptides and their chromatographic behavior as they have been deduced from synthetic peptides.' It has been found that in all cases the chromatographic behavior corresponded with that which would be expected of the peptide. This criterion was also valuable in drawing conclusions as to the nature of the peptides in an inseparable mixture: for example, if serine and alanine were found as N-terminal amino acids and threonine and proline as the other amino acids of the peptides, the peptides in such a mixture could only be ser-pro and ala-thr because ser-thr and ala-pro would separate easily. Quantitative Determinations by Spectrophotometry.The quantitative determination of all DNP-amino acids and DNP-peptides has been carried out spectrophotometrically in glacial acetic acid. The spectra of all DNP-amino acids and DNP-peptides with the exception of those of DNPproline, DNP-hydroxyproline and their peptides exhibit a main maximum at 338-343 mp in this solvent. Hence.
the molecular extinction coefficient of a compound with this maximum has been assumed to be 1.61 X l o 4 liters per mole crn.6,1pa t the maximum except in the instance of lysyl peptides in which the value is assumed to be 3.22 X I O 4 because of the presence of the two D N P groups. The value 1.75 X lo4 was used for DNP-proline, -hydroxyproline and derivatives. The error introduced by the assumption of such average values probably does not exceed &lo%.
Results Initial Separation of Peptides on Dowex-50.The results of the large-scale chromatograms of the partial acidic and basic hydrolysates of gelatin are presented in Fig. 2 in which the optical densities, derived from application of the ninhydrin procedure to the fractions, are plotted against the fraction number. All of the zones in Fig. 2 are numbered; the components of each zone are indicated
July 5, 1954
SEPARATION OF PEPTIDES IN PARTIAL HYDROLYSATES OF GELATIN
3559
TABLE I QUALITATIVEAND QUANTITATIVEDATAON SOME PEPTIDES IN ACIDICAND BASICPARTIAL HYDROLYSATES OF GELATIN
ISO-
Number I
Peptide or amino acid Ala-ala
I1 I11 IV V
Ala-arg Ala-(arg,gly) Ala-asp Ala-gly
VI VI1 VI11 IX X XI XI1 XI11 XIV
XVI
Ala-lys Ala-pro Arg-(ala,gly) Arg-glY-glY Asp-arg ASP-(arg-gly) Glu-ala Glu-arg Glu-(arg.gly) Glu-gly GIy-ala
XVII XVIII
Gly-asp Gly-glu
XIX
Gly-pro
xv
xx XXI XXII XXIII XXIV
xxv
XXVI XXVII XXVIII XXIX
Gly-pro-ala Hypro-gly Leu (ileu ?)-glu Leu (ileu ?)-hypro LYs-glY Pro-(gly,lys) Pro-ser Pro-thr Ser-ala
xxx
Ser-arg Ser-gly
XXXI XXXII XXXIII XXXIV
Thr-ala Thr-gly Val-glu Val-gly
lated from zone n0.a
17 50,51 32 32 9 15,16 50 28 50 32 32 25 25 15 25 25 14,15 17 51 9,lO 15 49 18,19 52 19 15 49 19 16 28 28 50 50 16 50 29 15 49 15,M 13 17 19
Totald
pmoles Rank peptide in OF order amino of acid er de258 ~.~ Teasmg.
gelatinb
4.6 2.1 3.0 0.9 1.1 13.0 4.8 1.9 3.0 0.2 0.4 1.2 0.7 6.6 1.8 1.0 4.5 9.0 6.0 1.0 7.0 2.3 10.4 61.8 3.5 35.6 10.5 0.4 1.7 1.8 1.0 1.0 0.7 1.5 0.5 0.9 17.4 1.0 1.1 17.5 0.5 4.1
211.4
Ala ASP
Glu G~Y LYS Pro
Ser Thr
9 45 2 38 5 8 44 33 6
4 40 3 39
74' 32 26
1%
quantity
9 14 27 21 5 15 13 34 32 20 30 8 16 23 10 6
Percentage' of the total amount of amino acid in nelatin accounted for by amount of peptide isolated Leu Gly Hypro (ileu) Lys Pro Ser Thr Val Ala Arg Asp Glu
-
3.5 1.6 1 . 1 2.4 0.3 0.7 1.0 0.4 5.0 1.8 0.7 1.1 0.1 0.2 0.3 1.0 1.1 0.6 0.7 2.5 1.5 0.8
33 18 17 25 26 29 19
2.4 0.9
>O
