ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 107, 23-29 (1961)
Studies XXVI. Digestion
on of Ovine
GUNNAR From the Hormone
Research Laboratory, Received
ASD CHOW HA0
Unitrer,sit!J of Cnlifornio, January
IVarious fractions of lact,ogenic hormone with difierent mohilities in starch gel electrophoresis have been digested with rhymotrypsin and trypsin. So differences in the digestion rates were found when the enzymic reaction was followed in the pH stat bJ recording the alkali uptake during digestion. Z;o significant differences were encountered when the peptide maps of the digested samples were compared. The number of bonds hydrolyzed by the enzyme as calculated from the alkali uptake during digestion was in agreement with the rate of alkali uptake during dinitrophenylation of the digested samples. No significant difierences in the amounts of ether-soluble NH2-terminal I>NP-amino acids were found in the digested and dinitrophenylated fract,ions. These results indicate a very close st,ructural resemblance between the various fractions.
rate of the digestion can be followed quantitatively, valuable information for selecting the proper conditions of digestion for future structural determinations is also obtained. The results of such studies on the various fractions of lactogenic hormone are reported in this paper.
Ovine lactogenic hormone (prolactin) prepared according to the method previously described (1) has been shown to consist of several components (Z-4). By means of chromatography on DEAE-cellulose, it was possible to separate the hormone into four fractions (4): A1 , A, B, and C, of which A, represents chiefly monomeric material. These fractions cannot be differentiated by amino acid and terminal residue analyses. It was deemed of interest to investigate further the question of chemical identity or difference in connection with these prolactin fractions, by the use of additional techniques. Digestion with proteolytic enzymes such as trypsin and chymotrypsin, followed by chromatography and electrophoresis to obtain a two-dimensional peptide map of the products, represents a sensitive method for detecting differences in the chemical composition of proteins. If the enzymic reactions are performed under conditions such that the
Purified lactogenic hormone was prepared as described in references (5) and (11, with the modifications reported in reference (4). This material will be designated prolactin. Oxidized prolactin was prepared as dcscribed in reference (6). The different fractions of prolactin were prepared according to reference (4j. They will be designated Fraction A, Fraction A, , Fraction B, and Fraction C. The protein content of the samples was calculated from nitrogen determinations, and all calculations were based on corrected values. DIGESTIOX
1 This work has been supported in part by a grant (A-6097) from the National Institutes of Health of the United States Public Health Service.
In a typical experiment, 20.0 mg of lactogenie hormone was mixed with 2.0 ml of wa33
. . . ..
. . .. 0
FIG. 1. Alkali uptake during pH stat. 170 solution of Fraction
Hours digestion with chymotrypsin Al Enzyme:hormone ratio
ter and the mixture was titrated in the pH stat (the Combitrator 3-D, Methran Ltd.) at 32°C with 0.0200 N sodium hydroxide to bring the pH to 8.5, resulting in complete solution of the protein. A solution (0.10 ml) of chymotrypsin (Armour, lot no. 381092) containing 2.0 mg per milliliter (enzyme: hormone ratio 1: 100) was then added with a blow-out pipet, and the alkali uptake during the reaction was recorded. The reaction was allowed to proceed for 12 hours. After the reaction was completed, the mixture was transferred quantitatively to a weighed test tube and immediately frozen and lyophilized. The alkali uptake as a function of time during chymotryptic digestion of Fraction A1 is shown in Fig. 1. The corresponding curves of all the other fractions had the same characteristics as that of A1 , indicating that there were no differences among them in the rate of the enzymic reactions. With oxidized prolactin, digestion proceeded at a higher rate (5.7 bonds broken in 5 minutes, 9.2 bonds broken in 15 minutes). Table I shows the total alkali uptake during complete chymotryptic digestion of prolactin and the various fractions; the table also shows the estimated number of peptide bonds split, calculated on the basis of an assumed average pK value of 7.5 at 25°C for the a-amino groups liberated, and of the fact that the pK is lowered 0.0257 per degree as the temperature is raised (7, 8). It is evi-
of Fraction A1 , as recorded by = 1: 100. Temp. 32”C, pH 8.5. TABLE
OF LACTOGENIC HORMONE WITH CHYMOTR~PSIN Estimated Alkali uptake number of (moles per mole bonds split per of protein) mole of protein (see text1
Prolart in Fraction A Fraction A1 Fraction I? Fraction C Oxidized prolactin a See reference
17.8 19.0 16.6 16.7 19.2 16.3
17.9 18.1 20 8
dent from Table I that) chymotrypsin splits about 19 peptide bonds, which is higher than would be expected on the basis of the aromatic residues (4) present in the molecule. Therefore, cleavage at other bonds in the chain must also have taken place. This was also reflected in the peptide maps of the digested fractions, which showed a fairly large number of spots (Fig. 2). There were, however, only small differences involving minor spots between the maps of the different fractions, differences which were not considered significant. Peptide maps were made according to the technique of Icats et al. (9).
The results of dinitrophenylation (7) of the intact and digested fractions are shown in
b FIG. 2. (a) Peptide map of chyrnotryptic. digest, of prolactin: chrcmwtography nol-acetic arid-water i4:l:Sj; electrophoresis in pyricline (0.042 :Il)-acetic buffer, pH 3.5, 38 volts/cm; 100 rnirlut.es, Whatman XMM ptlper. (1)) f’cptide digest of prolactin. Conditions: see Fig. 2a.
in n-butaacid (0.5 Jr) nup of tryptic
Table II and Fig. 3. The difference in alkali uptake during dinitrophenylation of intact and digested samples should be proportional to the number of NHZ-terminal groups liberated during digestion. The estimated number of amino groups has been calculated on the same basis as above with respect to pK value and temperature. The results, shown in Table II, are in agreement with the number of bonds broken as calculated from the alkali uptake during digestion (Table I). Determination of ether-soluble dinitrophenylated (DNP)-amino acids (10) in the digested and dinitrophenylated fractions showed no significant differences in this respect between Fraction Ai and the other fractions, as can be seen from Table III. The figures are not corrected for losses during TABLE
UPT~IKE DURING DINITROPHEN~L.~TION OF LMTOGENIC HORMONE BEFORE AND AFTER DIGESTION WITH CH~MOTRYPSIN
Estimated number of released N-terminal a.&no groups (see text)
30.4 30.4 31.0 28.2 31.0 30.2
51.3 50.9 50.2 47.6 53.2 43.6
20.9 20.5 19.2 19.4 22.2 13.4
18.9 18.5 17.3 17.5 20.0 12.4
Alkali uptake (moles per mole of protein) Preparation
-IProlactin Fraction Fraction Fraction Fraction Oxidized lactin
A A1 B C pro-
hydrolysis and chromatography and thus do not represent absolute values, but since they all suffer from the same errors they are valid for the purpose of comparison of the different fractions. DNP-arginine was in all cases qualitatively detected in the aqueous phase. DIGESTIOK WITH TRYPSIN Lactogenic hormone, 20.0 mg, was mixed with 2.0 ml of water and the mixture was titrated in the pH stat with 0.0300 N sodium hydroxide at 37°C to make a pH of 8.5; 0.10 ml of a 0.15 n/r solution of calcium chloride was then added (final Ca++ coiicentratioii, approximately 7 X 1O-3 M). When the pH h ad again reached 8.5, 0.10 ml of a solution of trypsin (Worthington, lot no. TRL6122) containing 2.00 mg per milliliter was added and the alkali uptake during the reaction was recorded. After about 2-4 hours, another 0.10 ml of the same solution of trypsin was added to make a final enzyme: hormone ratio of 1:50. After about 20 hours, the reaction mixture was quantitatively transferred to a weighed test tube, frozen, and lyophilized. The alkali uptake as a function of time for the digestion of Fractions A, , A, B, and C was identical, indicating that all fractions were hydrolyzed at the same rate, with the exception of oxidized prolactin which was digested at a higher rate (11.8 bonds in 2 minutes, 13.4 bonds in 5 minutes, and 15.4 bonds in 1.5 minutes). Table IV shows the total alkali uptake and the estimated number of bonds broken during 20 hours of hyI
FIG. 3. Alkali uptake during dinitrophenylation of chymotryptic digest of Fraction A, , as recorded by pH stat. 10.00 mg of digested hormone, 1.5 ml 1.5 M KCl, 0.1 ml FDNB, temp. iO”C, pH 8.0.
From precipitated DSP-peptides
From ether-soluble DNP-peptides /
Aspartic acid Glutamic acid Swine Threoninc C:lycine Alanine Yaline Leurine IMysine
2.1 0.3 1.3 0.4 0.1 1.0 0.5 1 0.0 1 0.7
2.1-3.0 0.1-0.5 1.2-l. (5 0.4-0.0 0.3-0.5 1.0-1.2 0.s0.G 0.8-0.9 0.bO.li
‘1 Residues per mole of pwtein. raphy. ‘I Range values.
0.1 0.1-0.2 0.1 O.li-0.7 0.1-0.2 Figures
drolysis of prolactin and the various fractions with trypsin. The uuulber of bonds broken. which was calculated as described above ‘for the chymotrypic digestion, esceeds the sum of the number of arginine aud lysine residues (-l). This is also reflected in the peptide maps where the number of spots is also higher (see Fig. 3). There were, however, no significaut differences in the maps of the different fractions. It is interesting to note that about half the number of peptide bonds broken are hydrolyzed during the first 30 minutes, after which tinle the reaction slows down. Doubling the enzyme conceutration after 2-3 hours has very little effect on the rate of the reaction. Results of dinitropheuylation of the tryptic digests are preseuted in Table V. The uumber of KH2-terminal groups calculated from the alkali uptake during diuitropheuylation are iu agreement with the number of bonds broken during digestion, just as described above in the case of the chynlotryptic digestion. The KH.!-terminal residues in the digests arc shown in Table VI; the figures are not corrected for losses during hydrolysis arid chromatography. In all instauces, DIil’-arginine was detected in the aqueous phases. 1~ISCUSHIOX
Recording the alkali uptake during dinitrophenylation of the digested proteiu serves
for losses during
0.5 1 .:3 0.6 2.1 0.0
0.G0.5 1. l-l .:I 0.hO.li 1.3-l.fi ~ O.wl.T
Estimated number of bonds split per mole of protein
21.1 21.3 21.0 20.8 21.6 21.2
23.0 22.11 22.0 22. 1 23.2 32.8
~)I~~ITI~O~,HEN~L.\TION HORMONE AFTER I~IGEXTIUN WITH TKYPSIN
Alkali uptake (moles per molr of protein)
/ l~:stimatud number of released N-termmal amino groups lye text)
__ Prolactin Fraction Fraction Fraction Frartion Oxidized la&in
-4 ril B C pro-
0.5 1.2 O.-l 1 ci 0.0
Alkali uptake holes per mole of protein)
A ill B C prolactin
AI,K.II,I IJPTAKE OF L.\CTOGEXIC
OF L.~CTOGENI(I HORMONE WITH TRYPSIN
Prolactin Fraction Fraction Fraction Fraction Oxidized
.~~ 52.9 51.8 55.9 51.6 56.9
2’2.5 23.5 24.9 23.4 1 25.9 22.2
20.-l 21.4 22.6 21.3 23.5 20.2
From precipitated DNP-peptides
Aspartic acid Glutamic acid Serine Threonine Glycine Alanine Leucine Dityrosine
Frxion 1.2 1.5 0.9 0.3 0.8 1.7 2.8 1.4
ACID RESIDUES IN TRYPTIC DIGESTS DETERMINED
-i From ether-soluble DNP-peptides
Fractions* Oxidized A, B, and C prolactin 1.2-2.4 1.3-1.6 0.7-1.2 0.2-0.4 0.~0.8 1.3-1.7 2.2-3.0 0.8 1.1
OF LACTOGENIC HORMONE
1.1 1.3 0.7 0.2 0.8 1.8 2.6 1.6
0.5 0.2 0.3 0.1 0.3 0.3 0.7
Fractions* Oxidized A, B, and C prolactin 0.2-0.5 0.1-0.2 0.1-0.3 0.1 0.2-0.5 0.2-0.3 0.5-0.7 0.1-0.4
0.2 0.1 0.1 0.1 0.3 0.1 0.6 -
SUITI Fraction A1 1.7 1.7 1.2 0.4 1.1 2.0 3.5 1.5
Fraction’ Oxidized A, B, and C prolactin ___~ 1.6-2.7 1.4-1.8 0.9-1.1 0.2-0.5 0.8-1.1 1.5-1.9 2.9-3.4 1.0-1.4
1.3 1.4 0.8 0.3 1.1 1.9 3.2 1.6
u Residues per mole tography. * Range values.
as a check on the number of broken bonds determined on the basis of alkali uptake during enzymatic digestion in the pH stat. The correspondence between the values obtained by both methods is good, considering the relatively low accuracy (approximately f.5 %,) of the graphic determination of the alkali uptake, which involves extrapolation of the end slope of the pH stat curve to zero time, and also in view of the fact that the values representing the number of amino groups released are calculated from the difference between alkali uptake during dinitrophenylation of intact and digested samples. When oxidized prolactin is digested with chymotrypsin, the number of NH*-terminal amino acids calculated from alkali uptake during dinitrophenylation is lower than the number of bonds broken, as determined by alkali uptake during digestion. This phenomenon might be due to changes, caused by oxidation, in the pK values of peptides containing serine as NH?-terminal amino acid, for the yield of serine in the subsequent end group determination is also low. The results of quantitative determination of NHS-terminal amino acids in the digested samples suffer from fairly large errors due to destruction during hydrolysis and losses during chromatography. The sum of h-H,terminal amino acid residues in the chymotryptic digest of Fraction A1 is 10.1 (Table III) and in the tryptic digest, 13.1 (Table
for losses during
VI). Based on the number of bonds broken, as determined by the alkali uptake during digestion and dinitrophenylation, this represents about a 62 % over-all yield of Nterminal amino acids. All the fractions investigated gave the same yield (except the chymotryptic digest of oxidized prolactin) . It is well known (11) that the yields of DNPamino acids in acid hydrolysis of DNP-peptides show considerable variation, depending upon the structure of the peptides; indeed, in some cases (7) they can be as low as 10 %, even when the DNP-amino acid itself is stable under the same conditions. Since NH*-terminal arginine has not been determined, the yields obtained can be considered reasonable. It is interesting to note that although all the fractions of lactogenic hormone contain 9 threonine residues (4), the yield of DNPthreonine is only 0.4 residues per mole. It seems likely, therefore, that only one peptide in both the chymotryptic and tryptic hydrolyzates had threonine as the X-terminal amino acid. Since it is known (4) that the NHgterminal residue of all the fractions is threonine, this peptide must be derived from the N-terminal portion of the molecule. The results, reported herein, of enzymic digestion of the various fractions of lactogenie hormone with chymotrypsin and trypsin show no significant differences between the various fractions, which is a further indication of the previously (4) noted close
structural similarity between them. A recent study (12j has also shown that these fractions camlot be differentiated on the basis of biological activities. It is known that the various prolactin fractions represent differences in degree of aggregation of the hormone. There may also be differences in helical structure or conformation of the protein molecule, differences which cannot be detected by the methods used in the previous investigations (3, 4), as by the method employed in this study. In what other respects they may differ is not known at present. REFERENCES 1. COLE, R. D., .ixr) LI, C. H., d. &of. (‘hem. 213, 197 (1955). 2. COLE, R. D., AND LI, C. H., birch. Biochem. Uiophys.
‘78, 392 (1958).
3. SQUIRE, P. G., ST.UU~I.*N, H., .uw LI, C. I-I., J. Rid. (‘hem. 233, 1389 (1963). 4. SLCYSEI~, JI., .JND LI, C. H., Arch. Hiochem. Biophys. 104, 50 (1964). 5. LI, C. H., (:Ex’Hu-IsI~, I. I., DIXON. J. I)., LEVY, A. L., .\ND HMRIS, J. I., .I. Hid. (‘hem. 213, 171 (1955). Ii. LI, C. H., J. /?io/. (‘hem. 227, 157 (1957). 7. LEVY, A. L., .txn LI, C. H., .I. Bid. (‘hem. 213, 487 (10553.
il. ill., DXEYER, W. J., AND ANMNRES, C. B.. ./. J3iol. (‘hem. 234, 2897 (1959). 10. LEVY, A. I,., .\‘nture 174, 126 (1951). 11. FR.\EN~~EI,~C~R-~~.~T, H., HMRIS, J. I., .\SIJ LEVY, A. L., dlefhods ~iochm. iina/. 2,3(if) (1955). 12. LI, C. H., .\NU FLITS, J). S., Gen. CON/~. Eutlocrinol. (in press). 9.