the purification of nerve growth factor from bovine seminal plasma

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257. No. 14, Issue of July 25. pp. 8541-8548, 1982 Prrnted m U S . A

The Purification of Nerve Growth Factor from Bovine Seminal Plasma BIOCHEMICAL CHARACTERIZATION AND PARTIAL AMINO ACID SEQUENCE*

(Received for publication, July 20, 1981)

Gregory P. Harper#, Robert W. Glanvillel, and Hans Thoenen$ From the +Department of Neurochemistry, Max Planck Institute for Psychiatry, and the YDepartment of Connective Tissue Research, Max Planck Institute for Biochemistry, Am Klopferspitz 18a, 8033 Martinsried/Munich, Federal Republic of Germany

Nerve growth factor (NGF), a protein regulating the development and function of certain neural crest deriv- atives, has been purified from bovine seminal plasma, an extremely rich source of the protein. Around 10 mg of pure NGF (yield, 10-20%) can be isolated from 10 g of lyophilized seminal plasma (around 100 ml of semen). The behavior during purification indicates that, like the NGF in the mouse submandibular gland, bovine NGF exists as a high molecular weight complex that dissociates at extremes of pH to reveal a smaller subunit having NGF biological activity. The isolated low molecular weight form of bovine NGF is a dimer of noncovalently linked polypeptide chains (MF - 15,000 on sodium dodecyl sulfate-polyacrylamide gels), with an isoelectric point of 9.5-10. These properties differ from those of low molecular weight (&subunit) mouse NGF, which comprises two noncovalently linked peptide chains of M, = 13,256 (from sequence studies), and which has an isoelectric point of 9.3. The amino acid sequence of the NH2-terminal 26 residues of bovine NGF has been determined and found to be similar to, but not identical with, that of mouse NGF. Thus, residues 3, 9, and 18, which are threonine, methionine, and valine, respectively, in mouse NGF, are serine, arginine, and isoleucine in bovine NGF.

Nerve growth factor is a protein that regulates the development and normal function of sensory and sympathetic neurons and of adrenal chromaffin cells (for review, see Thoe- nen and Barde, 1980). Until recently, the only known source of mammalian NGF was the submandibular gland of the adult mouse, and the NGF purified from this tissue has been extensively characterized (Server and Shooter, 1977). Recently, however, NGF’ has also been detected in appreciable amounts in the prostate glands of the guinea pig (Harper et al., 1979) and in those of the rabbit and bull (Harper and Thoenen,

* This work was supported by the Deutsche Forschungsgemein- schaft, Grant T h 270/1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “nduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 T o whom reprint requests should be addressed. ’ The abbreviations used are: NGF, nerve growth factor, with the

following prefixed abbreviations: HMW-, high molecular weight; LMW-, low molecular weight; B-, bovine. 7 S and p refer to standard preparations of the HMW- and LMW- forms, respectively, of mouse NGF (see Server and Shooter, 1977); PI, isoelectric point; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography; bis, N,N’-methylenebis-acrylamide; TEMED, N,N,N’,N’-tet- ramethylethylenediamine; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); PTH, phenylthiohydantoin; HPTLC, high performance thin layer chromatography; BU, biological unit of NGF.

1980b). Although all these mammalian NGFs appear to have the same biological effects, at least in the classical assay system of cultured dorsal root ganglia from 8-day chick embryos (Levi-Montalcini et al., 1954; Harper et al., 1979; Harper and Thoenen, 1980b), they exhibit substantial immunological differences (Harper et al., 1979; Harper and Thoenen, 1980a and 1980b), presumably reflecting differences in their biochemical structures.

As the NGF in mouse submandibular glands is secreted exclusively into the saliva and not into the circulation (Wal- lace and Partlow; 1976; Murphy et al., 1977; Suda et al., 1978), it was expected that the NGF in the prostate glands would be secreted into the seminal plasma. Preliminary experiments showed that this is indeed the case, and, moreover, that bovine seminal plasma is an extremely rich source of NGF. The NGF from this source has therefore been purified, and some of its biochemical properties are reported here. The amino acid sequence of the NH2-terminal part of the molecule also has been determined. I t is hoped that comparative studies of mammalian NGFs will provide information on the significance of the various biochemical features of the NGF molecules for their biological and immunological properties.

MATERIALS AND METHODS”

RESULTS

Distribution of NGF in Mammalian Seminal Plasmas- The results of biological assays of NGF in mammalian seminal plasmas are given in Table I. Bovine seminal plasma contains extremely high levels of NGF (approximately 0.7 mg of bovine NGF (BNGF)/ml of semen, based on the specific activity of purified BNGF reported below), while sheep and goat seminal plasmas contain relatively low amounts of the protein. No NGF could be detected in the seminal plasmas of humans or pigs. The biological activities of all the NGFs detected were completely inhibited by affinity column-purified antibodies (Stoeckel et al., 1976) to mouse submandibular gland NGF, this test justifying their identification specifically as nerve growth factors (Harper and Thoenen, 1980a).

Purification of NGF from Bovine Seminal Plasma- Whole, undiluted bovine semen was stored in liquid nitrogen until required. Thawed semen was diluted with ice-cold distilled water (1:l or 12) and centrifuged at 20,000 x g for 60

of “Results” and “Discussion,” Figs. 5 and 6, and Table 111) are Portions of this paper (including “Materials and Methods,” parts

presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnif.ying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Hockville Pike, Bethesda, MD 20814. Request Document No. 81M-1733, cite authors, and include a check or money order for $6.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

8541

8542 Purification of Nerve Growth Factor from Bovine Seminal Plasma

min at 4 "C to sediment the spermatozoa. The pale yellow, slightly opalescent supernatant was lyophilized and stored at -70 "C; around 95 mg of lyophilized seminal plasma (specific activity, 2 BU of NGF/pg of lyophilized material) was obtained per ml of semen.

5-10 g of lyophilized seminal plasma were usually processed for each preparative run, corresponding to around 10-20 X 10'' BU of NGF (-50-100 mg of NGF; E 100% yield). The seminal

TARIX I Distribution of nerve growth factor in mammalian seminal

plasmas The biological unit (BU) of NGF is defined as the amount of

material per ml of tissue culture medium that stimulates the optimal outgrowth of nerve fibers from an explanted dorsal root ganglion dissected from 8-day-old chick embryos and cultured in a standard plasma clot system (Levi-Montalcini et al.. 1954).

Source NCF/ejaculale RIJ

Bovine seminal plasma 860,000 Sheep seminal plasma 28 Goat seminal plasma" 14 Human seminal plasma <0.80 Porcine seminal plasma <73

Bovine prostate gland" 26.000' Mouse submandibular gland 11,ooo'

NGF/rnl semen RIJ

185,000 I5 21

<0.22 <0.45

550" 56,000''

" Figures for NGF levels refer to goat seminal plasma after dialysis against water and centrifugation to remove insoluble material. The apparent levels of NGF before this treatment are lower and incon- sistent, possibly due to the presence of inhibitory factors, but it is also likely that this treatment causes some loss of endogenous NGF activity, by adsorption to the dialysis bags or to the precipitate.

" Figures from Harper and Thoenen (1980b). Figures refer to the NGF levels in BU per organ. for comparison.

" Figures refer to the NGF levels in BU per g of tissue, wet weight, for comparison. The value for the bovine prostate gland is artificially low, as a lot of other tissue has to be dissected to ensure all the disseminated prostate gland in this species is included.

Figures from Harper et al. (1980).

plasma was dissolved in 200 ml of 20 mM phosphate buffer, pH 6.8, and stirred for 15 min at 4 "C to allow the solution to clarify. The solution was loaded onto a column of DEAE- cellulose (2.6 x 35 cm) equilibrated in the same buffer. The material passing straight through this column was then loaded onto a column of carboxymethyl (CM)-cellulose (2.6 X 50 cm) connected directly to the outflow of the DEAE-cellulose column, and also equilibrated in the same buffer (flow rate, -70 ml/h). The two columns were washed extensively with the phosphate buffer and all material passing through them was concentrated by vacuum dialysis; this material contained essentially all the recoverable NGF activity (specific activity, -10 BU of NGF/pg of lyophilized material; yield, -50%).

The concentrated NGF-containing fraction was dialyzed against 0.1 M sodium citrate buffer, pH 3.0, containing 0.4 M NaCI (2 X 5 liters of buffer, 4 "C, 12-24 h) and then loaded onto a sulfopropyl (SP)-Sephadex C-25 column (2.6 X 35 cm) equilibrated in the citrate/NaCl buffer (flow rate, -70 ml/h). The column was washed very extensively with the citrate/ NaCl buffer until the absorbance of the eluate had returned to an absolutely stable base-line. This lengthy washing is essential for a successful purification. The column was washed with 0.1 M sodium citrate buffer, pH 3.0, to remove the NaCl, and then eluted with 50 mM Tris-HC1 buffer, pH 9.0, until the absorbance had again returned to an absolutely stable base- line. Finally, the column was eluted with a linear NaCl gradient (0 to 0.5 M NaCI; total volume, 2 liters) in 50 mM Tris- HCI, pH 9.0, buffer. Pure bovine NGF eluted as the onlv major peak, a t around 0.25 M NaCl. The NGF fraction was concentrated by vacuum dialysis, desalted by dialysis against distilled water (2 X 5 liters, 4 "C, 24 h), lyophilized, and stored at -70 "C (specific activity, 200-500 BU of NGF/pg of protein; yield, 10-20%).

The SP-Sephadex column was finally washed prior to re- use with 25 mM ethanolamine-NaOH buffer, pH 11.5, containing 2 M NaCl. This buffer eluted a significant amount of protein, though little NGF (-1% of the activity loaded).

7 8 9 10 11 12

FIG. 1. SDS-polyacrylamide gel electrophoresis of chromatographic fractions during the purification of bovine seminal plasma NGF. Trac/z I , crude bovine seminal plasma (270 pg); Track 2, material binding to, and subsequently eluted from, the DEAE- cellulose column (50 pg); Track -3. material binding to, and subsequently eluting from, the CM-cellulose column (150 pg); Trach 4, material loaded onto the SP-Sephadex column (270 pg); Track 5, material passing straight through the SI'-Sephadex column (50 pg); Tracir 6, material eluted by 50 mM Tris-HCI, pH 9.0, from the SI'- Sephadex column (270 pg); Track 7. material eluting at low NaCl concentration during the gradient elution of the SI'-Sephadex column (270 pg); Track 8, material eluted by 25 mM ethanolamine-NaOH, pH 11.5, containing 2 M NaCI, from the SI'-Sephadex column (270 pg); Trach 9. calibration proteins, specifically, phosphorylase h (subunit

M, = 94,000). bovine serum albumin (66,200). ovalbumin (43, OOO), bovine carbonic anhydrase B (30,OOO). soybean trypsin inhibitor (21,000). and tr-lactalbumin (14,400) (around 7 pg/band). In other gel tracks (not shown), myoglobin (17,500). lysozyme (14,400). horse cytochrome c (12,4001, and mouse NGF (13.256 and 12.358; see "Discussion") were also used as markers; Tracks 10 (270 pg) and 11 (7 pg) , purified bovine NGF from the SI'-Sephadex column; Track 12, purified mouse NGF (7 pg); only the partially cleaved, 1 IO-residue chain can be seen clearly in this gel (see "I>iscussion"). It should be noted that such heavy loads as shown in Trach 10 tend to overem- phasize the proportion of contaminants in the preparation, as material diffuses out of the polyacr.vlamide gel from the overcongested RNCF band during staining and destaining.

Purification of Nerve Growth Factor from Bovine Seminal Plasma 8543

SDS-Polyacrylamide Gel Electrophoresis-Purification of the bovine NGF was routinely followed using SDS-polyacrylamide gel electrophoresis in 15%. gels. Fig. 1 shows the band pattern for the various fractions during the procedure, and for the final bovine NGF preparation. The protein purity of the latter was judged to be 295% from overloaded gels. Only one major band was evident, with or without treatment of the protein with P-mercaptoethanol, and this treatment did not detectably change the position of the BNGF band. In contrast, our purified mouse NGF (Suda et al., 1978) usually showed two major bands (see “Discussion”) with the lower band strongly predominating; again, the positions did not alter following treatment with P-mercaptoethanol. The subunit of bovine NGF had a higher apparent molecular weight than either of the two mouse NGF bands (13,256 and 12,378 from sequence data; see “Discussion”). Calibration of the gel with

1 2 1 2

FIG. 2 (left) . Isoelectric focusing of bovine and mouse NCFs in polyacrylamide gels. The figure is an enlargement (hence the apparent broadness of the bands, which were in fact very sharp) of the basic region of a pI 3-10 gel, showing the three bands of mouse NGF (Track 1; 40 pg) and the more basic, single band of bovine NGF (track 2; 40 pg). The gel was rather overloaded in order to demonstrate the weakest mouse NGF band (see Moore et al., 1974), and to detect any weak extra bands in the bovine preparation. The proteins used to calibrate the gels are not shown, but the following were run: pepsin (PI 2.9), amyloglucosidase (3.5). ferritin (4.35). soybean trypsin inhibitor (4.55). ovalbumin (4.60). bovine serum albumin (4.8), /i”lactoglob- ulin A (5.2). bovine carbonic anhydrase B (5.85). human carbonic anhydrase B (6.55). myoglobin (6.85, 7.35). lentil lectin (8.15, 8.45. 8.651, chymotrypsinogen A (8.8, 9.2. 9.6). trypsinogen (9.3, mouse NGF (9.1. 9.2. 9.3) horse cytochrome c (9.4). bovine cytochrome c (10.25), and lysozyme (10.75).

FIG. 3 (right). SDS-polyacrylamide gel electrophoresis of purified bovine NCF stained for glycoproteins using the periodic acid-Schiff reagent method. Both tracks contain 270 pg of purified bovine NGF. Track 1 was stained for glycoproteins, and Trach 2 was stained for proteins with Coomassie brilliant blue. The arrowheads mark a and d , top and bottom, respectively, of the gels; h, center of overloaded bovine NGF band, which also tended to smear upward on these gels; c, carbohydrate staining migrating with the bromphenol blue dye front. An approximate relationship was derived between the Schiff reagent staining intensity and the carbohydrate content of glycoproteins by running samples of commercial preparations of the following purified glycoproteins: cr,-acid glycoprotein (orosomucoid; 40‘5 carbohydrate); fetuin (23%); horseradish peroxidase (16‘y); ribo- nuclease B (117); avidin ( 1 l‘?); fibrinogen (5% ); transferrin (4?); ovalbumin (34); and 1)Nase I (3%).

marker proteins of known molecular weight gave an apparent molecular weight for the BNGF peptide chain of 15,000 f 250 (the molecular weight was determined in one or more tracks of 10 independent gels).

Gel Filtration in 6 M Guanidine Hydrochloride-Purified bovine and mouse NGFs were incubated in 6 M guanidine hydrochloride, in 20 mM phosphate buffer, pH 6.5, for 24 h at 4 “C and then analyzed on a Sephacryl S-200 column (2.6 X 95 cm; flow rate, 25 ml/h) equilibrated with the same buffer and calibrated with commercial preparations of pure proteins. 6 M guanidine hydrochloride destroys all noncovalent struc- ture of the proteins, so the observed apparent molecular weights refer to the single polypeptide chains (see “Discussion”); both preparations gave only one major peak, corresponding to apparent M , = 12,930 for bovine NGF and 12,250 for mouse NGF (cf 12,378 according to the sequence data for the predominant cleaved chains; see “Discussion”).

Isoelectric Focusing-Isoelectric focusing in polyacrylamide gels of purified mouse NGF yielded three distinct bands of isoelectric points 9.3, 9.2, and 9.1, corresponding to around 45, 45, and lo%, respectively, of the total protein loaded (as judged by relative staining intensities; Fig. 2; see “Discussion”). In contrast, bovine NGF gave only one band (Fig. 2) corresponding to an isoelectric point of 9.5-10.0 (determined in several tracks each of four independent gels).

Carbohydrate Analysis-Analysis by gas-liquid chromatography of carbohydrate moieties within the purified bovine NGF preparation yielded the following data (sugar levels in micrograms of sugar/mg of protein): mannose, 12.46; fucose, none detectable; galactose, 23.36; glucose, 27.70; N-acetylglu- cosamine, 8.80; and N-acetylgalactosamine, 2.40 (all values k 5%). The total sugar content was thus around 7.5% by weight.

TABLE I1 Amino acid composition of bouine nerve growth factor

Amino acid

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine‘ Valine Methionine” Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan“

14.8 10.2 10.6 12.6

9.1 7.1

7.1 3.9

11.6 2.5 6.7 8. I 4.5 6.9 4.2

10.1 7.2 3.1

11 14 11 8 2 5 8 6

13 1 5 3 2

4 8

3

- I

-

Total 140.3 I18

“ Calculated on the basis of a peptide M, = 15.000 (see Fig. 1). The composition shown is an average from several hydrolyses of different preparations of BNGF. Only for serine was a consistent change (a decrease) seen with increasing time of hydrolysis, so the average value is extrapolated to zero time.

“Calculated from the sequence data (Angeletti and Rradshaw, 1971) for the intact 118-residue peptide chain of mouse NGF. ‘ Determined as S-aminoethylcysteine. Spectrophotometric deter-

minations with DTNB indicated that all these residues are involved in disulfide bridge formation.

‘’ Determined in the presence of 0.1% (v/v) /I-mercaptoethanol. ‘’ Determined spectrophotometrically by the method of Edelhoch

(1967).

8544 Purification of Nerve Growth F a c t o r from Bovine Seminal Plasma

mouse

bovine

g u i n e a p i g

Naja naja

mouse

bovine

g u i n e a p i g

Naja n a ~ a

1 5 10 NH -ser-ser-thr-his-pro-val-phe-his-met-gly-glu-phe-ser-val- 2

n . n NH -ser-ser-ser-hls-pro-val-phe-his-arg-gly-glu-phe-ser-val- 2 I I I I

1 5 1 0 NH -ser-ser-thr-his-pro-val-phe-his-met-gly-glu-phe-ser-val- 2

7 NH -g lu-asp-h is -pro-va l -h is -asn- leu-g ly-g lu-h is -pro-va l - 2 I I 1 1 I 7

NH -g lu-asp-h is -pro-va l -h is -asn- leu-g ly-g lu-h is -pro-va l - 2 I I 1 1 I

1 5 2 0 2 5 -cys-asp-ser-val-ser-val-trp-val-gly-asp-lys-thr...

1 2 0 2 5

U ile-ser-Val- ? -Val- ? -asp- ? - t h r . . .

1 5 2 0 n 2 5

P -cys-asp-ser-val-ser-val-trp-val-ala-asp-lys-thr...

...- t r p - ?-I ... ...- v a l - l y s - t h r ... U

FIG. 4. Amino acid sequences of the NHP-terminal portions of the NGFs from bovine seminal plasma, mouse submandibular gland, guinea pig prostate gland, and Naja naja venom. The figure gives the known sequences for mouse, guinea pig, and N. naja (cobra) NGF and the assigned sequence for bovine NGF. Homologous residues are enclosed in boxes. The data for mouse NGF are from Angeletti and Bradshaw (1971); those for guinea pig NGF are from Chapman et al. (1981) and Rubin and Bradshaw (1981), and those for cobra venom NGF are the un- ambiguously defined residues given by Hogue-Angeletti et al. (1976).

However, periodic acid-Schiff reagent staining of bovine NGF after SDS-polyacrylamide gel electrophoresis demonstrated a virtual absence of stain over the (overloaded) NGF band, whereas the staining intensities of some contaminating bands were sufficient to account for the amounts of sugar detected in the gas-liquid chromatographic experiments (Fig. 3). In particular, heavy carbohydrate staining was seen at the bottom of the gel, corresponding to an area devoid of detectable amounts of protein (stained by Coomassie brilliant blue). Possible sources of this material include fragments of the carbohydrate-based ion exchangers used for purification (see Fischer, 1980), carbohydrate fragments, e.g. from reproductive tract mucins (which contain up to 80% carbohydrate, readily susceptible to cleavage under very mild treatments; Montgom- ery, 1970), and polysaccharide moieties (e.g. fragments of glycosaminoglycans; Lindahl and Hook, 1978) binding noncovalently to proteins. The above level of carbohydrate in the sample, therefore, does not contradict the protein purity (395%)) determined by SDS gel electrophoresis (see above). Essentially similar results were obtained when glycoproteins were stained with the highly sensitive dansyl hydrazine method (Eckhardt et al., 1976); a small amount of fluorescence seen at the heavily overloaded position of the bovine NGF band was due to noncovalent binding of the fluorescent agent, as it was also seen at the same intensity when the periodate oxidation step was omitted (see Eckhardt et al., 1976). The carbohydrate staining elsewhere on the gel was, however, specific. Furthermore, treatment of bovine NGF with a broad spectrum of exoglycosidases (including neuraminidase) had no detectable effect on the position of the NGF band on SDS- polyacrylamide gel electrophoresis, whereas removal of any carbohydrate moiety from the NGF molecule would be expected to increase its electrophoretic mobility. Finally, BNGF was not able to bind to a concanavalin A-Sepharose 4B column, a group-specific adsorbent for many glycoproteins (see “Materials and Methods” for details).

Amino Acid Composition-The amino acid composition of bovine NGF is listed in Table 11, where it is compared with the known composition of purified mouse NGF. Spectropho- tometric determinations with DTNB indicated that there are

no free sulfhydryl groups in BNGF. Amino Acid Sequence Determination-The amino acid

sequence of the NH2-terminal portion of the bovine NGF molecule, as determined in three runs of an automatic sequenator using different preparations of the protein, is given in Fig. 4. Quantitative data for the sequenator runs are given in Table 3 (Miniprint). The reported sequence of residues 1-9, and in particular the identifications of residues 3 and 9 (see “Discussion”), were supported by the isolation of a peptide from a tryptic digest of BNGF, whose amino acid composition corresponded to the first 9 residues of the molecule.,”

DISCUSSION

Distribution of Seminal Plasma NGF-As expected, the production of NGF by various prostate glands is followed by its secretion into the seminal plasma; NGF has now been detected in the seminal plasmas of the bull, sheep, and goat, but not in those of humans or pigs. The extremely high levels of NGF in bovine seminal plasma (860,000 BU/ejaculate) contrast with the much lower levels previously detected in the bovine prostate gland itself (26,000 BU/organ; Harper and Thoenen, 1980b). More recent studies4 have indicated that the seminal vesicles and the ampullary glands, as well as the prostate glands, contribute NGF to bovine seminal plasma.

Purification of NGF from Bovine Seminal Plasma-The purification scheme developed in the present investigation yielded bovine NGF of 295% purity, as judged from SDS- polyacrylamide gel electrophoresis and at an overall recovery of around 10-20%. The specific activity of the final preparation was 2-5 ng/BU, which is identical with the specific activity of purified mouse NGF in our biological assay system. Around 10 mg of pure protein can be isolated from 10 g of lyophilized seminal plasma (100 ml of semen).

The purification scheme for BNGF is derived from that developed by Mobley et al. (1976) for the rapid isolation of mouse submandibular gland NGF. This is based on the fact that mouse NGF normally exists as a high molecular weight

’ G. P. Harper, R. W. Glanville, and H. Thoenen, unpublished data. M. E. Schwab, G. P. Harper, and H. Thoenen, manuscript in

preparation.


complex, called 7 S NGF, of M, - 140,000 and of isoelectric point 5.1. The complex is only stable between pH 5 and 8 and is further stabilized by the presence of zinc ions (Server and Shooter, 1977). This complex passes through a fist CM-cellulose column at pH 6.8, while basic proteins in the gland extract bind to the column and are thus removed. Subse- quently, the active fraction is acidified to pH 4. This causes the 7 S NGF complex to dissociate, revealing the highly basic (p1 9.3) p-subunit (Mr = 26,512), the only subunit of the 7 S complex having NGF biological activity (Server and Shooter, 1977). This basic, low molecular weight mouse NGF therefore binds to a second CM-cellulose column at pH 4 and, being the most basic protein remaining in the fraction, is the last protein to be eluted from the column following changes of pH and ionic strength.

The fact that this scheme is essentially effective for the isolation of bovine NGF indicates that bovine NGF shares some of the properties of mouse NGF, but certain differences are evident. The NGF activity in crude bovine seminal plasma certainly all exists as a high molecular weight complex (HMW- BNGF), as in preliminary experiments (at pH 6.8-7.5) it eluted from Sephadex G-100 or Sephacryl S-200 gel filtration columns (data not shown) only at positions exactly analogous to those observed on these columns for mouse 7 S NGF (Varon et al., 1967; Bocchini and Angeletti, 1969; Stach et al., 1977). However, unlike mouse 7 S NGF (Varon et al., 1967; Stach et al., 1977), HMW-BNGF does not bind to DEAE-cellulose at pH 6.8. Furthermore, preliminary experiments showed that, unlike mouse 7 S NGF, HMW-BNGF does not dissociate into its component subunits at pH 4.0, even in the presence of 1 mM EDTA to sequester putative zinc ions stabilizing the complex. Thus, all the bovine NGF activity passed through a CM-cellulose column similar to that used by Mobley et al. (1976) (pH 4.0, in 0.4 M NaCl). The bovine complex has to be acidified to pH 3.0 before it dissociates. Interestingly, the NGF in guinea pig prostate glands (Harper et al., 1979) also exists as a high molecular weight complex, which dissociates at pH 3.0 but not at pH 4.0 (Chapman et al., 1981). The low molecular weight bovine NGF subunit, corresponding to the p- subunit of mouse NGF, is revealed as a highly basic protein (PI 9.5-10) that binds firmly to SP-Sephadex at pH 3.0. As other basic proteins have been removed by the earlier CM- cellulose step at pH 6.8, pure LMW-BNGF can readily be isolated following changes of pH and ionic strength.

The other difference from the procedure described for mouse NGF (Mobley et al., 1976) is the essential incorporation of an initial DEAE-cellulose column at pH 6.8. This removes the majority of the acidic proteins from the crude seminal plasma before the HMW-BNGF is dissociated.

Biochemical Characterization of Low Molecular Weight Bovine NGF-The biochemical characterization of the low molecular weight form of bovine NGF reveals several differences from the known properties of the mouse p NGF molecule (Server and Shooter, 1977). Mouse NGF is a dimer (Mr = 26,512; PI 9.3) of two identical and noncovalently linked polypeptide chains of 118 residues (monomeric M, = 13,256). However, this intact form of the NGF molecule is only obtained using certain purification procedures. If other procedures are used, the NGF molecules are proteolytically cleaved to variable extents, losing NHp-terminal octapeptide sequences and COOH-terminal arginine residues. Neither cleavage has any effect on the biological activity of mouse NGF. The loss of the octapeptide can be seen on 15% SDS-polyacrylamide gels (Mobley et al., 1976; Server and Shooter, 1977), and this explains the usual appearance of two bands (intact 118- and cleaved 110-residue chains) in our purified preparation of mouse NGF (Suda et al., 1978; a substantial

majority of the chains are in fact cleaved). The loss of the COOH-terminal arginine residue from one polypeptide chain alters the isoelectric point of the native dimer from 9.3 to 9.2; the loss of this arginine residue from the second chain pro- duces a species of PI 9.1. Pure mouse NGF can therefore, as in the present case, yield three bands on isoelectric focusing (Moore et al., 1974; Server and Shooter, 1977). Purified guinea pig NGF also yields three bands on isoelectric focusing (PI 8.5 k 0.5; Chapman et al., 1981) but only one band on SDS- polyacrylamide gel electrophoresis (apparent molecular weight identical with that of the 118-residue chain of mouse NGF; Harper et al., 1979; Chapman et al., 1981; Rubin and Bradshaw, 1981).

The results of the present experiments show that bovine NGF probably has a longer peptide chain (Mr = 15,000 from SDS gel electrophoresis) and a higher isoelectric point (9.5-10) than mouse (or guinea pig) NGF. Furthermore, no evidence was seen for the loss during this particular purification procedure of terminal residues or peptides, resulting in detectable changes in molecular weight or isoelectric point.

The higher apparent molecular weight of bovine NGF in SDS gel electrophoreses, compared to that of mouse NGF, probably cannot (see “Results”) be attributed to the former molecule being a glycoprotein (Weber and Osborn, 1975). However, for proteins of molecular weight less than around 15,000, the binding of SDS does not always completely mask the inherent charge and conformation of the protein (Weber and Osborn, 1975). It cannot therefore be entirely excluded that the apparently higher molecular weight of bovine NGF in SDS gel electrophoreses, compared to that of mouse NGF, is due to the higher isoelectric point of the bovine protein (Weber and Osborn, 1975). This possibility is to some extent supported by the results of gel filtration in the presence of 6 M guanidine hydrochloride. According to this procedure (Fish et al., 1969; Belew et al., 1978) which is not influenced by the isoelectric point of the proteins, the molecular weights of the polypeptide chains of bovine and mouse NGFs are barely distinguishable. However the resolution and accuracy of SDS- polyacrylamide gels is generally superior to that of such denaturing gel filtration columns, so it is the molecular weight of the bovine NGF subunit as determined in the electrophoretic experiments that has been adopted at the present stage of characterization.

The data presented in detail in the Miniprint of this paper indicate that bovine NGF, like mouse NGF, is a dimer of the polypeptide chains seen in SDS-polyacrylamide gels. As p- mercaptoethanol has no effect on the band position of BNGF on SDS gel electrophoresis, thereby excluding the existence of disulfide bridges between the two BNGF peptide chains, these two chains of BNGF can only be associated by noncovalent forces, as is the case for mouse NGF. The SDS gels would indicate an approximate molecular weight for the native bovine NGF dimer of around 30,000.

Amino Acid Composition and NH2-terminal Sequence- The main differences between the amino acid compositions of bovine and mouse NGFs (Table 11) are increases in BNGF in the proportions of aspartate, glutamate, proline, glycine, leucine, and tyrosine residues, and a decrease in the number of threonine residues. Native BNGF contains no free sulfhydryl groups, SO all cysteine residues in BNGF are involved in disulfide bridges; as p-mercaptoethanol treatment has no effect on the behavior of BNGF in SDS-polyacrylamide gel electrophoresis (see above), these must all be intrachain bridges, as in mouse NGF (Greene et al., 1971). However, bovine NGF contains only two intrachain disulfide bridges per peptide chain, in contrast to the three bridges per chain seen in mouse NGF (Angeletti and Bradshaw, 1971). Interest-

8546 Purification of Nerve Growth Factor from Bovine Seminal Plasma

ingly, Chapman et al. (1981) found only two disulfide bridges per peptide chain of guinea pig NGF, though Rubin and Bradshaw (1981) reported three.

The NH1-terminal amino acid sequence of BNGF (residues 1-26; Fig. 4 ) is very similar to the known sequences of mouse (Angeletti and Bradshaw, 1971) and guinea pig (Chapman et al., 1981; Rubin and Bradshaw, 1981) NGFs. However, clear differences were indicated at positions 3, 9, and 18, which are threonine, methionine, and valine, respectively, in mouse and guinea pig NGFs, but serine, arginine, and isoleucine in bovine NGF.

Clearly the NGF sequence is highly conserved in mammals, a t least at the NH, terminus; the closer homology between the two rodent NGFs than with bovine NGF might be expected from the evolutionary distance between these three species. I t is perhaps surprising that the sequence of the NH2- terminal octapeptide is so conservative, as it is known that this peptide is not required for the biological activities so far delineated for mouse NGF (Mobley et ai., 1976; Server and Shooter, 1977).

Acknowledgments-We are very grateful to the following people and organizations for supplying us with seminal plasmas: Dr. H. Kupferschmied, Centre d’Insemination de Neuchhtel, Switzerland; Dr. P. Summermatter, Besamungsstation Biitschwil, Switzerland; Dr. J . Luginbiihl, Besamungsstation Oberdettigen, Switzerland; Dr. 0. Haeger, Priif- und Besamungsstation Munchen-Grub, West Germany. H. Dieringer, Department of Connective Tissue Research, Max Planck Institute for Biochemistry, performed the chromatographic sugar analyses.

REFERENCES Angeletti, R. H., and Bradshaw, R. A. (1971) Proc. Natl. Acad. Sci.

Belew, M., Fohlman, J., and Janson, J.-C. (1978) FEBS Lett. 91, 302-304

Blackburn, S . (1978) in Amino Acid Determination: Methods and Techniques (Blackburn, S., ed) 2nd ed, pp. 7-37, Marcel Dekker, New York

Bocchini, V., and Angeletti, P. U. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 787-794

Chapman, C. A,, Banks, B. E. C., Vernon, C. A,, and Walker, J . M. (1981) Eur. J. Biochem. 115,347-351

Eckhardt, A. E., Hayes, C. E., and Goldstein, I. J. (1976) Anal. Biochem. 73, 192-197

Edelhoch, H. (1967) Biochemistry 6, 1948-1954 Edman, P. (1970) in Protein Sequence Determination (Needleman,

Fischer, L. (1980) Gel Filtration Chromatography, 2nd ed, pp. 1-269,

Fish, W. W., Mann, K. G., and Tanford, C. (1969) J. Biol. Chem. 244,

Fohlman, J., Eaker, D., Karlsson, E., and Thesleff, S. (1976) Eur. J.

U. S. A. 68, 2417-2420

S. B., ed) pp. 211-255, Springer-Verlag, New York

Elsevier/North Holland, Amsterdam

4989-4994

Biochem. 68,457-469

Neurobiology 1 , 3 7 4 8 Greene, L. A., Varon, S., Patch, A,, and Shooter, E. M. (1971)

Habeeb, A. F. S. A. (1972) Methods Enrymol. 25B, 457-464 Harper, G. P., and Thoenen, H. (1980a) J. Neurochem. 34, 5-16 Harper, G. P., and Thoenen, H. (1980b) J. Neurochem. 34,893-903 Harper, G. P., Barde, Y.-A., Burnstock, G., Carstairs, J. R., Dennison,

M. E., Suda, K., and Vernon, C. A. (1979) Nature 279, 160-162 Harper, G. P., Pearce, F. L., and Vernon, C. A. (1980) Dev. Biol. 77,

391-402 Hogue-Angeletti, R. A., Frazier, W. A., Jacobs, J. W., Niall, H. D.,

and Bradshaw, R. A. (1976) Biochemistry 15, 26-34 Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 Lasky, M. (1978) in Electrophoresis ’78 (Catsimpoolas, N., ed) pp.

Levi-Montalcini, R., Meyer, H., and Hamburger, V. (1954) Cancer

Lindahl, U., and Hook, M. (1978) Annu. Reu. Biochem. 47, 385-417 Lottspeich, F. (1980) Hoppe-Seyler’s 2. Physiol. Chem. 361,

Margolis, J., and Kenrick, K. G. (1968) Anal. Biochem. 25, 347-362 Mobley, W. C., Shenker, A., and Shooter, E. M. (1976) Biochemistry

15, 5543-5552 Montgomery, R. (1970) in The Carbohydrates (Pigman, W., Horton,

D., and Herp, A,, eds) 2nd ed, Vol. IIB, pp. 627-709, Academic Press, New York

Moore, J . B., Moblev, W. C., and Shooter, E. M. (1974) Biochemistry

195-210, Elsevier, New York

Res. 14,49-57

1829-1834

13,833-840 MurDhv. R. A,. Saide. J. D.. Blanchard. M. H., and Young, M. (1977)

Pioc: Natl. Acad. Sci. U. S. A. 74,2330-2333

Eur. J . Biochem. 32,569-575

25, 155-159

._ .

Pearce, F. L., Banthorpe, D. V., Cook, J. M., and Vernon, C. A. (1973)

Pignatti, P.-F., Baker, M. E., and Shooter, E. M. (1975) J. Neurochem.

Prehm, P., and Scheid, A. (1978) J. Chromatogr. 166,461-467 Raftery, M. A., and Cole, R. D. (1966) J. Biol. Chem. 241, 3457-3461 Ronne, H., Anundi, H., Rask, L., and Peterson, P. A. (1979) Biochem.

Rubin, J . S., and Bradshaw, R. A. (1981) J. Neurosci. Res. 6, 451-464 Server, A. C., and Shooter, E. M. (1977) Adu. Protein Chem. 31,

Slater, G. (1969) Anal. Chem. 41, 1039-1041 Stach, R. W., Wagner, B. J., and Stach, B. M. (1977) Anal. Biochem.

Biophys. Res. Commun. 87, 330-336

339-409

I

83, 26-32 Stoeckel. K.. Gannon. C.. Guroff, G., and Thoenen, H. (1976) J .

Neurocheh. 2< 1207-1211 Studier, F. W. (1973) J. Mol. Biol. 79, 237-248 Suda, K., Barde, Y.-A., and Thoenen, H. (1978) Proc. Natl. Acad.

Thoenen, H., and Barde, Y.-A. (1980) Physiol. Reu. 60, 1284-1335 Varon. S.. Nomura, J., and Shooter, E. M. (1967) Biochemistry 6,

Sci. U. S. A. 75,4042-4046

2202-2209 Wallace. L. J.. and Partlow. L. M. (1976) Proc. Natl. Acad. Sei. U. S.

I .

A. 73,4210-4214 Weber, K., and Osborn, M. (1975) in The Proteins (Neurath, H., Hill,

R. L.. and Boeder. C.-L., eds) 3rd ed, Vol. I, pp. 179-223, Academic Press, New York


SUPPLEMENTARY MATERIAL P o R

THE PURIFICITION OF NERVE GRGUTH FACTOR FROM glVINE SEMINAL PLASMA: BICCHEllICIL CHARACTERIZATION AND PARTIAL AMINO-ACID SEQUENCE

Gregory P. Harper, Robert W. Clanville and Hans Thoenen by

MATERIIL AND MTTHODS

Ilaterrals - DEhE-cellUlo~e 52 and CH-cellulose 52 were Obtaxned from WhatMniSP-Sephadex C-25. Sephadel G100, Sephacryl S200 and concana- valln A-Sepharose 4B were Obfalned from Phamcia. Clrrler ampholytes and equipment for flat-bed IBOeleCtrZC foculling were svpplled by LKB; other chemcala for gel electrophoresis and l-lectric focuslng were obtalned from Blo-Rad. Marker protelns for the callbration of electrophoretic and chromatographic procedures were obtarned from Pharmacxa, slgma, Serva and Boehrmqer; they are listed for mdlvi-

chlorlde 168 -Lab- grade from Kerck or .Pu-. grade from Flub. and dual procedures in the corresponding Figure legends. Guanidine hydro-

the buffered eolutlons -re therefore purified by the lethod Of Pohlman et a1 11976) to reduce UV-absorblnq Contaminants. Seminal plasmas *ere obtalned froa the sources llsied ln the .Acknowledgments. section; human samples were those determined to be "0-1 in hospital fertlllty ClIn~cs. U O U S e NGF was purified by the ethod Of Boachinl and lngelettl 119691. as modifled by suda et a1 11978).

Isoelectric Pacvsrnp - Polyacrylaolde gel iswlectric facuelng lflnal concentration 5% polyacrylemlde) was carried out in an LKB aultlphor Bystem. easentlally as recommnded I" LKB Applicatlon Note 250. The gel solution was prepared wzth sllght mdlflcatlons: 3.0-3.6 ml LKB Ampholines lfor PI

Ampholxne pH 9-11, and 2.8 nl Amphaline pH 3.5-10- for PI range 7.8-10: range 3-10: 0.2 ml Amphollne pH 1-6. 0.2 ml klnpholine pS 5-7. 0 .4 m1

0.6 m1 Amphollne pH 1-9. and 2.4 Dl Ampholine pE 6-111: 10 nl stock 801ution of 30% acryldmlde, 0 . 8 % bis; 12 p1 TWED; 180 111 a-oiy. persulphate solntmn 10.1 q l m l l and x 2 ml (PI range 3-10) or 16.8 ml IpI range 7.8-101 water contamlng 7.5 g sucrose. Protein s01ut~ons 12-4 mglml ~n 20 .II phosphate buffer, pH 6.8, or, for NGPs. in 0.111 acetate buffer PB 5.01 were app~led via f l ~ t e r paper strzps (capacity c. 20 "1). -1s were'rm at 4-C, llth a water-cooled base-plate, at constant power (24 watts; LXB PQwer Pack 21031, 1500 V m a x m u m voltage, 80 U lnltral Current for 90 min - 3 h

tIIChloroaCetlC acld solution 160 m n ) , washed in 2 changes of 551 solvent (PI range 3-101 Or for 3 h (PI range 7.8-10). Gels &re fixed In 12.58

for SDS-gels. The pH gradrent was determmed using Mrker proteins of ( s e e SDS-gels) for 3 0 mln each, and then stained and destalned an described

known lsoelectrlc polnts (see legend to Fig. 2).

Glycoproteins were also analysed by perrdrc acld-Schxff's reagent stain- Lng of SDS-polyacrylanlde gels, Yhlch r r e performed all described above. After electmphozesle. gels were f u e d in 12.51 aqueou. CrIehloroacetic

eachl, lncvbated at 4.C In 0.2P aqueovs perIodiC acid solution 145 rinl acld for 60 mln, rlnaed ln 2 changes of 7 . 5 8 aqueous acetic acid 130 =in

vlth Schlff's reagent ISlgmal for 45 n ~ n . The .taming intensity was and, vlrhovr rashlng out the perlodlc acid, stained at 4-C in the dark

allowed to develop in 109 aqueous acetic acld for 2 h at room temperature. 1" the dark, before gels were photographed n t h a green filter.

SDS-palyacrylanlde g e l s were also atained for glycoprotelne ~ a l n g milnor modlflcarlons of the hlghly sensltlve flUoreBCent aethod of ECkhardt et a1 119161, based on the oxidation of sugar residues rlth periodic acld. followed by coupllng of the resulting aldehyde mleties with dansyl hydrazine.

Bovlne NGP -019 further analysed on SDS-polyacrylamide gels . using in parallel Coolassie Brlllldnt Blue staining for proteine and priodlc acid- Schrff's reagent starnlnq for ~1YCOD~Otelns. followrnm treatment wlth a

IC-D-mannose Or4-D-q1YcOBe residues, a sample of bovine seminal plasma Rs a flnal test of whether BNGF 1s a glycoprotein contalnlng the c-mn

was passed Over d column Of concanavalin A-SephaZOBe 48 equll~brated

and CaCl to malntaln the blndlng capaclty of the lectin. The fra rlons In 50 mu Trls-HCI buffer, pH 6.8, contalning 1 MI of each of MnCla,-ngc12

passlng Zhrough the ~DlYmn, and those blndlng and elutable by 50 m.(-D- methylglucoslde or.(-D-r%ethylmannoslde ISlgmaI were analysed for their NGF content uslng the hlologlcal assay.

were obtarned fra) acid hydrolysee m the presence of 0.18 lv/v1 8-mr- captoethanol, to Counter OxidatLon to methlonine sulphorlde (Blackburn, 19781. cystelne 188 determined a18 S-anlnoethylcysteine IRaftery and Cole, 1 9 6 6 1 , after the following treatment Of BNGF: l i l BNGP was d18- solved In 1 K Tns-ECI buffer, pH 8.0. cantalnlng 20 m!t dithioerythrl- tal IMerckI and 8 M urea IMerckl (37.C. 16 hl; (ill ethyleneiune

dark); 11uI excess reagents were removed by dlalysis against water 12" Iserva) was added to a final concentration Of 0.5 n 120Dc, 4 h, in the

the dark1 L" Spectrapor No. 6 bags lmolecular weight exclusion limlf

groups In BNGF were determined spectrophotopetrlcally using 5.5"dlthio- around 3500). and the derlvatlred proteln was lyophlll*ed. Free sulphydryl

b~sl2-nitrobenrolc acid) (DTNB: S l g Y l in the presence of 2% SDS and 0.5 mglml EDTA IHabeeb, 1 9 7 2 ) . The tryptophan content Of the BNGP pro- tela m s determined spectrophotometrically by the method of Edelhoch 11961).

AmLno-acld Seouence Determinations - Mlno-acld sequence. were determined for 10-50 "mole. BNGF per run uarng a Beckman liquld-phase sequencer

Mass.). A protein guadrol programme 10.5 U quadroll was used 1890 B 890 B or 890 c fitted wlth P6 AutoConverter* ISequOiDdt Inc. watertorn,

progr- 060 215, model 890 C progrnnm 12 29 741 and the derlvatlred

phenylthiohydsntoin IPTEI amino-acids. using alcoholic ACI as dencrrbed amino-aclds produced by the sequencers were .on-line' converted to

in the Autoconverter wnval. The PTB amino-aolds r r e Identifled u-ing HPTU: and EPLC. BPTLC plates Ipre-Coated slllca gel 60 P 254, Merck) Were wed with BOlvent systems deacrlbed by E d m n 11970) . An HPLC Bystem lmdel SP 8000. Spectra Physlce), equpped with a reverse-phase C o l m IRP-18. 5 pm Ultrasphere., Becknannl. was used to separate PTB amlno-acida under conditions that have recently been &c.crlbed ~n detail ILottapeich, 1980).

Table 3: purntitatzve Data for N-terminal Sequence Determlnatlon Of BNGF

* Amino-acld zdentlfred Yleld l-l)"b'c'd

1 2 3 4 5 6 1 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 21 25 26

b

d

8548 Purification of Nerrle Growth Factor from Bovine Seminal Plasma

1 2 3 4

P l q . 5. Gradlent gel electrophoresis of native NGP DrOtelnS Track I : 10 p q pmrlfled mouee NGP; Track 2: 40 pg purlfred bovine NGP: Track 3: 160 p q plrlfled mouse NGP; Track 4: 160 pg purlfled bovine NGP. Tracks 1 and 2, and tracks 3 and 4, were N n In two dlfferent gels. The

w t h those on the other gel, but only wlth the marker proteln tracks p s l t l o n s of the var .10~8 NCP bands cannot therefore be dlrectly compared

lnot shown1 from the sa- gel laee text). The sllght dlstortlon of the lower bands in Cracks 1 and 2 IS due to some uneven ehrinkaae of the gel durlnq destamlng. The follow~ng marker protelns e r e u&d to calibrate che gels: ovalbumn lnatlve mlecular welght 43,0001, horseradish peroxidase 140,000). 140,0001, a-lactoglobulln A 135,0001, DNase I 131,000l.

ChymtrypsLnogen A 125,0001, trypamogen l24.5001, myoglobin 117,500~. bcvlne carbonlc anhydrase B 110,OOOl. human carbonic anhydrase B 129.700).

ly~oryme 114,4001 and cytochrome c 112.4001. In track 4 , the heavy BNGP band towards the top of the gel 1s accompanied by three contaminant bands lof natlve molecular weights around 46>300; 43,400 and 12.700l. a r e readlly dlstlnqulshed ln the 0clq~na.l gels than ~n thls photograph. The

particulate matter lodged ~n the loading well. relaclvely hlgh stamhng intensity at the cop of track 4 168 due to some

I

Pig. 6. HPLC- el flltration of Purifled b o Y ~ n e NGF The elution pr%llee (at 206 nm to take ad-f the Increased sen- Sltivlty at thxs vavelength; LIB AppIIcacIon Note 3151 on proteln colwnn 1-125 IYoter 's l , rhlch separates protems of m l e c u l a r relghts between 2,000 and 80,000. ace given for the or lg lna l BNGP prepararlon l a ) , and for the subsequent re-chronaroqgraphy of the two maln peaks Ib

The column buffer was 50 11111 phosphate, pH 6.0, and the flow-rate was and c, and then 6-9 for the Wcond re-chmmdtogrdphx steps: see t e x t ) .

0.5 mllun. The scale bars correspond to: labaorbancel 0.002 for pro-

profiles. The broad arrow on each profrle marks the loadlng of the flles a-c and 0.001 for profllea d-q; and lelutron vel-I 4 ml for a11

column, lhlle the two longer arrows Lndlcate the elution vo lums of the two orlglnal peaks 11" l a ) ) . In profalea e and f the earller peak can

peak eluted slqhtly later than expected fro11 the lnltlal profile 1111: no longer h d&atinguLshed. Generally, on re-chromatqraphy, the second

thls LS probably a consequence of loadlnq much larger volumes. Pssenrlally s l m l a r proflles bere also ObCaLned w l n q 200 mM phosphate buffer,

acetate buffer, pH 4.0, c o n t l l l n l n q 0.5 or 1.0 n NdCl. The follomnq pH 7.0: 50 mM phosphate buffer. pH 6.0 Contalnlnq 1 I NaCl; or 50 mn

prmelns r e r e w e d LO callbrare the column: aldolase lnaclve m l e c u l a r relght 158,0001, bovlne serum albmln 166,2001, lentil lectln l52,OOOl. ovalbumin 143.000). horseradlsh peroxadase 1 4 0 , 0 0 0 ~ . 8-lacroglobul~n A

NGP 124.7501, trypBLn09en 124,5001, soya bean trypsln tnhlblror 121,0001. 135,0001, DNase I 131,OOOl. bovine carbonlc anhydrase b 1 1 0 , 0 0 0 ~ . mouse

myoglobrn 117,5001, lpozy14 114,4001, and Cytochr- c 112,4001.

the purification of nerve growth factor from bovine seminal plasma

Documents