Reprinted from
Proc. Natl. Acad. Sci. USA
Vol. 76, No. 11, pp. 5601-5605, November 1979
Biochemistry

Purification and partial characterization of human
lymphoblastoid interferon

(lower molecular weight component/immunoabsorbance/amino acid composition)
KATHRYN C. ZOON, MARK E. SMITH, PAMELA J. BRIDGEN, DOROTHY ZUR NEDDEN, AND
CHRISTIAN B. ANFINSEN

Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205
Contributed by C. B. Anfinsen, August 24,1979

ABSTRACT  One component of human lymphoblastoid in-
terferon obtained from Namalwa cultures induced by Newcastle
disease virus has been purified to a specific activity of 2.5 X lo*
interferon units per mg of
amino acid analysis). A sin rotein (protein content based on
parent molecular weight o f e polypeptide species with an ap-
            18,500 comigrating with the anti-

viral activity was observed by sodium dodecyl sulfate/polyac-
rylamide gel electrophoresis. Preliminary amino-terminal se-
quencing results sup
species is essentially rl ort the conclusion that the interferon
             omogeneous.

The purification and characterization of human interferon are
currently under investigation because of its clinical potential
as an antiviral and antitumor agent (1). Several groups have
reported the isolation of homogeneous forms of mouse (2,3),
human leukocyte (4,5), and human fibroblast interferons (6,
7). Only minute quantities of these apparently homogeneous
materials are available for chemical analyses due to the small
amounts of interferon produced by induced cells as well as losses
incurred during the purification. Thus, only limited data on the
amino acid compositions and sequences of interferons have
been reported (4,8,9). Recent improvements in the large-scale
culture of Namalwa cells and the subsequent induction of in-
terferon with Newcastle disease virus, strain Bl, have made
relatively large quantities of interferon continuously available
(10-12). This is a report on the purification of a component of
human lymphoblastoid interferon and on its amino acid com-
position.

MATERIALS AND METHODS

Production of Lymphoblastoid Interferon. The procedures
for the culture of Namalwa cells and the induction of interferon
have been described (10-12). A portion of the crude interferon
used in this study was supplied by Litton Bionetics under Na-
tional Cancer Institute Contract NOl-CO-75380. This material
was received as resuspended trichloroacetic acid precipitate
as described (11). Newcastle disease virus, strain Bl, was pre-
pared by Jack Campbell as reported (10).

Interferon Assay. The antiviral activity of interferon was
determined by a modification of the micro method described
for rabbit kidney cells (13). Th' 1s method uses low passage pri-
mary culture human foreskin fibroblasts with vesicular sto-
matitis virus as the challenge. All interferon units are expressed
with reference to the National Institutes of Health human in-
terferon standard G-023-901-527.

Preparation of Anti-Interferon Serum. Sheep were im-
munized with lymphoblastoid interferon in complete Freund's
adjuvant over a period of 4 months, following an injection
schedule described previously (14). The interferon used was
partially purified to a specific activity of 1-5 X 105 units/mg

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of protein by immunoabsorbant affinity chromatography using
sheep anti-leukocyte interferon coupled to Sepharose 4B. A
fraction enriched in gamma globulins was produced from the
serum by repeated precipitation with ammonium sulfate at 50%
saturation. The gamma globulin fraction was then repeatedly
passed over an "impurities column" (fetal calf serum, allantoic
fluid with Newcastle disease virus, and homogenized buffy coat
bound to Sepharose 4B) to enhance the specificity for anti-
interferon (14). The anti-interferon was then coupled to
Sepharose 4B (24-36 mg of protein per ml of swollen Sepha-
rose). The typical binding capacity of the anti-interferon was
in the range of 4.0-16.0 X 1Oa interferon units per mg of cou-
pled protein.

Preparation of Interferon from Uninduced Namalwa
Cells. Namalwa cultures were grown and diluted as described
(10, 11). The culture supernatant was precipitated with tri-
chloroacetic acid and the purification steps described for in-
duced cultures were followed.

Protein Determination and Amino Acid Analyses. Protein
concentrations were determined by the method of Lowry et
al. (15) with bovine serum albumin as a standard or by amino
acid analyses. The analyses were done in the laboratory of P.
E. Hare, Geophysical Laboratory, Carnegie Institute of
Washington, Washington, DC, using high-performance liquid
chromatography and fluorescence detection of o-phthalal-
dehyde amino acid derivatives (16, 17). Proline was measured
by gas chromatography as described (18), except that trifluo-
roacetic anhydride was used rather than pentafluoropropionic
anhydride. Cysteic acid and methionine sulfone were measured
after performic acid oxidation (19, 20). Tryptophan was de-
termined after hydrolysis in 6 M HCl/4% (wt/vol) thioglycolic
acid (21).

PURIFICATION

Concentration of interferon from cell culture medium by
precipitation with trichloroacetic acid and the subsequent re-
moval of the trichloroacetate by gel filtration on Sephadex G-25
have been described (11). All columns and collection tubes or
vessels used after the Sephadex G-25 column were plastic or
siliconized glass.

Affinity Chromatography on Anti-Interferon-Sepharose.
The anti-lymphoblastoid interferon columns (275-770 ml) were
equilibrated with phosphate-buffered saline, pH 7.4 (Pi/NaCl).
The eluant from the previous step (Sephadex G-25), repre-
senting 33.2-88.2 g of protein and 0.5-2 X 1Oa units of inter-
feron, was applied to each column and washed with 8-24 liters
of Pi/NaCl at 6oC. The columns were washed (25oC) with
500-1000 ml of Pi/NaCl and 250-500 ml of McIlvaine's ci-

Abbreviations: NaDodSOd, sodium dodecyl sulfate; PJNaCl, phos-
phate-buffered saline (4 mM KHzP04/15 mM NazHP01/133 mM
NaCI, pH 7.4) containing 0.05% NaNs; CBB R-250, Coomassie brilliant
blue R-250.

5601


5602  Biochemistry: Zoon et al.

trate/phosphate buffer, pH 5.0 (22). The absorbed interferon
was eluted with 500-1000 ml of McIlvaine's citrate/phosphate
buffer, pH 2.4-2.6. The interferon was stored at,-70oC until
enough material was accumulated for the next purification step.
For subsequent use the columns were washed first with 250-500
ml of PJNaCl, then with 250-300 ml of Pi/NaCl containing
4 M guanidine hydrochloride to remove any remaining bound
proteins, and finally with l-l.5 liters of PJNaCl.

Large-Scale Concentration of Affinity-Purified Interferon.
The interferon, eluted at pH 2.4-2.6 from the immunoabsor-
bant affinity columns, was pooled and concentrated on a Pel-
licon cassette ultrafiltration system (Millipore) PT series, with
an ultrafilter having a nominal molecular weight limit of 10,000
and 5 square feet (0.46 m2) of surface area.

Sephadex G-150. Concentrated interferon (0.5-5.5 X 10s
interferon units) in 75-100 ml was adjusted to pH 3.5 with 1 M
NaOH, applied to a Sephadex G-150 column, and eluted with
McIlvaine's citrate/phosphate buffer, pH 3.5, containing 0.05%
NaNs at 4-6oC.

SP-Sephadex. Operations were carried out at 6oC. All buffers
were McIlvaine's citrate/phosphate buffers and contained
0.02% NaNs.

The pooled Sephadex G-150 fractions containing the antiviral
activity were concentrated to 300-450 ml by ultrafiltration,
using the Millipore Pellicon cassette system. The concentrate
was then applied to a SP-Sephadex column (1.5 cm X 5 cm) that
had been equilibrated with pH 3.5 buffer. The column was
washed at a flow rate of 1.3 ml/min, first with pH 3.5 buffer
and then with pH 4.4 buffer. A subsequent wash with pH 5.5
buffer eluted approximately 90% of the interferon. A final wash
with pH 7.0 buffer followed by 0.1 M NaaHP04 buffer at pH
8.0 was required to remove the remaining bound interferon.

Treatment of Interferon with Glycosidase Mixture. Most
of the carbohydrate moiety of the interferon was removed by
a mixture of glycosidases isolated from a strain of Streptococcus
(Diplococcus) pneumoniae, type 1, prepared by S. Bose (23).
The glycosidase preparation (2.8 mg of protein per ml) con-
tained fl-galactosidase (9.4 units/ml), N-acetyl-fl-galactos-
aminidase (38 units/ml), and neuraminidase (1.4 units/ml)
(24-26). The interferon (pH 5.5) fraction of the preceding
SP-Sephadex chromatography was adjusted to pH 6.0 and 5 ~1
of the glycosidase mixture was added per mg of protein. In-
cubation proceeded for 4-5 hr at 37oC with shaking.

L-Tryptophyl-L-Tryptophan-Affi-Gel 10 Chromatography.
Affi-Gel 10 (lyophilized) was purchased from Bio-Rad. After
the treatment of the interferon preparation with the glycosidase
mixture described above, the material was adjusted to pH 7.0
with 1 M NaOH and applied to the column at a flow rate of 1.3

Proc. Natl. Acad. Sci. USA 76 (1979)

ml/min at 6oC. The column (11.3 cm in height; volume, 22.7
ml) was first washed with 100 ml of Pi/NaCl(6"C), and then
with 60 ml of PJNaCl containing 1 M NaCl(23"C). The ab-
sorbed interferon was eluted with 60 ml of PJNaCl containing
1 M NaCl and 75% (vol/vol) ethylene glycol (23oC) and col-
lected in an equal volume of 0.1% sodium dodecyl sulfate
(NaDodSOJ. The interferon eluate was dialyzed against 6-liter
volumes of 0.05% NaDodSOd first for 16 hr (6oC) and then
twice for 4 hr (25oC).

Preparation of the Interferon for NaDodSOd/Polyacryl-
amide Gel Electrophoresis. After elution from the L-trypto-
phyl-L-tryptophan-Affi-Gel 10 column and dialysis against
0.05% NaDodSOd, the interferon was concentrated to 0.5-1.0
ml by ultrafiltration at 6oC in an Amicon cell equipped with
a PM10 membrane. The sample was adjusted to 0.05 M Trip
WC1 at pH 6.8, 10% (vol/vol) glycerol, 1% NaDodSOa, and
0.002% bromophenol blue and incubated for 2-3 hr at 25oC
prior to electrophoresis.

NaDodS04/Polyacrylamide Gel Electrophoresis. Poly-
acrylamide gel electrophoresis was performed by using a
modification of the Laemmli system (27,28). The electropho-
resis units and sample well forming-combs were purchased
from Hoefer Scientific Instruments (San Francisco, CA).
Acrylamide and bisacrylamide were purchased from Bio-Rad
and NaDodSOd from Gallard-Schlesinger Chemical Corp.
(Carle Place, NY). Preparative gels were 1.5 mm in thickness
and 24-27 cm long (five wells). Protein standards of a molecular
weight range of 68,000-13,700 [bovine serum albumin (68,000),
ovalbumin (43,000) soybean trypsin inhibitor (21,500) myo-
globin (16,000) and lysozyme (13,700)] were applied in one
well on each slab. Approximately 200-400 pg of protein was
applied to each of the remaining wells and the gels were
subjected to electrophoresis for 16 hr at 11 mA per slab at 25"C,
using a constant temperature bath (Neslab, Portsmouth, NH).
The gels-were stained for 3-4 hr in 25% (vol/vol) isopropa-
nol/lO% (vol/vol) acetic acid/O.O25% Coomassie brilliant blue
R-250 (CBB R-250), destained with 10% acetic acid with four
to five changes over a 3- to 4-hr period, and washed twice with
deionized water. The CBB R-250-stained components mi-
grating between the soybean trypsin inhibitor and myoglobin
standards were excised and eluted with Pi/NaCl/O.l% Na-
DodS04 at 37oC for 16-18 hr. The volume of the elution buffer
varied from 2 to 6 ml, depending on the amount of gel to be
extracted. The extracted gel was removed by passing each
fraction through a Quik Sep column (Isolab Inc., Akron, OH),
which was then washed with 1.5 ml of the elution buffer. The
eluates were assayed and stored at -70oC. Analytical gels were
0.75 mm thick and 9 cm long.

                Table 1.  Purification of human lymphoblastoid interferon*
                          Units recovered Protein,  Specific activity, Recovery range
           Step                  x 10-s       w   units X 10m6/mg   per step, %

  Sephadex G-150                         128        60.6          2.1        40-60
  Ultrafiltration                          128        60.6          2.1        go-100
  SP-Sephadex C-25+                        81        20.1          4.0        65-100
  Glycosidase mixture treatment                 81        20.4          4.0         100
  L-Tryptophyl-L-tryptophan-Affi-Gel 10           72         5.1         14         84-87
  NaDodSOJpoIyacrylamide gel electrophoresis
   Peak fraction*                          20        0.080       250         27-31
    Total                               40        ND        ND         55-63
ND, not determined.
* In the experiment described here, total recovery of interferon from trichloroacetic acid precipitate (7.8 X 10s units) to
the peak fraction of the gel is approximately 3%. Modifications in storage and handling procedures described in the Results
have increased the overall recovery to approximately 10%.
+ pH 5.5 eluate only.
t The amount of interferon, estimated by amino acid analysis, recovered in the polypeptide species with the apparent mo-
lecular weight of 18,500.


Biochemistry: Zoon et al.

hoc. Natl. Acad. Sci. USA 76 (1979)   5603

RESULTS

A human lymphoblastoid interferon with an apparent molec-
ular weight of 18,500 has been purified to a single component
by NaDodS04/polyacrylamide gel electrophoresis and has a
specific activity of 2.5 X 10s units/mg of protein. Its production,
concentration by precipitation, and gel filtration on Sephadex
G-25 have been described (11). The subsequent immunoab-
sorbant-affinity chromatography step was modified by sub-
stituting anti-lymphoblastoid interferon for anti-leukocyte
interferon (11). The purification using either antibody was
essentially the same. Three to 10% of the antiviral activity did
not bind to the column. An average of 70% was eluted with
McIlvaine's citrate/phosphate buffer, pH 2.4-2.6, and 20% or
less was eluted with 4 M guanidine hydrochloride in Pi/NaCl.
The interferon eluted at pH 2.4-2.6 had a specific activity of
l-8 X lo5 units/mg of protein (lOO- to 800-fold purification
over the trichloroacetic acid precipitate) and was antigenically
the leukocyte subspecies. It was stored at -70oC until 2-5 X
lo8 units were accumulated. The interferon was then thawed,
pooled, and concentrated by ultrafiltration using the Pellicon
cassette system. The concentrate was fractionated by gel fil-
tration on Sephadex G-150. An elution profile is shown in Fig.
1. Less than 5% of the antiviral activity was associated with
proteins eluting at the void volume. Approximately 40-60% of
the antiviral activity was recovered in the volume eluted be-
tween 3800 and 5000 ml. This pooled eluant had a protein
concentration of 23-27 pg/ml.

Interferon eluted from the Sephadex G-150 column was
concentrated by ultrafiltration with no loss of antiviral activity.
A summary of the purification is shown in Table 1. We have
experienced losses of antiviral activity up to 60% in partially
purified interferon preparations when protein at concentrations
below 100 pg/ml was stored at 6oC or -70oC in plastic bottles
or tubes. Collection of interferon fractions with these low pro-
tein concentrations in siliconized glass bottles followed by im-
mediate concentration using ultrafiltration (Pellicon cassette
system previously described) has virtually eliminated these
losses. Thus, the overall recovery of the interferon species with
a molecular weight of 18,500 is 5-10% rather than the 3% re-
ported in Table 1 for a batch that had incurred these adsorption
losses.

After gel filtration on Sephadex G-150 the material was
further purified by ion-exchange chromatography using SP-
Sephadex C-25. Essentially 100% of the interferon activity was
bound to the SP-Sephadex column at pH 3.5. Less than 1% of

FIG. 1.  Purification of lymphoblastoid interferon by Sephadex
G-150. The immunoabsorbant affinity eluates (3 X lo8 units, 867 mg
of protein) were concentrated, adjusted to pH 3.5, and applied to the
column (46.5 cm in height, 6200 ml in volume). Fractions (12-13 ml)
were collected at a flow rate of 60 ml/hr at 4-6OC. The peak fractions
of interferon contained 1.5 X lOa units and 52.3 mg of protein.

the interferon was eluted at pH 4.4. Approximately 90% of the
recovered interferon was eluted at pH 5.5 and 10% at pH 7.0.
Fig. 2, lanes 1 and 2, shows the CBB R-250-stained polypeptide
patterns obtained with interferon-containing fractions eluted
from Sephadex G-150 and SP-Sephadex columns, respectively.
Analytical NaDodS04/polyacrylamide gels of the SP-Sephadex
fractions showed that several polypeptide species migrating in
the region of the antiviral activity were removed by the ion-
exchange chromatography (results not shown). Treatment of
the interferon preparation with the glycosidase mixture resulted
in complete recovery of the antiviral activity. No component
of the glycosidase preparation appeared to comigrate with the
interferon polypeptide species when analyzed by Na-
DodS04/polyacrylamide gel electrophoresis.

The dipeptide L-tryptophyl-L-tryptophan, bound to a
solid-phase support, has been used in the purification of human
leukocyte interferon (29). This chromatography was effective
in purifying lymphoblastoid interferon. However, the high

M,
x 1o-3

68d

43-h

21.5-b

16+

13.7*

__1.'

   .

"b".

           1       2        3        4

FIG. 2.  Analytical NaDodS04/polyacrylamide gel electrophoresis
of interferon from induced and uninduced cultures after several steps
in the purification. Lanes l-3 show the interferon from Newcastle
disease virus-induced cells and lane 4, interferon from uninduced cells.
Marker proteins designated by arrows on left are described in Ma-
terials and Methods. The arrow on the right indicates the component
corresponding to the antiviral activity. Lane 1, Sephadex G-150
fraction; lane 2, SP-Sephadex fraction, pH 5.5; lanes 3 and 4, ethylene
glycol fractions of L-tryptophyl-L-tryptophan-Affi-Gel 10.


5604  Biochemistry: Zoon et al.

concentration of ethylene glycol necessary to elute the inter-
feron also appeared to cause a loss of antiviral activity. There-
fore, the interferon eluted with 75% ethylene glycol was col-
lected in an equal volume of 0.1% NaDodSOd. The reduction
of the ethylene glycol concentration and the addition of Na-
DodS04 stabilized the interferon. The stabilization of interferon
with low concentrations of NaDodSOd was also observed with
fibroblast interferon and leukocyte interferon (6,30,31). Fig.
2, lane 3, shows an analytical NaDodSOd gel of the interferon
preparation after L-tryptophyl-L-tryptophan-Affi-Gel 10
chromatography.

In order to support that the polypeptide with the molecular
weight 18,500 associated with the antiviral activity was in fact
related to induction, interferon was prepared from uninduced
cultures, which had been shown to produce low levels of in-
terferon constitutively (32). The supernatant of 200 liters of
uninduced Namalwa cells contained 2.5 X 105 units of inter-
feron, which represents 0.25% of the amount obtained with a
virus-induced culture. The specific activity of the interferon
from the uninduced culture after gel filtration on Sephadex
G-25 was 4 units/mg of protein. The purification of this in-
terferon was analogous to that of the interferon from induced
cultures. Fig. 2, lane 4, shows the CBB R-250-stained pattern
of the ethylene glycol-eluate of the L-tryptophyl-L-tryptophan
column. The intensity of the stained component with a mo-
lecular weight of 18,500 from the uninduced culture was
greatly diminished. This correlates with the difference in the
specific activities of the two ethylene glycol fractions, the
partially purified interferon from uninduced cells having a
specific activity of 2.5 X 10s units/mg of protein.

Fig. 3 shows a typical preparative NaDodSOb gel, and the
comigration of the 18,500 component with antiviral activity.
The gel fractions corresponding to this active polypeptide
species were excised, eluted, and analyzed by analytical Na-
DodS04/polyacrylamide gel electrophoresis as shown in Fig.
4, lane 2. The amino acid composition of the polypeptide is
shown in Table 2. The analyses were performed with l- to 3-ng
samples, which were analyzed directly or dialyzed twice against
6-liter volumes of PJNaCl containing 0.1% NaDodSOd and

68,000      2 1,500 18,000
I    I I

                     x
                    5.;

FIG. 3.  NaDodS04/polyacrylamide gel electrophoresis of partially
purified interferon. Interferon (1.9 X lo7 units, 1.2 mg of protein) was
applied to the gel. Total recovery of interferon was 9.7X lb6 units with
5.0 X lo6 units in the 18.500 saecies. The CBB R-250 nolvaentide
pattern on the top of the figure represents one lane of-preparative
gel.

Proc. Natl. Acad. Sci. USA 76 (1979)

M,
x 1o-3

68-t

                 1            2

FIG. 4.  Analytical NaDodSOJpolyacrylamide gel electrophoresis
of the 18,500 species of lymphoblastoid interferon. Lane 1, molecular
weight standards: 2.5 fig of bovine serum albumin (68,000) and 5.0 pg
each of soybean trypsin inhibitor (21,500) and myoglobin (16,000).
Lane 2, 2.2 pg of 18,500 species derived from peak fraction of Na-
DodS04 preparative gel (see Table 1 and Fig. 3).

then twice against l-liter volumes of 0.01% NaDodS04 prior
to analysis. Unhydrolyzed samples were analyzed to monitor
for possible contamination and the quantity of each amino acid
detected was less than 10% of that found in the hydrolyzed
samples. These levels were subtracted from the values observed
with the hydrolyzed samples. The value obtained for methio-
nine sulfone is a minimum value because of losses in oxidation
(20). Sequencing data on the amino-terminal region of this
component obtained in collaboration with M. Hunkapiller and
L. Hood of the California Institute of Technology, Pasadena,
CA, indicate an essentially homogeneous polypeptide (to be
published). A second polypeptide representing less than 10%
of the total amount of protein was detected during the first few
steps of sequencing of one preparation; however, with a second
preparation, a single sequence was obtained. The quantity of
each amino acid phenylthiohydantoin derivative released
correlated with the amount of protein analyzed, strongly in-
dicating that this component of lymphoblastoid interferon is
not blocked at its amino terminus and is the protein being se-
quenced.


Biochemistry: Zoon et al.

      Table 2.  Amino acid composition of human
         lymphoblastoid interferon

    Amino acid           Residues per
     residue          155 amino acid residues*

      Asx                   14.0
      Thr                   7.5
      Ser                   10.0
      Glx                   25.6
      pro+                   10.2
     GUY                   10.0
      Ala                    10.3
    cyst                    1.7
      Val                    7.2
     Mets                   1.1
      Ile                     6.5
      Leu                   16.7
     Tyr                    3.6
      Phe                    6.7
      His                     4.1
     LYS                    9.8
     Arg                    9.0
     Tv                     0.6
* On the basis of a molecular weight of 18,500, the number of amino
acid residues is 155. The number of residues of each amino acid
represents an average of four determinations, except for Pro, Cys,
Met, and Trp.
+ Measured by gas chromatography; two determinations.
* Measured after performic acid oxidation as cysteic acid; two de-

terminations.

g Measured after performic acid oxidation as methionine sulfone; one
determination.

DISCUSSION

A single component of human lymphoblastoid interferon has
been purified over lOO,OOO-fold to homogeneity. The success
of its purification can be attributed to recent developments in
the large-scale production of lymphoblastoid interferon (11,
12). The preparation of large quantities of crude interferon has
made it possible to purify sufficient quantities of homogeneous
18,500 polypeptide to permit the initial characterization
studies.

The purified interferon is enriched in hydrophobic amino
acids and is very similar in composition to the mouse and human
fibroblast and leukocyte interferons (4, 8, 9). All these inter-
ferons are characterized by large quantities of leucine, aspartic
acid/asparagine, and glutamic acid/glutamine. Attempts to
identify the amino terminus of human lymphoblastoid inter-
feron by dansylation were unsuccessful primarily because of
contaminating amino acids present in the purified interferon
preparation (SS), and initial sequence studies had shown mul-
tiple phenylthiohydantoin derivatives in the first two cycles.
Subsequent cycles had yielded essentially one sequence. In
recent interferon preparations, the contaminants were removed
and a single sequence for the amino-terminal region was ob-
tained.

Sequencing studies on the purified interferon, using both
liquid and solid-phase sequencers, are not yet complete. A
comparison of the biological properties as well as the amino acid
sequences of the purified human lymphoblastoid, leukocyte,
and fibroblast interferons, and mouse interferons should soon
be possible. In addition, it is hoped that the imminent avail-
ability of large amounts of purified lymphoblastoid interferon
will permit more extensive examination of the antitumor,
antiviral, immunosuppressive, and growth inhibition effects
reported for partially purified interferon.

      Proc. Natl. Acad. Sci. USA 76 (1979)   5605

We thank Dr. P. E. Hare for his expert advice and assistance in
performing the amino acid analyses in his laboratory.

1.

2.

3.

4.

5.

6.
7.

8.

9.

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11.

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17.

18.

19.
20.
21.

22.
23.

24.

25.

26.
27.
28.

29.

30.

31.

32

33

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