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ANALYTICAL BIOCHEMISTRY 136, 7-23 (1984)

REVIEW

A Picomole Protein and Peptide Chemistry: Some


Applications to the Opioid Peptides’

STANLEY STEIN* AND SIDNEY UDENnUENDt


*Department of Molecular Genetics and tRoche Institute of Molecular Biology,
Roche Research Center, Nutley, New Jersey 07110

Technical advances that were introduced in tabolism. These proteins could be readily iso-
the late fifties led to the important discoveries lated in the milligram to gram range, providing
in protein chemistry of the sixties and sev- micromole quantities for assay. Most protein
enties. Some of the more important of these chemists still deal with micromole quantities.
techniques were chromatography (column and On the other hand, endocrinologists, immu-
thin-layer), gel electrophoresis, sequencing nologists, and molecular biologists are now
procedures, and the amino acid autoanalyzer. finding it necessary to deal with far smaller
The latter procedure, developed by Stanford amounts of active polypeptides, typically pi-
Moore, William Stein, and their colleagues, comole quantities.
represents the first real quantitative chemical More resourceful scientists were able to start
procedure in the field of protein chemistry. with huge amounts of tissue to obtain suffi-
Moore and Stein really put protein chemistry cient quantities of an active substance present
on a stoichiometric footing. They also pro- in trace amounts for characterization. For ex-
vided their fellow scientists with the instru- ample, in their pioneering research, Li (2) uti-
mentation and technology to permit them to lized tens of thousands of beef and hog pi-
study proteins as chemical entities (1). All the tuitaries to isolate the pituitary hormones,
above procedures made it possible to isolate while Guillemin (3), Schally (4), and their col-
and characterize enzymes, structural proteins, leagues required hundreds of thousands of beef
immunoglobulins, pituitary hormones, hy- hypothalami for the isolation of releasing fac-
pothalamic-releasing factors, and a host of tors. The obvious need for simpler and more
other biologically active polypeptides. While sensitive methods has led to many important
newer instrumentation and reagents were in- new advances. Taken together, these advances
troduced from time to time, protein- and pep- comprise a new protein and peptide chemistry.
tide-isolation techniques did not change dra- This report will present details of the new
matically until recently. The need for newer polypeptide chemistry mainly as it has de-
methods arose because of the changing inter- veloped in our own laboratories.
ests of biologists. The methods introduced in Over the past several years we have devel-
the fifties and sixties were designed to deal oped a technology (instrumentation and pro-
with the more abundant body proteins, he- cedures) to permit the characterization of
moglobin, albumin, globulins, and the en- small amounts of biologically active polypep-
zymes involved in digestion and energy me- tides (nanomoles or less) that can be isolated
from gram quantities of fresh tissues. For this
’ This article is dedicated to the memory of Stanford purpose, fluorescent procedures were intro-
Moore. duced that are applicable at the picomole level

7 0003-2697184 $3.00
Copyright 0 1984 by Academic Press. Inc.
All rights of reproduction in any form reserved.
8 STEIN AND UDENFRIEND

for monitoring polypeptides in column ef- All amino acids, with the exception of the
fluents, for measuring polypeptide concentra- imino acids proline and hydroxyproline, react
tion at each step of purification, for quanti- with fluorescamine. For amino acid analysis
tative peptide mapping (of enzymatically or the imino acids are converted to fluoresca-
chemically generated fragments), and for mine-reactive compounds, as described below.
amino acid analysis. These procedures are In polypeptides the reactive moieties are the
ideally suited for high-performance liquid t-amino groups of lysine residues and the (Y-
chromatography, which has revolutionized amino group of the amino-terminal residue.
both analytical and preparative separations. Polypeptides that have no lysine residues and
Microsequencing is carried out by two differ- with a blocked amino-terminus (e.g., N-for-
ent procedures, automated Edman degrada- myl, N-acetyl, pyroglutamyl, or prolyl) will
tion and time-course analysis of the release of not be detected with fluorescamine unless they
amino acids by an exopeptidase. The above are first subjected to hydrolysis. Fortunately,
methodology has been extensively applied by this situation is uncommon.
us to the isolation and characterization of
opioid peptides. Other notable studies from Monitoring of Column Efluents
our Institute include the isolations of human
For preparative applications a column ef-
leukocyte (5) and fibroblast interferon (6),
fluent is split so that only a small portion is
thymic peptides (7), and a series of antigen-
taken for reaction with fluorescamine, while
specific suppressor proteins from T-lympho-
the remainder is preserved for further study.
cytes (8).
A diagramatic sketch of a column-monitoring
In 197 1 our laboratory reported a procedure
instrument with this stream-splitting capacity
for detecting polypeptides fluorometrically
(12) is illustrated in Fig. 1. The stream-sam-
with a reagent composed of ninhydrin and
pling valve that is used is highly reproducible
phenylacetaldehyde (9). From studies on the
and reliable and the proportion of column
mechanism of that reaction Weigele and co-
effluent taken for detection is readily adjust-
workers (10) developed the reagent fluores-
able over a wide range. The limit of sensitivity
camine. This reagent yields the same fluoro-
of the instrument approaches 10 pmol of
phor as is obtained with ninhydrin and
amino groups (i.e., a protein with 10 amino
phenylacetaldehyde but by a more efficient
groups/m01 is detectable at a level of 1 pmol).
mechanism. Fluorescamine has become a key
Thus, for chromatography at the nanomole
component of our methodology because of
level a few percent of the column effluent is
it’s many inherent advantages (11). When it
more than adequate for fluorometric detec-
is added to an aqueous solution of a com-
tion. Absorption at short wavelengths (typi-
pound with a primary amino group it reacts
cally 206-2 16 nm) approaches the sensitivity
irreversibly and almost instantaneously to
of fluorescamine for monitoring columns and
form a relatively stable, intensely fluorescent
with less complicated instrumentation. How-
product. Excess reagent is hydrolyzed almost
ever, the lack of specificity of absorption in
as rapidly to nonreactive, water-soluble by-
the far-ultraviolet subjects it to non-peptide
products. What is most important is that flu-
interference and sharply limits the buffers and
orescamine and its hydrolysis products are
solvents that can be used during purification.
nonfluoresent so that the fluorescence pro-
duced is directly proportional to the concen-
High-Performance Liquid Chromatography
tration of the amino compound. Fluoresca-
mine is therefore an ideal reagent to add to Several years ago we began to apply the
column effluents on a continual basis for on- newly emerging technique of HPLC to the
line monitoring of amino acids and polypep- isolation of polypeptides. HPLC is carried out
tides. on rigid, small, and uniform sized (3- 10 pm)
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY

FLIJOROMETER

FIG. 1. Schematic illustration of the system used to monitor high-performance liquid chromatograms.
The sampling valve transfers aliquots of column effluent as a predetermined rate into the detection system.
The aliquots are picked up by a stream of water, and mixed with borate buffer (pH 9.3) and fluorescamine
in acetone. The generated fluorescence is measured in a flow cell and recorded. The fraction of column
effluent used for fluorescence assayto that collected is readily adjustable from 1 to 5% for isolation purposes
and to essentially 100% for analytical purposes such as amino acid assay.

beads (usually derivitized silica) that are tightly 17 residues, respectively, with the latter having
packed into a steel column. Although the same just one additional residue of leucine at its
principles of separation apply as in conven- carboxyl-terminus. Even more dramatic is the
tional column chromatography, the individual resolution of naturally occurring endorphin
components elute from the HPLC column as (M, 3500) from its synthetic analog which
extremely sharp peaks, resulting in markedly has a leucine ,instead of a methionine at
improved separations. A column 25 cm in position 5.
length with a diameter of 4.6 mm separates We have used reverse-phase HPLC for the
with an efficiency equivalent to 15,000 theo- purification to homogeneity of polypeptides
retical plates. The efficiency of conventional ranging in size from a few to several hundred
chromatographic columns of the same di- amino acid residues. In this technique the
mensions would be on the order of 100 theo- polypeptides partition between a hydrophobic
retical plates. Preparative HPLC runs are typ- stationary phase and a mobile phase. For elu-
ically complete within 1 to 4 h and, as a result tion, a gradient of increasing propanol, at
of the improved resolution, fewer steps are constant pH (3 to 6), has been used most fre-
required for the purification of a polypeptide quently. Pyridine-acetate buffer is used to
to homogeneity. Figure 2 demonstrates the maintain a constant pH and also to overcome
resolution of a series of opioid wptides that unwanted hydrophilic interactions of the
are actively being studied today. It should be polypeptides with the silica matrix. The pyr-
noted that (Y-and y-endorphin contain 16 and idine-acetate, propanol-water eluant system
10 STEIN AND UDENFRIEND

7
, ,oom

6- Leu5-&-Endorphr

FRACTION NUMBER

FIG. 2. Separation of opioid peptides on HPLC. A Lichrosorb RP- 18 column was run at room temperature
at 40 ml/h. Initial and final eluants both contained 0.5 M formic acid adjusted to pH 4.0 with pyridine.
The sample, containing 2 nmol of each endorphin and 10 nmol of each enkephalin, was injected in 1 ml
of the initial buffer and 6% of the effluent was monitored.

has the important advantage of being com- have demonstrated that silica beads of large
pletely volatile. Monitoring of column ef- pore size are required as supports for the re-
fluents by uv absorption is not practical‘with verse-phase HPLC of large polypeptides ( 14).
these eluents due to the high absorbancy of Proteins such as the subunit chains of collagen
pyridine. However, these eluents do not in- (A& 90,000) and even mixtures of intact col-
terfere in the fluorescamine-based column- lagen (A4,280,000) have been successfully re-
monitoring system. It should be noted that solved by HPLC. We have also demonstrated
most commercial HPLC instruments are that with large proteins slower flow rates are
equipped only with ultraviolet detection. The required than for amino acids and small pep-
most frequently used procedure for the elution tides (15). The equilibration of solute between
of peptides with such instrumentation utilizes the mobile and stationary phases that is re-
a gradient of acetonitrile in aqueous trifluo- quired to obtain optimal resolution takes lon-
roacetic acid (13). With high-purity solvents ger with the more slowly diffusing proteins.
this solvent system is compatible with both The literature from many HPLC manufac-
short-wavelength ultraviolet and fluorescence turers suggests column runs of 10 to 20 min
detection. This mixture of solvents is also vol- for proteins. While one can resolve peaks of
atile. some standard proteins in such a short time,
The sequential use of reverse-phase chro- much longer runs are required to optimally
matography with certain modifications has resolve the large number of components that
been successfully applied to the purification are present in a complex tissue extract. Op-
of many polypeptides. Several types of reverse- timal resolution of mixtures of polypeptides
phase columns, containing such functional requires hours of chromatography rather than
groups as octyl, octadecyl, phenyl, diphenyl, minutes.
and cyanopropyl, each with its own specificity, Reverse-phase HPLC has not yet been
are available. Furthermore, chromatography widely applied to enzymes, probably because
with the same support and gradient but at a of the fear of their instability in the organic
different pH can resolve a mixture of poly- solvents that are used for elution. However,
peptides in an entirely different manner. We many enzymes are surprisingly stable to re-
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY 11

verse-phase HPLC. Regnier et al. (16) have due to the programmed addition of N-chlo-
used acetonitrile and TFA’ for chromatog- rosuccinimide, which is used to convert the
raphy of alkaline phosphatases. In our labo- imine, proline, to the primary amine, 4-ami-
ratory placental alkaline phosphatase has been nobutyraldehyde. It should be noted that the
purified by HPLC on cyanopropyl-substituted chromatographic separation is based on the
columns (300 nm pore size) utilizing a l-pro- original procedure of Spackman et al. (l), with
panol-pyridine acetate mixture for resolution but minor improvements with respect to the
(N. Ezra, personal communication). Chy- quality of the chromatography support and
motrypsin and trypsin have also been purified the purity of the buffers. The fluorescamine
by HPLC (17). amino acid analyzer has also been adapted to
Some enzymes may lose biological activity amino sugar analysis, again in the picomole
when exposed to organic solvents. In such range (19). We have also adapted the proce-
cases ion-exchange HPLC can be used. The dure for assay of neutral sugars in the picomole
HPLC version operates on the same principles range (Moschera, personal communication).
as conventional ion-exchange columns but A few hundred picomoles of a protein are
sharper peaks are obtained. The preparation adequate for replicate assays of amino acids,
of ion-exchange HPLC supports suitable for amino sugars, and neutral sugars. We applied
polypeptides was pioneered by Regnier and this methodology to show that the sugar con-
co-workers (18). Two of their advances were tent of pro-opiomelanocortin varies from one
to use large-pore silica as the matrix and to animal to another in the same species. Samples
chemically coat the silica with an “organic isolated from some individuals contained no
skin” (glycophase) to minimize nonspecific amino sugar, while samples from others
adsorption of the protein to the silica. A variety yielded a second pro-opiomelanocortin peak
of anion- and cation-exchange columns are with several equivalents of carbohydrate per
now commercially available. High-sensitivity mole. Proenkephalin from bovine adrenal
monitoring of the effluents of such columns medulla apparently contains no amino sugar
by ultraviolet absorption at short wavelengths as determined from assay of the enkephalin-
can present problems due to ultraviolet-ab- containing (EC) peptide most likely to have
sorbing contaminants in the salt solutions used such substitution (20).
for development. Monitoring at 280 nm is less Another reagent, o-phthalaldehyde, is also
sensitive but still useful. Fluorescamine mon- used for fluorescent amino acid analysis at the
itoring is virtually unaffected. picomole level by postcolumn reaction (21).
Precolumn reaction of amino acids with o-
Amino Acid Analysis phthalaldehyde is even more sensitive and has
been used in our laboratory for certain ap-
An instrument similar to the one shown in
plications (i.e., carboxypeptidase Y sequenc-
Fig. 1 has been used for many years in our
ing, see below) (22). The sensitivity achievable
laboratories as an automated amino acid an-
with either reagent is about lOO-fold greater
alyzer. Using postcolumn reaction with flu-
than with the standard ninhydrin-based an-
orescamine, amino acid analysis can be per-
alyzers optimized for high sensitivity.
formed at the picomole level for all the amino
A unique property of fluorescamine is that
acids found in proteins ( 19). A chromatogram
it gives little fluorescence with ammonia (ca.
of a standard calibration mixture of amino two to three orders of magnitude less than
acids ( 100 pmol) is shown in Fig. 3. The base-
with an amino acid). This is of great impor-
line shift associated with the proline peak is
tance in the fluorescamine amino acid ana-
’ Abbreviations used: TFA, trifluoroacetic acid, SDS, lyzer, since traces of ammonia are generally
sodium dodecyl sulfate; PTH, phenylthiohydantoin; EC, present in column eluents. This insensitivity
enkephalin-containing. to ammonia makes it possible to perform
W

24 48 72
TIME (mlnutasl

1 I I
24 48 72 96 120 144
TIME minuted

FIG. 3. Chromatography of a standard mixture containing 100 pmol of each amino acid (A) and of the
control buffer mixture (B).

12
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY 13

amino acid analysis of a protein present in a amines are present, as in the earliest stages of
stained band on an electrophoretic gel. In this a purification procedure. This is true for all
procedure (23) the stained protein band is ex- protein methods. However, an intermediate
cised from the gel and hydrolyzed in acid gel-filtration step prior to the fluorescamine
(without extraction of the protein from the reaction can separate the polypeptides from
gel), and the hydrolysate is applied to the col- low-molecular-weight amines. The utility of
umn. The large amount of ammonia liberated the automated fluorescamine method is based
from the polyacrylamide gel during hydrolysis on the advantages of being sensitive in the
does not interfere with the analysis of the basic nanogram (picomole) range, applicable to
amino acids as it does with either ninhydrin polypeptides of any size, reproducible, and
or o-phthalaldehyde detection. simple to perform. Since it takes only 2 min
per assay, many samples can be assayed in a
Quantitative Assay of Peptide and Proteins short period of time.

During the purification of any polypeptide Criteria of Homogeneity


it is necessary to monitor specific activity (bio-
The goal of purification is to obtain the
logical activity per unit of polypeptide) at each
desired polypeptide as a single component.
step as an index of the efficiency of the pu-
There are many indices of the attainment of
rification. This requires a bioassay for the nu-
homogeneity. Changes in specific activity
merator and estimation of total polypeptide
during purification diminish as homogeneity
for the denominator. Proteins are widely as-
is approached, and the attainment of constant
sayed by the Lowry method, by Coomassie
specific activity on continued purification is
blue binding, and by ultraviolet absorption.
one of the most important criteria of purity.
However, all three procedures require several
This should be paralled by the attainment of
micrograms (nanomoles) of material and are
a single sharp peak on HPLC and by a single
not even applicable to small polypeptides. It
band on SDS-gel electrophoresis (for larger
should be noted that in the mixtures char-
polypeptides). It should be noted that Coom-
acteristic of tissue extracts during purification,
assie blue, which is widely used for staining
measurements of total protein are arbitrarily
protein gels, is not sufficiently sensitive to be
compared to a standard. Each of the assays
used in microchemistry. The more recently
depends on some characteristic, i.e., aromatic
introduced silver-staining procedure (24) is the
amino acids for the Lowry method and for
one that we have used in our studies. It is
ultraviolet absorption. We have devised an
readily applied with a sensitivity in the nano-
alternative procedure to assay polypeptides of
gram range.
all sizes in the nanogram (picomole) range.
One “bypasses” the HPLC column in the flu-
Quantitative Peptide Mapping
orescamine-monitoring system (Fig. 1) and
injects samples (microliter volumes) directly A protein yields a characteristic mixture of
into the stream of buffer. The fluorescence peptides when treated with a protease. When
produced is proportional to the amount of the peptides obtained by treatment of a large
polypeptide injected and is related to the free- polypeptide with trypsin are subjected to two-
amino content, mainly lysine. As shown in dimensional chromatography and/or electro-
Fig. 4, several nanograms of a protein or small phoresis the resulting chromatograms or elec-
peptide are sufficient for assay and fluores- trophoretograms are referred to as tryptic maps
cence is proportional to concentration up to and provide valuable structural information.
at least 10 pg. The fluorescamine assay for These maps have generally been developed by
polypeptides is subject to error when amino treatment of paper or thin-layer plates with
acids or other low-molecular-weight primary ninhydrin, or for more sensitivity with flu-
14 STEIN AND UDENFRIEND

PROTEIN hancqroms) PROTEIN (mbcrogroms)

FIG. 4. Bypass analysis of protein concentration with the septum injector. Varying volumes (2.5, 5.0,
7.5, and 10 ~1) of bovine serum albumin (10 ng/Fl on letI and 100 ng/pl on right) were injected at 2-min
intervals. The recorder tracings of the individual injections are shown in the center and the plots of average
peak height versus amount of protein are shown on thp sides (Ref. (18)).

orescamine. However, such procedures are not acid residue (as the PTH or phenylthiohy-
quantitative and preparative applications are dantoin derivative) from the amino terminal
difficult. The high resolution of HPLC makes of the polypeptide. The PTH derivatives are
it possible to resolve all the peptides in a tryptic identified by HPLC with ultraviolet detection
digest of even the largest protein. Furthermore, (26). Recently, a commercial instrument,
the method is quantitative and can be applied based on a new design for automated Edman
to picomole quantities of proteins. An appli- degradation (27) has become available for se-
cation of HPLC tryptic mapping with flu- quencing at the lOO-pmol level. (Applied
orescamine detection to the opioid peptides Biosystems, Foster City, Calif.). The imme-
is presented in a subsequent section. Peptide diate popularity of this instrument signals an-
mapping by HPLC, utilizing ultraviolet de- other major advance in protein analysis.
tection and a reverse-phase column with a Automated Edman degradation by the
gradient of acetonitrile in aqueous trifluora- above instrumentation can generally yield se-
cetic acid, is most commonly used (13). quences of 20-30 residues. However, certain
residues in a sequence can seriously hinder
further degradation. For this and other reasons
Microsequencing
alternative methods are desirable. Sequential
Polypeptides of any size can be submitted exopeptidase digestion with fluorescent amino
to sequence analysis by the Edman degrada- acid analysis has been found quite useful for
tion procedure (25). In collaboration with sequencing from the carboxyl terminus (22).
J. E. Shively we have used an automated In this procedure about 300 pmol of a poly-
“spinning cup” instrument that was modified peptide is treated with carboxypeptidase Y and
for microsequencing (26). One nanomole of aliquots (10%) are withdrawn at various time
peptide is sufficient in this instrument. Each intervals for amino acid analysis. The pre-
cycle of chemical reaction removes an amino ferred method of analysis has been a procedure
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY 15

in which the aliquot of digest is treated with studies are presented as illustrations of the
o-phthalaldehyde and the fluorophors ob- above methodology.
tained are resolved by reverse-phase HPLC. The enkephalins were first isolated from
This approach is especially suited for smaller porcine brain in 1975 (28). Shortly thereafter
peptides. Sequencing can be carried out from it was recognized that the same pentapeptide
the amino terminus in a similar manner using sequence is present in a pituitary protein, p-
an aminopeptidase. lipotropin (M, 10,000) (29). &Endorphin, a
35004.4, fragment of /3-lipotropin, and the
Cloning and cDNA Sequencing smaller fragments (Y- and y-endorphin were
subsequently shown to contain the enkephalin
Until recently sequencing proteins was car-
sequence at their amino termini (30). All the
ried out entirely by chemical means. Large
above studies required thousands of pituitaries
peptides and proteins cannot be entirely se-
obtained from large animals (sheep, beef, hogs,
quenced in their intact state but are generally
or camels). We turned our attention to the
cleaved into fragments prior to sequencing.
opioid peptides for a number of reasons. First,
Obtaining a complete sequence of even an
we realized that elucidation of the physiology
average-sized protein by such methods takes
and pharmacology of these substances would
several months. With the advent of molecular
require studies on small laboratory animals.
genetics it has become possible to prepare and
The rat has long been used in studies of mor-
sequence the DNA equivalent of a protein,
phine pharmacology. If such studies were to
cDNA, in a few weeks. This does not mean
be carried out in the rat, one had to make
that protein chemistry is obsolete. Micropro-
certain that the enkephalins and endorphins
tein chemistry must now be used in partner-
in the rat are chemically identical to those in
ship with cDNA cloning and sequencing. They
are a powerful combination. It should be kept
in mind that a newly discovered biological TABLE 1
activity in a tissue extract cannot be readily
cloned until something is known about it. The AMINO ACID COMPOSITIONS
more information that can be obtained about &Lipotropin &Endorphin
a polypeptide the easier it is to clone. Partial Amino
sequences obtained from a protein can be used acid Rata Sheep b Rat” Sheep b
to construct synthetic oligodeoxynucleotide
probes provided the isolated protein is pure. Asx 7 2 2.1 2
Thr 4 4 3.0 3
The latter information can be most important Ser 4 5 1.6 2
in isolating a specific mRNA, particularly if Glx 12 12 3.1 3
it is a rare species. It is also important in de- Pro 10 5 1.3 1
tecting the specific clones. Information from GUY 8 8 2.8 3
chemical sequencing is essential for locating Ala 4 13 2.1 2
CYS 0 0 0 0
the amino and carboxyl termini of a hormone Val 4 2 1.2 1
within a prohormone sequenced by cDNA. Met 2 2 0.9 1
Finally, chemical peptide-sequencing data Ile 2 2 1.9 2
provide a check on DNA sequencing. Leu 1 6 2.2 2
Tyr 3 3 1.1 1
Phe 4 3 2.2 2
Application to the Opioid Peptides His 3 2 0.9 1
Elucidation of the structure of proenke- LYS 12 10 5.1 5
Aa 5 5 0 0
phalin and the products of its processing in
our laboratories made use of alI the procedures ’ Determined by multiple analysesof 24-h hydrolysates.
discussed above. Details of some of those ’ From published sequence data.
16 STEIN AND UDENFRIEND

A IO g the larger animals. Another purpose of the


1
study was to elucidate the pathway of enke-
06 E
1 06 2
phalin biosynthesis. We began a systematic
0 3.0 5 isolation of the various opioid-related peptides
2 04 Y
20 J
in rat pituitaries using a radioreceptor assay
i for opioid peptide activity. Two nanomoles
0.2 1.0 t3
I of pure &lipotropin, the lO,OOO-Da precursor
00 5
0 20 40 0 9 of the endorphins, was isolated from 40 rat
pituitaries (3 1). This was sufficient for replicate
amino acid analyses, for determination of
molecular weight, and for other studies. Six
I nanomoles of P-endorphin were isolated from
a 200 rat pituitaries. The amino acid analyses
d
r in Table 1 indicate that rat P-endorphin has
f the same structure as the previously charac-
2
2 terized sheep /?-endorphin. However, rat ,8-
2.0 $
-
[M;T]ENK [L

FRACTION NUMBER

160

120

60

40

0 42 I50 I66
TIME (min)

FIG. 5. Purification of proopiocortin from a single camel FIG. 6. Chromatography of opioid peptides in Fraction
pituitary. The frozen pituitary was homogenized in dilute V from adrenal medulla. A deproteinized extract from
acid in the presence of protease inhibitors. Initial fmc- seven adrenal glands was applied to an Ultrasphere ODS
tionation of the extract was carried out on Sephadex G- column (5 pm, 4.6 X 250 mm) and eluted at 30 ml/h
100 (A) Fractions 21-30 were combined, concentrated with 0.5 M formic acid/O.4 M pyridine, pH 4.9, with a
under reduced pressure, and purified further on Sephadex gradient of I-propanol: 0% (12 min) followed by a 3-h
G-75 (not shown). Chromatography of the peak fractions linear gradient of 5-202. Fractions ( 1.O ml) were collected,
of opioid activity from the G-75 column was performed and aliquots (75 ~1) were lyophilized and analyzed by the
on a Lichrosorb RP-8 column (B). Rechromatography of radioreceptor-binding assaydirectly (A) or after digestion
fraction 25 (B) on the RP-8 column was performed under with carboxypeptidase B (13). The numbered peaks (I-
modified elution conditions(C). Fraction 50 (C)was shown 9) indicate identified opioid peptides. The elution positions
to be homogeneous by gel electrophoresis, tryptic peptide of synthetic [Metlenkephalin and [Leulenkephalin are
mapping, and specific activity (33). shown in (A) (38).
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY 17

L
0
TIME (mid

FIG. 7. High-performance liquid chromatography of peak IV from bovine adrenal medulla on Lichrosorb
RP-18. The column (10 pm, 4.6 X 250 nm) was eluted at 30 ml/h with 0.15 M formic acid/O.4 M pyridine
buffer, pH 4.0, using a linear gradient of 1-propanol from 0 to 20% for 120 min. Approximately 2% of
the column effluent was directed to the fluorescamine-monitoring system. Aliquots of each fraction (1.5
ml) were analyzed by the radioreceptor-binding assay before and after digestion with trypsin. The active
fractions are labelled A-J (40).

lipotropin differs considerably from species to reasons, it is more generally referred to as pro-
species. opiomelanocortin.
During our studies on rat pituitary extracts Although the above series of peptides con-
we demonstrated the presence of a larger pre- taining the [Metlenkephalin sequence had
cursor of /3-lipotropin (31,32). When fl-lipo- been isolated from the anterior pituitary gland,
tropin or any of the endorphins is digested there was no proof that they represented in-
with trypsin, the fragment P-lipotropin 6 l-69 termediates in the biosynthesis of [Met]-
is generated. This nonapeptide contains enkephalin, particularly in tissues other than
the [Metlenkephalin sequence and is the the pituitary. A number of observations, in-
only tryptic fragment that contains opioid cluding the absence of equivalent precursors
activity. A high-molecular-weight component of [Leulenkephalin in the anterior pituitary,
(-30,000) from an extract of rat pituitaries led us to search elsewhere for the true enke-
was prepared by chromatography on Sephadex phalin precursor. Based on immunocyto-
G-75. This protein extract was digested with chemical observations of large amounts of en-
trypsin and the mixture of peptides was ap- kephalin-like material in adrenal medulla (35),
plied to a reverse-phase column. Fractions we looked for and discovered a new family of
were collected and assayed for opioid activity, EC peptides in this tissue (36). With the aid
which was found only at the elution position of our fluorescamine-HPLC methodology,
corresponding to that of the synthetic nona- isolation and characterization of most of the
peptide. This -30,000 Da precursor, which EC peptides in adrenal extracts were accom-
we named pro-opiocortin, was subsequently plished within 2 years. In all these studies no
purified to homogeneity in nanomole amounts more than 25-50 g of bovine adrenal glands
(Fig. 5) from a single camel pituitary (33) and were used in any one experiment.
subjected to amino acid assay. Pro-opiocortin Chromaffin granules from bovine adrenal
contains within it the sequence of corticotro- glands were homogenized in dilute acid con-
pin, MSH, as well as &lipotropin, as was first taining protease inhibitors and subjected to
demonstrated by Mains et al. (34). For obvious chromatography on G-75. Aliquots of the
18 STEIN AND UDENFRIEND

60

I I
0 30 60 90
TIME (min)

FIG. 8. Fractions F-G and H-l obtained from RP-18 chromatography of peak IV (Fig. 7) were each
applied to a Spherisorb CN column (5 pm, 4.6 X 250 mm). Each column was eluted at 30 ml/h with 0.5
M formic acid/O.4 M pyridine buffer, pH 4.0, using a linear gradient of I-propanol from 12 to 28%. Aliquots
were subjected to radioreceptor assay following treatment with trypsin (A) F-G, (B) H-I (40).

fractions were then treated with trypsin and all sizes were observed, ranging from approx-
carboxypeptidase B to release internal enke- imately 20,000 Da to the pentapeptides them-
phalin sequences for bioassay. EC peptides of selves. Many of the larger peptides contained

TABLE 2
OVERALL PURIFICATION OF THE 12,600-D EC PEPTIDE FROM BOVINE ADRENAL MEDULLA

Volume Activity Protein Specific activity Recovery Purification


Procedural step (ml) (nmol)” bg) (nmol/mg) (%I (-fold)

Supematant 1200 285 12,900 0.02 100 1


Isolated chromaffin
granules 50 203 430 0.47 70 23
Sephadex G-75 182 108 6.7 16 37 782
Lichrosorb RP- 18 5.5 73 1.74 42 25 2047
Ultrasphere Cls 4.2 61 0.93 66 21 3220
Spherisorb CN 4.0 59 0.76 78 20 3818
Diphenyl 4.0 56 0.72 78 19 3818
a In [Metlenkephalin equivalents.
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY 19

multiple enkephalin sequences (37). These apeptides and heptapeptides (Fig. 6). Each was
were arbitrarily divided by size into five fiac- purified to homogeneity and its structure de-
tions (I to V). Neutral extracts contained an termined by amino acid assay and sequencing
even larger EC peptide. Fraction V, containing (38,39).
the smallest EC peptides, was resolved by Fraction IV, when subjected to HPLC, re-
HPLC into many active peptides including vealed several EC peptides in the range of 2000
[Met]- and [Leulenkephalin, as well as hex- to 5000 Da. Each was subjected to further

Time, min

FIG. 9. Tryptic maps of the 9.6~kDa carboxymethylated EC peptide (A), the 12.6~kDa carboxymethylated
EC peptide (B), and the 3.8~kDa EC peptide (C). In each case, 4 nmol of the purified peptide were dissolved
in 200 ~1 of 0.2 M phosphate buffer, pH 8.2, and then digested with 1 pg of trypsin for 18 h at 37°C. After
digestion, the pH was adjusted to 4.0 with acetic acid and 2-mercaptoethanol was added to reduce methionine
residues that may have been oxidized. The ‘2SI-[Leu]enkephalin (approximately 2000 cpm), added as an
internal standard for each chromatography run, produced three reproducible peaks of radioactivity. These
are marked by arrows in each chromatogram. Chromatography was performed on an Ultrasphere ODS
HPLC column (5 pm particle size; 4.6 X 250 mm) using a linear gradient of I-propanol (- - -) in 0.9 M
pyridine acetate, pH 4.0, at a flow rate of 20 ml/h. Five percent of the column effluent was diverted to the
detection system. Aliquots of each fraction were also digested with carboxypeptidase B and assayed for
receptor-binding activity. The tryptic peptides are numbered according to their elution position. Those
from the 8.6~kDa EC peptide are designated by the symbol T9, those from the 12.6~kDa peptide by the
symbol T13, and those from the 3.8-kDa peptide by T4. Hatched bars, [Leu]enkephalin equivalents; (-),
relative fluorescence (41).
20 STEIN AND UDENFRIEND

TABLE 3
SUMMARY OF NONENKEPHALIN TRYPTIC PEPTIDES ISOLATED FROM THE
12.6.kDa, 8.6-kDa, AND 3.8-kDa POLYPEPTIDES

12.6-kDa ECP 8.6-kDa ECP 3.8-kDa ECP


Yield Yield Yield
Shared residues No. (%o) No., 6) No. (%)

Ala,Leu,Arg T13-2 (65) T9-2 (60) NP -


Asx,Thr,SerZ ,GlxZ ,Ala,CMC3 ,Tyr,Arg T13-3 (76) T9-3 (88) NP -
Thrt,Glx,CMC,Trp,Lys T13-4 (61) T9-4 (74) NP -
Ser,Glx3 ,Ala,Leu2 ,His,Lys T13-5 (76) T9-5 (76) NP -
Ser,Pro,Leu2,Lys T13-6 (11) T9-6 (10) NP -
Thr,Glx* ,LeuS,Lys T13-I2 (95) T9-9 (96) NP -
Asx,Thr,Ser2 ,Glx,Pro* ,Alaz ,Leul ,Lys T13-13 (73) T9-10 (82) NP -
Asxz ,Thr, ,Glx2 ,Proz ,Gly,Ala,CMC2 ,Leus ,Lys T13-14 (8) T9-1 I (10) NP -
Asxz ,Glx6 ,Pro,GlyX ,Ala,Tyr,Met,Va12 ,Leus ,Lysz T13-15 (45) NP - T4-5 (42)
A~~~,G1~~,Pro,Gly,,Ala,Tyr,Met,Val~,Leu~,Lys T13-16 (30) NP - T4-6 (34)
Asx2 ,Thr2 ,Ser,Glx* ,ProJ ,Gly,Ala,CMC2 ,Leus ,Lysz T13-17 (50) T9-12 (44) NP -
Asx2 ,Thrz ,Ser,Glxl ,Pro3 ,Gly,Ala2 ,CMC2 ,Leue ,Lysl!Am T13-18 (5) T9-13 (8) NP -
Note. Designations for the tryptic peptides are as in Fig. 9. NP, not present; CMC, carboxymethylcysteine (Ref.
(41)).

HPLC until homogeneity was achieved. De- 18,200 Da were also purified from bovine ad-
tails of the purification and characterization renal medulla. Purification of the 12,600-Da
of Peptide I (40) are shown in Figs. 7 and 8. EC peptide (41) is summarized in Table 2.
Peptide I was of interest for two reasons. It The attainment of constant specific activity
was the first example of an EC peptide that and a single sharp peak at the final steps of
contained more than one enkephalin se- HPLC are indicative of homogeneity. The
quence. It also contained within it a sequence peptide also yielded one band on SDS-gel
of unusually low degeneracy that was later electrophoresis. The molecular weight esti-
used to design the probe for cloning proen- mated from amino acid analysis was higher
kephalin cDNA (see below). than was subsequently established by partial
EC peptides of 5300, 8600, 12,600, and sequencing. The structure of the 12,600-Da
1 5 IO 15 20

Ser-Pro-His-Leu-Glu-A~p-Glu-Thr-Ly.-Olu-~eu-Gln-~y~-Arg-Tyr-Gly-Gly-Phe-Met-Arg

Peptide I
39

S...LlGG.UGG.AUG-GAZUACG... mRNA

ACC.ACC.TAC.CTG.ATGO 5, DNA primer


3’

FIG. 10. The amino acid sequence and corresponding mRNA nucleotide sequence of peptide I that was
used to derive the synthetic decahexanucleotide used as a primer for cloning proenkephalin cDNA. Note
the extremely low degeneracy of the codons. The synthetic nucleotide is shown at the bottom.
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY 21

EC peptide and its relationship to two other tryptic products of the 12,600-Da peptide were
EC peptides that we had isolated, 3800 and present in either the 3800- or 8600-Da EC
8600, were deduced by sequencing as well as peptide in exactly the same proportions. The
by quantitative tryptic mapping. Equimolar summary of these data in Table 3 demon-
quantities (4 nmol) of each of the three pep- strates the precursor-product relationship of
tides were digested with trypsin and the digests some of the larger and smaller EC peptides
subjected to HPLC (Fig. 9). It can be seen in adrenal extracts.
that, except for the enkephalin region, all Sixteen large- and intermediate-size EC

Glu cys

SW Cln Asp Cys Ala Thr Cys Ser Tyr Arg Leu Ala Arg Pro Thr Asp Leu

Asp Pro Leu Ala Cys Thr Leu Glu Cys Clu Gly Lys Leu Pro SW Leu Lys
__( ;- GCT TGC ACT CTG GAA TGT GAG GGG AAA CTA CCT TCT CTC AAG

Thr Trp Glu Thr Cys Lys Clu Leu Leu Gin Leu Thr Lys Leu Glu Leu Pro
ACC TCG GAA ACC TGC AAG GAG CTT CTG CAG CTG ACC AAA CTA GAA CTT CCT

Pro Asp Ala Thr Ser Ala Leu SW Lys Gin Clu Glu Ser His Leu Leu Ala
CCA GAT CCC ACC ACT GCC CTC AGC AAA CAC GAG GAG ACC CAC CTG CTT GCT

w\Tyr Gly Gly Phe Met[wdTyr Gly Glv Phe MetlslMet


AAC AAG TAC GGG GGC TTC ATG AAG CGG TAT GGG CGC TTC ATG AAG AAA ATG

Asp Glu Leu Tyr Pro Leu Glu Val Glu Glu Glu Ala Asn Gly Gly Glu Val
GAT GAG CTG TAC CCC CTG GAA GTG GAA GAA GAG GCA AAT CGA GGT GAG GTC

Leu Glylwg]Tyr Gly Gly Phe Met-Asp Ala Glu Glu Asp Asp
CTT GGC AAG AGA TAT GGG GGC TTC ATG AAG AAG GAT GCA GAG GAA CAT GAC

Gly Leu Gly Asn SW Ser Am Leu Leu Lys Glu Leu Leu Gly Ala Gly Asp
GGC CTG GGC AAC TCC TCC AAC CTG CTC AAG GAG CTG CTG GGA GCC GGG GAC

Gin Arg Glu Gly SW Leu His Gin Glu Gly Ser Asp Ala Glu Asp Val SW
CAG CGA GAG GGG AGC CTC CAC CAG GAG GGC ACT GAT GCT GAA GAC GTG AGC

wArg]Tyr Gly Glv Phe Met Arg Gly LeuLndSer Pro His Leu Glu
AAG AGA TAC GGG CCC TTC ATG AGA GGC TTA AAG AGA AGC CCC CAC CTA GAA

Asp Glu Thr Lys Glu Leu GlnwdTvr Gly Glv Phe MetmVal
GAT GAA ACC AAA GAG CTG CAG AAG CGA TAC GGG GGT TTC ATG AGA AGA GTG

Gly Arg Pro Glu Trp Trp Met Asp Tyr GlnlxtTVr Gly Gly Phe Leu
GGT CGT CCA GAG TGG TGG ATG GAC TAC CAG AAA AGG TAC GGT GGC TTC CTC

[m]Phe Ala Glu Pro Leu Pro Ser Glu Glu Glu Gly Glu Ser Tyr Ser
AAG CGC TTC GCC GAG CCC CTA CCC TCC GAG GAA GAA GGC GAA AGT TAC TCC

Lys Glu Val Pro Glu Met GlulArg]Tyr Gly Gly Phe Met Arg Ph*e*
ABC GAA GTT CCT GAA ATG GAG AAA AGA TAT CGA GGA TTT ATG AGA TTTTAAT
-
CCCCTTTCCCATCAGTGACCTGAAGCCCCAGCAAGCCTTCCTCTGCCCCCAGTGAAAGACTGCTGCG

CTGGTGTGTTGTATTGTCCCGAGTCGCTTGCATTATATAGTTGACTTGAGAGTCCAGATAATTAACT

ATACAACCTGAAAGCTGTGATCCCAGGTTCTGTGTTCTGAGAATCTTTAAGCTTTTAAATATTGGTC

TGTTGCAGCTGTCTTGTTTCCATGCTCAGTTTTTTGTTATCACTTTGTCCTTTATTTTTGACATAAT

GCCAATAAATGCCTACTTGTGTGTAGATATAACTAAAA-3'

FIG. 11. Complete nucleotide sequence and derived amino acid sequence for proenkephalin. The en-
kephalins are underlined, with the flanking basic amino acids. ‘Octapeptide, [Metlenkephalin-Ar$-Gly’-
Leu*. **heptapeptide, [Metlenkephalin-Arg6-Phe7. Note that the DNA sequence obtained from the clone
does’not cover the entire amino acid sequence of the largest ECP. The stop codon is underlined, as is the
sequence AATAAA in the 3’-untranslated region. (Ref. (43)).
22 STEIN AND IJDENFRIEND

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in Annual Review of Immunology (Paul, W. E.,
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shown in Fig. 11. Exactly the same structure 10. Weigele, M., DeBemardo, S. L., Tengi, J. P., and
Leimgruber, W. (1972) J. Amer. Chem. Sot. 94,
of proenkephalin was obtained from the cor- 4052-4054.
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15. Jones, B. N., Lewis, R. V., Paabo, S., Kojima, K.,
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meetings. At those lunches he discussed our Anal. Chem. 48, 1839-1845.
work, not his. He also visited our laboratories 17. Titani, K., Sasagawa, T., Resing, K., and Walsh,
K. A. (1982) Anal. Biochem. 123, 408-4 12.
and invited us to his. What is notable is that 18. Chang, S. H., Gooding, K. M.. and Regnier, F. E.
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him were in the late forties and fifties, for the 19. Stein, S., and Brink, L. (1981) in Methods in En-
other (S.S.) they were in the late sixties and zymology (Pestka, S., ed.), Vol. 79, pp. 20-25, Ac-
seventies. For over three decades, Stanford ademic Press, New York.
20. Kilpatrick, D. L., Gibson, K. D., and Jones, B. N.
Moore gave much of his time to encouraging (1983) Arch. Biochem. Biophys. 224, 402-404.
and advising young investigators. He was truly 2 1. Bohlen, P., and Mellet, M. ( 1979) Anal. B&hem. 94,
a statesman as well as a great scientist. 313-321.
PICOMOLE PROTEIN AND PEPTIDE CHEMISTRY 23

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