Note: Descriptions are shown in the official language in which they were submitted.
The present invention relates to delta-bilirubin and
compounds resembling delta-bilirubin. More particularly, the
present invention relates to processes for enriching delta-bilirubin
and the like in solution.
Bilirubins are breakdown products of heme, found
naturally in human serum. Four main types of bilirubin are
understood to exist, namely, unconjugated bilirubin (Bu), mono-
sugar-conjugated bilirubin, di-sugar-conjugated bilirubin and
delta-bilirubin.
Delta-bilirubin comprises bilirubin covalently linked
to albumin via a peptide bond between a propionic acid side chain
of the tetrapyrrole group of bilirubin, and the epsilon amino
group of a lysine residue in albumin. This lysine residue is
located between amino acid residues 97 and 224 in the N-terminal
half of the albumin protein. Delta-bilirubin is thus often referred
to as biliproteinl BP.
Delta-bilirubin has been tested for its ability to
protect various mammalian cell types against oxy-radical damage.
The results indicate a biomedical utility for delta-bilirubin.
Further, research indicates that the compound has utility in
retarding oxidative degradation of food products, drug
formulations, cosmetics and perfumes.
The isolation of delta-bilirubin from sera is
described by Lauff et al., in "Clinical Chemistry", 28: 629-637,
1982. This method involves a high pressure liquid
S chromatographic separation of delta-bilirubin from the other
bilirubin fractions existing in sera, specifically, unconjugated
bilirubin, mono-conjugated bilirubin and di-conjugated bilirubin.
K~saka et al., "Japanese Journal of Clin. Lab.
Automation", 11:88 92, 1986, describe a modified method of
extracting delta-bilirubin from icteric (i.e. jaundiced) sera.
These extraction methods provide a means for
isolating delta-bilirubin from all other types of bilirubin; however,
lS they do not provide a means for obtaining a relatively purified
solution of delta-bilirubin. The isolated delta-bilirubin fraction is
substantially free from other bilirubin compounds; however,
unbound albumin protein from the serum sample remains present
as a contaminant.
McDona~h et al. (J. Clin. Invest. 74:L763-770,
1984) describe the biosynthetic formation of a compound similar
to delta-bilirubin by incubating synthesized conjugated bilirubin
with albumin. Again, the product is obtained as a mixture with
albumin.
It is desirable to reduce the amount of peptide or
protein contamination of delta-bilirubin and similar compounds as
much as possible, in order to provide a biochemical material of
predictable, controlled constitution and properties and to minimize
the risk of undesirable biochemical side effects on use thereof.
However, many different methods for separation of delta-bilirubin
from albumin and other peptide compounds have been attempted,
but without great success.
Accordingly, it is an object of the present invention
to provide a novel and more efficient method for separating delta-
bilirubin and similar compounds from free albumin and peptides
present in the reaction solution in which the delta-bilirubin or
similar compound has been produced, thus providing an enriched
solution thereof.
The present invention provides a process whereby
very substantial quantities of albumin and other peptide
compounds can be removed from liquid mixtures thereof
containing delta-bilirubin or similar compounds (collectively
referred to herein as delta-bilipeptides as discussed below), to
provide a solution of delta-bilipeptide sufficiently enriched in
delta-bilipeptide so as to be biochemically useful and of
substantially constant and predictable composition.
The method is based upon the discovery that
albumin and similar proteins have chemical affinity for the
bilirubin nucleus to a sufficient extent that they will selectively
covalently bind delta-bilipeptide so as to extract it preferen~ially
from a mixture of delta-bilipeptide with albumin and albumin-like
peptides. Thus, the delta-bilipeptide can be attached to a
chromatographic column gel to extract covalently the albumin
peptide from the mixture whilst the delta-bilipeptide is merely
held to the column material more weakly, perhaps on an ion
S exchange, electrostatic attraction basis, or through other
conceivable forces such as weak affinity based interactions.
Thus, the delta-bilipeptide can be preferentially eluted from the
column to obtain an aqueous solution substantiall~ enriched in
delta-bilipeptide and substantially free from albumin peptides.
Conversely, an albumin peptide can be attached to the column
material to bond the delta-bilipeptide covalently whilst the
albumin peptide is held only by electrostatic attraction, so that the
albumin peptide can be preferentially eluted, and a solution
enriched in delta-bilipeptide can be obtained by subsequent
elution.
The discovery that albumin peptides and delta-
bilipeptides can be selectively separated in this way is surprising,
and not to be predicted from a consideration of the prior art or
the chemical nature of the substances involved. The molecular
size of the bilirubin nucleus is so small compared with that of
human serum albumin (molecular weight 546 v 60,000) that one
would not expect it to be sufficiently clearly located and identified
within a mixture of the two for affinity chromatographic
separation. This is especially so when, as in delta-bilirubin, the
bilirubin nucleus is already bound to a molecule of albumin. One
would expect it to be effectively hidden in the enfolded protein
chains, and effectively protected from chemical bonding thereby.
Surprisingly, this is not the case. There appears to be some
?
unusual feature in the stereochemistry of delta-bilirubin and other
delta-peptides which leaves the bilirubin nucleus available for
covalent a~finity binding to another albumin molecule or albumin
peptide.
The process of the present invention is thus useful
in separating natural delta-bilirubin from serum, to remove
therefrom substantially all of the serum proteins such as HSA
which would normally contaminate it. In addition, however, it
is useful in separating synthetically produced delta-bilirubin and
other delta-bilipeptides from albumin and other peptides which
may be used as reagents in synthetic preparation of delta-
bilipeptides and which may be present in delta-bilipeptide
solutions as unreacted reagents or reaction by-products.
The term "delta-bilipeptide" as used herein means
a compound which is either delta-bilirubin itself or which
resembles delta-bilirubin and has the general conformational
structure of delta-bilirubin, but in which the albumin portion is
either truncated or is replaced with a shorter amino acid sequence
of at least 6 amino acids but including the sequence -Lys-Glx-
Arg- at the point of covalent attachment to a propionic acid group
of the bilirubin nucleus, via the aforesaid -Lys- residue. In the
above sequence, Lys represents lysine, Glx represents glutamine
or glutamic acid and Arg represents arginine.
The term "albumin-like peptide" as used herein
means a peptide having an amino acid sequence corresponding to
that of human serum albumin, comprising at least about ~0 amino
acid units.
Synthetic methods for preparing such bilipeptides
are described and claimed in my co-pending U.S. patent
application serial number , filed on even date herewith,
and the disclosure of which is incorporated herein by reference.
The methods involve the use or production of peptides as reagents
or reaction products. Accordingly, the process of the present
invention is useful in connection therewith.
Moreover, a synthetic method for preparing delta-
bilirubin itself is based on site-specific reaction of unconjugated
bilirubin, pre-activated by reaction with Woodward's reagent K,
with human serum albumin. The method of the present invention
is useful in connection with extraction of the product from that
process also.
A preferred embodiment of the invention is
exemplified in the following drawing in which;
Figure 1 illustrates the chromatographic elution
pattern resulting from the enrichment of a delta-bilirubin sample
as descnbed in Example 3.
When albumin or a peptide having an albumin-like
amino acid sequence is covalently linked to the column material,
there is a marked preference for the binding of the delta-
bilipeptides in the li~uid mixture applied to the column, by
- 6 -
chemical affinity forming covalent bonds. The free propionic
acid group on the bilirubin nucleus is believed to react with an
amine group on the albumin or similar peptide linked to the
column. The other proteinaceous materials in the solution are
held to the column by weaker forces including but not limited to
electrostatic attraction. Consequently, initial elution of the
column, e.g. with water, produces a solution rich in albumin and
peptide material and substantially free from any delta-bilipeptides.
This elution is continued until the eluent water contains no
proteinaceous material, then the elution medium can be changed
to an ionic hydrolysis medium, whereupon the delta-bilipeptide is
eluted from the column. Suitable such ionic elutants include
aqueous sodium phosphate or potassium phosphate buffered to a
basic pH. Other suitable such buffers are described by Good,
"Biochemistry", vol. 5, page 467, 1966. Very weak, basic
buffered ionic eluents are preferred, e.g. 0.05 molar or less, in
dissolved salt, to avoid complications of subsequent salt emoval
from the product, but stronger buffers are also effective. The
solut;on so obtained is substantially enriched in delta-bilipeptide
and almost but not completely free from residual albumin or other
contaminating proteins or peptides.
In the preferred method of operating according to
the invention, an intermediate elution step is used, after the water
elution to remove the albumin and albumin residues. This
intermediate elution suitably uses methanol, ethanol proponal or
mixtures therof as eluent, and serves to extract further
proteinaceous material, especially denatured proteins, from the
column, without disturbing the covalently bonded delta-
bilipeptide. In this way, delta-bilipeptide contaminated with even
smaller quantities of residual proteins is finally obtained.
When delta-bilipeptide e.g. delta-bilirubin is
S covalently linked to the column material, the reverse situation
occurs, namely, there is afflnity covalent binding of the albumin
and/or other peptide constituents of the mixture to the column,
utilizing the free acid groups of the bilirubin attached to the
column. The delta-bilipeptide constituent of the solution is held
merely by forces. Then, initial elution with water yields an
enriched delta-bilirubin solution, and subsequent elution with
alkaline buffered sodium phosphate solution will remove the
proteinaceous material from the column subsequently. This
however, is a less preferred procedure, since it is likely to lead
to a solution containing larger quantities of contaminating albumin
and peptide residues than the former situation, where substantially
all such residues are removed prior to elution of the delta-
bilipeptide from the column.
The process of the present invention provides delta-
bilipeptides which are sufficiently free from albumin and peptide
residues, no matter how the delta-bilipeptide has been previously
obtained or synthesiæd, that it can be put to beneficial
biochemical use. The residual albumin and peptides are present
in such very small amounts that the delta-bilirubin is for practical
purposes biochemically useful, for administration to patients as a
cytoprotective agent, for use as a food or cosmetic antioxidant,
etc. It is also sufficiently pure that the results of so using it are
consistent, predictable and reproducible. Moreover, it is
sut`ficiently pure to permit its ùse as a starting matenal for further
chemical modification, e.g. enzymatic cleavage of delta-bilirubin
to form a delta-bilipeptide.
The nature of the chromatographic gel material is
not critical, provided that it is solid, inert, biochemically
acceptable and capable of ready derivatization to bond the
albumin peptide or delta-bilipeptide thereto. Any of the
commercially available chromatographic gels commonly used in
protein and peptide separations can be used. Commonly they are
crosslinked polysaccharides. Methods of attaching peptides to
them covalently are well known to those skilled in the art. Use
of carbodiimide as a coupling agent is one suitable and preferred
such method.
The present invention will now be described with
reference to the following specific, non-limiting examples.
EXAMPLE 1
Synthesis of Delta-Bilirubin usin~ Woodward's Rea~ent
The first step in synthesizing delta-bilirubin using
Woodward's Reagent is the formation of the Bilirubin-
Woodward's Reagent K Complex (BW). Based on the method of
Kuenzle et al. (J. Biol. Chem. 251:801-807, 1976), 116.8 mg or
0.2 mmol of unconjugated bilirubin (Sigma) were combined with
151.8 mg (0.6 mmol) of Woodward's Reagent K (Sigma), 20 ml
of acetonitrile (Aldrich) and 0 3 ml of tIiethylamine (Aldrich).
This mixture was stilTed fo- 45 minutes at 20C followed by an
,S 1l~
evaporation step conducted at 30C. To the evaporated mixture
was added 5 ml of distilled water.
Bilirubin-reagent conjugates were formed which
were isolated using a Sephadex G-25 column (2.5 X 7.5 cm).
The fractions were eluted with water at a flow rate of 50 ml/hr.
Human serum albumin (HSA) was reacted with the
bilirubin-reagent conjugates (BW). Conjugate 2.8~mol was
combined with 3.0~mol of HSA in 10 ml of degassed phosphate
bu~fer solution. EDTA was added to a final concentration of 1
mM and the pH was subsequently adjusted to 8.5 with imidazole
(11.0 mmol). The solution was stirred under nitrogen at room
temperature in the dark for 8 hours.
The obtained BW-delta-bilirubin monomer product
was purified by adding the reaction mixture to a Sephacryl S 100
column, followed by elution with phosphate buffer solution at a
flow rate of 50 ml/hr. The delta-bilirubin product was dialysed
exhaustively against water, washed and condensed with ultra-
filtration.
Finally, Woodward's Reagent was removed from
the BW-delta-bilirubin product. The product was subjected to a
phenyl Sepharose CL~B column for the purpose of removing
Woodward's Reagent K to form isolated delh-bilirubin. This was
accomplished by dissolving the lyophilized BW-delh-bilirubin
monomer in 2M ammonium sul~ate (40 mg/ml) and then adding
it to the phenyl Sepharose CL-4B column saturated in 2M
- 10-
ammonium sulfate. Distilled water was used to elute delta-
bilirubin which was then dialysed and lyophilized.
EXAMPLE 2
Enrichment of Synthesized Delta-bilirubin
Chemically synthesized delta-bilirubin produced as
described in Example 1 may be further purified following
synthesis by application to a column of CH-Sepharose
chromatographic gel to which human serum albumin (fatty acid
free) is covalently linked with carbodiimide. The CH-Sepharose
gel is prepared as follows:
(i) 10-15 g of dry CH-Sepharose (Pharmacia) is swelled in 1
liter of 0.5 M NaCl overnight.
(ii) The swollen gel is filtered and washed in 3-4 litres of 0.3
M NaCl.0
(iii) The pH of a solution containing 3 g of 3X
crystallized human serum albumin in 60 mL of
double-distilled water is adjusted to 4.5. This
solution is added to the gel.
(iv) A solution of ethyl-3, 3-dimethyl aminopropyl
carbodiimide HCl is prepared by adding 23 g of the solid
to 60 mL doubled distilled water. The pH is adjusted to
4.5 with 0.5 M NaOH.
;` t ~!
(v) The carbodiimide solution is added dropwise to the
gel/HSA solution over 20 minutes maintaining a pH of 4.5
for the 20 minute addition period as well as the subsequent
hour.
(vi) The mixture is stirred for 24 hours at room temperature
and then the gel is collected on a sintered glass funnel.
(vii) The gel is washed through 3 cycles, each cycle comprising
102 washes. The first wash is with 0.5 Iitres of 0.1 M
sodium acetate buffer, pH 4, plus 1 M NaC 1. The second
wash is with 0.5 liter of 0.1 M sodium borate buffer, pH
8.0, plus 1 M NaCl.
15(viii) The gel is further washed with 4 litres of double-distilled
water to remove any buffer salts and then the gel is
suspended in 120mL of double-distilled water and stored
at 04C until use.
Free, contaminating albumin was eluted with
double-distilled water. Denatured serum proteins were eluted
with 95% ethanol. Delta-bilirubin was eluted using 0.05 M to
0.1 M sodium phosphate buffer, pH 7.5 ~/-0.05.
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~
EXAMPLE 3
Enrichment of Extracted Naturally Occurrin~ Delta-Bilirubin
Delta-bilirubin isolated from pooled icteric sera
according to Lauff et al. (Clin. Chem. 28, 1982, p. 636) was
enriched using the column described in Example 2.
The delta-bilirubin samaple was added to the column
and eluted as shown in Figure 1. Initially, material was eluted
with double-distilled water and it was found to contain albumin
for the most part. This was confirrned by immuno-
electrophoresis and by the use of anti-HSA antibody. Once the
absorbance of the eluent at 430 nm decreased to zero, the column
was washed with 2-3 column volumes of 95% ethanol (B).
Turbid material containing some denatured serum proteins was
washed off the column with these ethanol elutions. When the
absorbance dwindled to zero, the column was eluted with a 0.05
M (0.1 mM) sodium phosphate buffer, pH 7.0-8.0 (C). The ratio
of absorbance at 280 nm to 430 nm over the elution of this peak
was 0.8: 0.9, and characterized the molar ratio of bilirubin:
albumin. The ratio of 0.8: 0.9 is substantially close to a ratio of
1:1. This is in contrast to the 0.05: 0.2 ratio obtained for
serum-isolated delta-bilirubin without enrichment.
EXAMPLE 4
Enzymatic Clea~a~e to Produce Albumin Peptides
Peptide fragments of albumin containing the
t~ipeptide sequence Lys-Glx-Arg (where Glx is either glutamine
- 13 -
or glutamic acid) are obtained by the cyanogen bromide cleavage
of human serum albumin in 70% formic acid under nitrogen.
Fragments containing residues 124-297 (from the N-terminus of
albumin) are isolated by chromatographic methods.
s
The isolated fragment is subjected to limited
proteolysis. Such limited proteolysis comprises incubation with
pepsin at a pH of between 2.0 and 3.0 for 1 hour at 37C. The
peptides are resolved on a Waters CN-propyl column. Further
proteolysis is effected with trypsin at pH 8 for 30 minutes. The
fragments are re-chromatographed on the Waters CN-propyl
column.
EX~MPLE S
Synthesis of Bilipeptides usin~ Woodward's Rea~ent
The suitable peptide fragments obtained according
to Example 4, which include the tripeptide sequence Lys-Glx-
Arg, are reacted with the bilirubin-reagent conjugates prepared as
described in Example 1. Thus 2.8~mol of conjugate is combined
with 3.0~mol of peptide in 10 ml of degassed phosphate buffer
solution. EDTA is added to a final concentration of 1 mM and
the pH was subsequently adjusted to 8.5 with imidazole (11.0
mmol). The solution is stirred under nitrogen at room
temperature in the dark for 8 hours.
The bilipeptide is purified by adding the reaction
mixture to a Sephacryl S 100 column, followed by elution with
phosphate buffer solution at a flow rate of 50 ml/hr. The
- 14-
? ~
bilipeptide is dialysed exhaustively against water, washed and
condensed with ultra-filtration.
EXAMPLE 6
Enrichment of Bilipeptides
The bilipeptides synthesized as described inExample
5 above may also be enriched using the column described in
Example 2.
Free, contaminating albumin is eluted from the
column with double-distilled water. Denatured serum proteins
are eluted with 95% ethanol. Bilipeptides are eluted using 0.05
M to 0.1 M sodium phosphate buffer, pH 7.5 +/-0.05.
