Process for purifying and stabilizing catechol-containing proteins and materials obtained thereby

A process is provided for purifying and stabilizing marine mussel polyphenolic proteins rich in 3,4-dihydroxyphenylalanine (dopa) and hydroxyproline (hyp) while obtaining high yields thereof. The process includes the steps of providing an acid soluble extract of the dopa-containing proteins, removing the low molecular weight material from the extract and reacting the remaining proteinaceous material with a water soluble borate at a pH of 7.0-9.0 to provide a soluble borate complex of the dopa-containing protein while precipitating impurities. The complex is separated from the precipitate and may be concentrated for storage or treated with an acetic acid solution and either concentrated or lyophilized and stored under an inert atmosphere. The purified proteins exhibit a dopa:protein index of purity ratio of at least about 0.10.

BACKGROUND OF THE INVENTION 
The present invention relates generally to polyphenolic proteins. More 
specifically it is concerned with a new and improved process for purifying 
and stabilizing catechol-containing proteins and with the stabilized 
proteinaceous materials obtained thereby. 
As mentioned in my publication, The Journal of Biological Chemistry, Vol. 
258, No. 5, pp. 2911-2915 (Mar. 10, 1983), several species of common 
marine mussels of the genus Mytilus secure themselves to solid substrates 
through a complex array of plaque-tipped collagenous byssal threads. The 
ends of these threads are rich in a polyphenolic adhesive substance that 
is mixed by the animal's foot with a curing enzyme (phenoloxidase) and a 
mucosubstance to provide a complex three-component natural adhesive 
system. The polyphenolic protein component of that system has been 
identified as a polymeric protein rich in 3,4-dihydroxyphenylalanine 
(dopa) (11 percent) and hydroxyproline (hyp) (13 percent). The amino acid 
composition of the polyphenolic proteins is reported by Waite and Tanzer 
in "Polyphenolic Substance of Mytilus edulis: Novel Adhesive Containing 
L-DOPA and Hydroxyproline" Science, Vol. 212, pp. 1038-1040 (1981). The 
disclosures within these publications are incorporated herein by 
reference. 
These polyphenolic proteins are unique in their ability to adhere to 
substrates under the environmentally adverse and turbulent conditions in 
which the mussels exist. This is significant since, typically, adhesives 
are adversely affected by the presence of water on the substrates being 
adhered. Water competes with the adhesive for the surface, tends to 
hydrolyze the adhesive, and frequently plasticizes the adhesive. 
Accordingly, it is usually required that the substrate surfaces being 
adhered to be substantially free from water or other aqueous impurities. 
As can be appreciated, such conditions are not always possible, 
particularly for bioadhesives used in medical and dental applications and 
employing a wide variety of substrates such as those encountered when 
gluing or restoring fractured hard tissue in the body such as bone, 
cartilage, teeth, ligaments, blood vessels and the like. 
The polyphenolic proteinaceous bioadhesive is also unusual in its superior 
strength characteristics which appear to be comparable to those achieved 
by synthetic cyanoacrylates. Since it can be applied to wet surfaces 
without prior drying, it may be considered to be superior to such 
adhesives. Further, the polyphenolic protein cures extremely rapidly, is 
nontoxic, and can be used in very fine or thin films exhibiting a 
coefficient of expansion similar to biological tissue. 
As reported in the publications mentioned hereinbefore, the polyphenolic 
protein consists of a rather large polypeptide chain having a molecular 
weight of about 110,000 to 140,000 in which seven amino acids account for 
about 80 percent of all the amino acid residues within the peptide. A 
particular decapeptide sequence is given and it is stated that the 
reported sequence and related sequences may be repeated as often as 75 
times in the polypeptide proteins. The reported presence of dopa and 
hydroxyprolines is unusual since dopa is only rarely encountered as a 
component of naturally occurring proteins and the hyroxyprolines are 
primarily associated with collagens having a high glycine content. 
The isolation of the polyphenolic proteins reported hereinbefore involves 
treatment of dissected phenol glands of numerous mussels with a neutral 
salt buffer followed by extraction of the protein with acetic acid. As 
reported, the extraction is effective in providing a reasonable amount of 
the polyphenolic proteins. However, the acid soluble material at this 
stage of isolation and purification has a limited shelf life. This is 
believed to be due to many factors including the presence of collagen and 
dopa's susceptability to facile oxidation to its quinone moiety. Dopa is 
an o-diphenol and readily forms quinones and semiquinones by photolysis, 
autoxidation and enzyme catalysis. Additionally, the protein is very 
sensitive to the presence of transition metal elements and tends to 
irreversibly coalesce with other proteinaceous materials still present 
within the acetic acid extract. It is known that other o-diphenols can 
chelate various metals, such as copper, iron, manganese, zinc, and nickel 
with high affinity. This characteristic is believed to contribute to 
sclerotization of the o-diphenol proteins. 
While ion exchange techniques have been attempted as a means of achieving 
greater purification, it is recognized that yields of the proteins 
deteriorate drastically as a result of the extensive adsorption of the 
proteins by the ion exchange medium. In fact, up to 70 percent of the 
applied polyphenolic proteins are not recovered when using this technique. 
Gel filtration of the proteins using a variety of chromatographic 
materials and buffers generally results in very low or negligible yields. 
Although some materials permit recovery of the protein, they typically 
provide a limited fractionation range and generally are not preferred for 
purifying the bioadhesive proteins. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, it has been found that the 
polyphenolic proteins can be both further purified and even stabilized 
under controlled pH conditions without the drastic yield reductions 
experienced heretofore. Due to the high conversion rate of the proteins to 
their stabilized form, it is now possible to retain significant quantities 
of the proteins for prolonged periods of time for subsequent utilization. 
The purified and stabilized material also facilitates isolation and 
storage of the protein in concentrated or dry form. 
These and other advantages are achieved in accordance with the present 
invention by providing a new and improved process for purifying and 
stabilizing polyphenolic proteins rich in catechol that includes the steps 
of providing an acid soluble extract of the catechol-containing protein, 
removing from the extract those acid soluble materials having a low 
molecular weight to provide an extract fraction of relatively higher 
molecular weight catechol-containing proteins, reacting the extract 
fraction with a water soluble borate at a pH in the range of 7.0 to 9.0 to 
form selective soluble borate complexes of the proteins, and separating 
the borate complexes while maintaining the pH of the solution within the 
range of 7.0 to 9.0 to permit stabilized storage of the protein and 
subsequent use thereof. The borate complexes also may be treated with 
acetic acid to reduce the pH thereof into the range followed by 
concentrating or lyophilizing the acid solution under an inert atmosphere 
to obtain the purified proteins in solid form. 
A better understanding of this invention will be obtained from the 
following description of the process including the several steps and the 
relationships of one or more of such steps with respect to each of the 
others, and the product resultant thereof which possesses the features, 
characteristic, compositions, properties, and relation of elements 
described and exemplified herein. 
DESCRIPTION OF A PREFERRED EMBODIMENT 
As mentioned hereinbefore, an acetic acid extract of the desired 
polyphenolic proteins can be obtained from various species of marine 
mussels of the genus Mytilus. The dissected phenol glands of the mussels 
are initially subjected to an extraction operation to remove the 
impurities and extraneous proteins therefrom. This takes place at a 
neutral pH in the presence of large amounts of a neutral or slightly basic 
buffer salt followed by gentle centrifugation. The buffer salt solution 
contains various protease inhibitors to prevent premature degradation of 
the proteins as well as materials to prevent enzymatic oxidation of the 
dopa residues prevalent in the proteins. The dissected and homogenized 
glands in effect simply undergo a washing procedure so as to remove the 
undesirable soluble impurities. After gentle centrifugation, the insoluble 
component is extracted with an acidic solution, such as a dilute acetic 
acid solution, in which the polyphenolic proteins are extremely soluble. 
The acid soluble proteins usually contain 20-40 micrograms of dopa per 
milligram of protein, the ratio of dopa to protein serving as an index of 
purification. This extraction procedure is fully described in the 
aforementioned publications and is preferably carried out at a cold 
temperature of less than about 10.degree. C.; that is at a temperature 
range of about 1.degree.-8.degree. C. and preferably at about 4.degree. C. 
In fact, all of the purifying techniques described herein are carried out 
at approximately the same low temperature unless otherwise stated. 
The acid soluble polyphenolic proteins extracted in this manner have been 
found to have an apparent molecular weight, as determined by experimental 
techniques, falling within the range of about 110,000 to about 140,000. 
However, collagenase digestion of the proteins has indicated that the 
molecular weight of the collagenase-resistant fragments falls within the 
lower end of that range. The proteins are believed to be made up of from 
60 to about 100 repeating units of a decapeptide formed from seven 
different amino acid residues. The sequencing of these residues is set 
forth in the first above-mentioned publication. Large amounts of dopa and 
hyp as well as the numerous hydroxyl groups in the polyphenolic proteins 
are believed to be largely responsible for the desired bioadhesive 
properties of the material, and it is believed that synthetic decapeptides 
of the same type or slight modifications thereof may be used to synthesize 
larger proteins possessing the desired adhesive capabilities of the 
naturally occurring materials. 
In accordance with the present invention, the acid soluble proteins in the 
acetic acid extract are treated to separate and remove the lower molecular 
weight materials within the acid medium. Preferably, this is carried out 
by dialyzing the supernatant acetic acid extract against large volumes of 
dilute acetic acid using dialysis tubing having a molecular weight cut off 
of about 50,000 or less. In this connection, the material sold by Spectrum 
Medical Industries under the name "Spectra Pore" having an exclusion limit 
of 50,000 may be used with good success. The nondialyzable or high 
molecular weight fraction is then separated for the subsequent complexing 
operation. This initial dialysis of the acid soluble proteins is conducted 
against at least 20 volumes of acetic acid and frequently against as much 
as 500 volumes or more. The acetic acid concentration may vary from as 
little as 0.5 percent up to 5.0 percent but preferably is within the range 
of about 1-2 percent. The dialysis progresses for a significant period of 
time; that is, sufficient time to assure substantial if not complete 
removal of the lower molecular weight materials; e.g., for a period of 
about 5 or 6 hours. 
As a result of this dialysis purification operation, it has been found that 
the total quantity of protein is reduced by at least 50 percent or more 
while the reduction in the amount of dopa present within the nondialyzable 
fraction is reduced by no more than about 10-15 percent. Thus, the 
remaining extract fraction clearly contains a significantly high amount if 
not most of the higher molecular weight dopa-containing proteins within 
the original proteinaceous material. 
As mentioned, the extract fraction is further purified through the 
formation of a stable borate complex at slightly alkaline pH conditions. 
The yield of the complex is about 90 percent and more within the narrow pH 
range of about 7.0 to 9.0 but falls off sharply outside this range. Yields 
of 95-98 percent can be obtained within the preferred pH range of between 
7.5 and 9.0. Accordingly, the treatment with the borate solution 
preferably takes place under these pH conditions in order to convert the 
highest possible amount of the polyphenol proteins to the borate complex 
moiety. This is significant for a number of reasons. As mentioned, the 
proteins are soluble in an acid environment but are normally insoluble 
under neutral and alkaline pH conditions. The borate complex of the 
dopa-containing proteins, however, stabilizes the proteins under the 
latter conditions. The o-diphenol materials exhibit intermolecular 
condensation and cross-linking reaction sites and are known to form 
covalent adducts and chelates of various metal ions. The borate complexes 
tend to stabilize these proteinaceous materials when most of the proteins' 
reactive sites are tied up by the borate. As mentioned, the fall-off in 
borate complex concentration is sharp on both sides of the pH range and 
therefore it is important to maintain the pH at or about 8.0-8.5. 
In accordance with the preferred technique, the complexing is achieved by 
transferring the nondialyzable acid soluble extract of high molecular 
weight to a solution containing a soluble borate salt such as sodium 
borate at a pH within the selected range of 7.5 to 9.0 and extensively 
dialyzing the extract. A large relative volume of borate solution is 
employed with the solution having a borate concentration of 0.05 mole to 
0.5 mole and preferably about 0.1 to 0.2 mole. The proteins are dialyzed 
for an extended period of time at the desired low temperature of less than 
10.degree. C. and preferably about 4.degree. C. As a result of the 
prolonged dialysis, a white precipitate is formed in the nondialyzable 
fraction. This material is believed to be composed of uncomplexed proteins 
that are alkaline-insoluble; that is, insoluble under the dialysis 
conditions employed. The precipitate is believed to be predominantly 
collagen. 
The nondialyzable complex fraction and the precipitate are subjected to 
centrifugation at about 15,000 to 50,000 x g for a brief period of time, 
up to about one hour, and the supernatant liquid is carefully separated 
from the white precipitate and collected. It has been found that while the 
total protein within the borate solution has been reduced to less than 10 
percent of the initial quantity of proteinaceous material, the yield of 
dopa is within the range of about 70-80 percent of its initial quantity 
and the index of purification as measured by the dopa/protein ratio of at 
least about 0.10, thus indicating a highly purified state when considering 
the decapeptide structure believed to be the repeating unit of the 
bioadhesive proteinaceous material. 
As can be appreciated, slight improvements in the purity of the desired 
component from each purification step can be achieved by repeating the 
various purification operations and pooling the desired components. 
At this stage, the purified borate complex may be concentrated for storage 
and subsequent use. However, it is absolutely necessary that the required 
pH conditions be maintained and also that the borate molarity be 
maintained at a level greater than the molarity of the dopa by at least 
about 5-10 percent. 
Alternatively, the purified borate complex may be reacidified and either 
concentrated or lyophilized in an inert atmosphere. The reacidification 
can readily be accomplished by redialyzing the borate complex against one 
or more changes of a 1 percent acetic acid solution. The material 
collected after a prolonged period of time such as six hours can be either 
stored cold in the 1 percent acetic acid solution, concentrated or 
lyophilized to obtain the purified proteins in their solid form. This can 
be achieved in accordance with known techniques under an inert atmosphere 
such as nitrogen. 
The purified polyphenolic proteins obtained by this process can be analyzed 
for their amino acid content and in accordance with known techniques will 
produce a composition after acid hydrolysis containing 75-95 residues of 
dopa per 1,000 residues detected. Additionally, gel electrophoresis 
reveals that the impurities in the material are only visible upon massive 
overloading, suggesting that the impurity level within this gel is less 
than 10 percent. Further, as mentioned, reduction in the impurity level 
can be achieved by recycling the material through repetitive purification 
steps. However, some reduction in the yields of the materials are 
inevitable by recycling and are not preferred since the low impurity 
content of the material resulting from the process is usually of a 
satisfactory character. 
As mentioned, the purified dopa-containing polypeptide contains repeated 
amino acid decapeptide sequences. Sequenator analysis reveals a sequence 
of NH.sub.2 
[alanine-lysine-proline-serine-tyrosine-hyp-hyp-threonine-dopa-lysine] 
COOH. Dopa is detected mostly at position nine penultimate to the carboxyl 
terminus, although significant amounts are also present with tyrosine at 
position five. The two hyps are located next to one another at positions 
six and seven, although additional hyp is present with proline at position 
three. The hyp is mostly 4-hydroxyproline although significant 
3-hydroxyproline also occurs at position seven. 
In order that the present invention may be more readily understood, it will 
be further described with reference to the following specific example 
which was given by way of illustration only and is not intended to be a 
limit on the practice of the invention.

EXAMPLE 
About 300-400 specimens of the fresh mussel of the species Mytilus edulis 
were obtained and the phenol glands therefrom were removed by dissection 
at -20.degree. C. The total weight of the glands thus obtained was about 
40 grams. Of this amount, 8 grams were measured as protein and 0.08 gram 
was measured as dopa thus providing an index of purification of 0.01. The 
dissected glands were homogenized to a puree in 10 volumes of 1.0 M sodium 
chloride, 0.05 M Tris (pH 7.5) with 0.025 M EDTA, 10 mM N-ethylmaleimide, 
1 mM phenylmethylsulfonyl fluoride, and 1 mM potassium cyanide at 
4.degree. C. The puree was centrifuged at 1,000 x g for five minutes and 
the supernatant liquid was disgarded. The solid pellet was resuspended in 
5 volumes of 5.0 percent acetic acid and rehomogenized for two minutes. 
The resultant puree then was centrifuged for one hour at 40,000 x g. The 
supernatant liquid was collected and found to contain 3.4 grams of protein 
and 0.07 grams of dopa thereby indicating an index of purification of 
0.021. 
The supernatant liquid containing the acid soluble proteins was dialyzed 
against 100 volumes of 1 percent acetic acid for five hours and then 
transferred to 100 volumes of a 0.2 M sodium borate solution at a pH of 
8.5 for another six hours. After this period, a white precipitate was 
observed in the nondialyzable fraction which was then centrifuged at 
40,000 x g for 30 minutes, and the supernatant liquid was carefully 
collected. The collected material was analyzed and found to have a 
dopa-protein ratio of 0.10 with a recovery of 0.60 grams of dopa and 0.6 
grams of protein. Based on these results and the lack of measurable 
impurities, the impurity level is believed to be less than 10 percent. The 
amino acid analysis produced a composition after acid hydrolysis of 75-95 
residues of dopa per 1,000 residues detected. The yield of the purified 
product was 75 percent based on the initial and final dopa determinations 
as compared with less than 30 percent yield based on earlier methods. 
A portion of the supernatant liquid was redialyzed against two changes of 
400 volumes of 1 percent acetic acid for six hours. The nondialyzable 
material was collected and subjected to gel electrophoresis in an 
acid-urea buffer to identify the polyphenolic protein. A portion of this 
material was then stored cold while a second portion thereof was 
lyophilized to a dry powder under nitrogen. 
As will be apparent to persons skilled in the art, various modifications, 
adaptations, and variations of the foregoing specific disclosure can be 
made without departing from the teachings of the present invention.