Removal of metal ions from aqueous solution

A method of removing heavy metals from aqueous solution, a composition of matter used in effecting said removal, and apparatus used in effecting said removal. One or more of the polypeptides, poly (.gamma.-glutamylcysteinyl)glycines, is immobilized on an inert material in particulate form. Upon contact with an aqueous solution containing heavy metals, the polypeptides sequester the metals, removing them from the solution. There is selectivity of poly (.gamma.-glutamylcysteinyl)glycines having a particular number of monomer repeat units for particular metals. The polypeptides are easily regenerated by contact with a small amount of an organic acid, so that they can be used again to remove heavy metals from solution. This also results in the removal of the metals from the column in a concentrated form.

BACKGROUND OF THE INVENTION 
This invention relates to biochemistry and environmental protection. This 
invention is the result of a contract with the Department of Energy 
(Contract No. W-7405-ENG-36). 
Waste streams containing heavy metals, such as cadmium and copper, are 
generated in many industrial operations. The metals are toxic to animal 
and plant life and must be removed before the waste streams are discharged 
into the environment. Also, there are many sites where water containing 
heavy toxic metals has been dumped; these sites must be cleaned up or, at 
minimum, the sites must be stabilized to prevent the waste from migrating 
to contaminate more of the environment. 
Cadmium is an example of a toxic metal which must be excluded from the 
environment, it accumulates readily in living systems and, in humans, has 
been implicated as the cause of renal disturbances, lung insufficiency, 
bone lesions, hypertension, and cancer. The U.S. Environmental Protection 
Agency limits the amount of cadmium which may be present in drinking water 
to 10 parts per billion. Cadmium containing waste streams are generated in 
such industrial operations as zinc refining, battery manufacturing, 
electroplating, and pigment manufacturing. 
BRIEF DESCRIPTION OF THE INVENTION 
This invention is a method of removing heavy metals from aqueous solution, 
a composition of matter used in effecting said removal, and apparatus used 
in effecting said removal. One or more of the polypeptides, poly 
(.gamma.-glutamylcysteinyl)glycines, is immobilized on an inert material 
in particulate form. Upon contact with an aqueous solution containing 
heavy metals, the polypeptides sequester the metals, removing them from 
the solution. There is selectivity of poly 
(.gamma.-glutamylcysteinyl)glycines having a particular number of monomer 
repeat units for particular metals. The polypeptides are easily 
regenerated by contact with a small amount of an organic acid, so that 
they can be used again to remove heavy metals from solution. This also 
results in the removal of the metals from the column in a concentrated 
form. 
In a broad embodiment, the present invention is a method of removing heavy 
metals from an aqueous solution comprising contacting an aqueous solution 
with a solid substance comprised of water-insoluble polymeric material to 
which is attached molecules of poly (.gamma.-glutamylcysteinyl)glycines 
for a time period effective for metals to become attached to said poly 
(.gamma.-glutamylcysteinyl)glycines and separating said aqueous solutions, 
which is depleted of metals, from said solid substance.

DETAILED DESCRIPTION OF THE INVENTION 
A poly (.gamma.-glutamylcysteinyl)glycine molecule of this invention is a 
polypeptide which consists of a chain of monomer repeat units having the 
amino acid glycine attached to it. Each monomer repeat unit consists of 
two amino acids, glutamate and cysteine, joined by a peptide, or gamma, 
bond. The poly (.gamma.-glutamylcysteinyl)glycines are frequently 
represented by (Glu-Cys)nGl, where n is equal to the number of monomer 
repeat units. In experimentation associated with this invention, poly 
(.gamma.-glutamylcysteinyl)glycines having 2, 3, 4, and 5 repeat units 
have been produced and it is expected that the number of repeat units 
which may be utilized in the practice of this invention will range up to 
10 or more. 
The poly (.gamma.-glutamylcysteinyl)glycines of this invention have an 
affinity for the heavy toxic metals cadmium, copper, and zinc and will 
combine with these metals in water solution. In addition to these three 
metals, which have been the subject of experimentation, it is expected 
that poly (.gamma.-glutamylcysteinyl)glycines will also have an affinity 
for lead, mercury, and nickel. Certain poly 
(.gamma.-glutamylcysteinyl)glycines are selective for particular metals. 
It has been demonstrated by experimentation that the poly 
(.gamma.-glutamylcysteinyl)glycine having two repeat units will tend to 
sequester cadmium in preference to the other metals. Also, the compound 
having three repeat units favors copper. It is expected that additional 
selectivities exist, such that knowledge of the aqueous stream to be 
treated will allow a particular poly (.gamma.-glutamylcysteinyl)glycine to 
be selected. If two metals are to be removed, two or more poly 
(.gamma.-glutamylcysteinyl)glycines may be used. On the other hand, it may 
be desirable to remove only one metal from a stream containing two or more 
metals. In this case the poly (.gamma.-glutamylcysteinyl)glycine which is 
selective for that metal would be used. 
Since the poly (.gamma.-glutamylcysteinyl)glycines are soluble in water, 
they must be immobilized, or made insoluble. This is done by attaching the 
poly (.gamma.-glutamylcysteinyl)glycine molecules to an inert polymeric 
material, which is usually in the form of beads, which may range from 0.01 
to 20 mm or more in diameter. Any relatively inert polymer which has 
chemical sites to which will attach the free amino group of the glutamate 
group on one end of the chain may be used. A polysaccharide has been used 
in the experimentation. 
The solid substance consisting of the polymeric material with the poly 
(.gamma.-glutamylcysteinyl)glycines attached is placed in a container, 
such as an elongated vertical cylindrical vessel, and the aqueous 
metal-containing solution is allowed to flow through the solid substance 
by gravity. When the solid substance has reached its capacity to absorb 
metals, it is regenerated, that is, prepared for reuse in removing metals, 
by passing a small quantity of a high molecular weight organic acid 
through it. For example, 3 ml of oxalic acid was sufficient to regenerate 
a column which had removed metals from 1000 ml of solution. Large organic 
oxides having a low pH and weak chelating properties, such as citric acid 
and maleic acid, may be used. 
The polypeptides of this invention may, conceivably, be prepared by 
chemical means, but are most advantageously prepared by culturing certain 
substances in the presence of a heavy metal. Though a particular metal is 
used to stimulate production of poly (.gamma.-glutamylcysteinyl)glycines, 
the material produced has affinities for other metals, as described above. 
The following paragraphs describe a portion of the experimentation which 
was accomplished. 
Suspension cell cultures of Datura innoxia have been selected for 
resistance to different concentrations of CdCl.sub.2 using a stepwise 
selection protocol. Variant cell lines retain the ability to grow in 
normally toxic concentrations of Cd after growth in its absence for more 
than seven hundred generations. Resistance to Cd correlates with synthesis 
of large amounts of small cysteine-rich Cd complexes. 
Cadmium tolerant Datura innoxia cell cultures are grown in a standard plant 
cell culture media containing 250 .mu.m CdCl.sub.2 for at least 48 hours, 
with shaking at 30.degree. C. Growth under these conditions results in the 
synthesis of large amounts of poly(.gamma.-glutamylcysteinyl)glycines, 
which accumulates within the cells. 
To extract the polypeptides, the cells are washed once in an ice-cold 
buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM KCl, 1.5 mM MgCl.sub.2 and 
20 mM 2-mercaptoethanol. Cells are collected from the buffer by 
centrifugation at low speed for 10 minutes, resuspended in the same 
buffer, and broken open with a homogenizer. The resulting extract is 
centrifuged for 15 minutes at 15,000.times.g to remove insoluble material 
and the resulting supernatant is passed through a Sephadex G-50 (fine) 
column, which separates the polypeptides from the majority of the 
remaining cellular material. Fractions collected from the column which 
contain the polypeptides are identified by the presence of bound cadmium. 
These fractions are pooled and the polypeptides are washed and 
concentrated by ultrafiltration. The resulting preparation is used as a 
source of poly(.gamma.-glutamylcysteinyl)glycines to be attached to 
Sepharose beads. 
The amount of polypeptide present is determined by assaying for the amount 
of sulthydryl groups present using an Ellman's reaction. Sepharose 4B 
(Sigman Chemical Co., St. Louis, Mo.) is washed in a large excess of 
triple distilled water. The hydrated Sepharose is then suspended in an 
equal volume of 5 M potassium phosphate solution and chilled on ice. 0.4 
volumes of a solution containing 100 mg/ml CNBr (cyanogen bromide) in 
distilled water is added dropwise over a period of two minutes. The 
suspension is shaken gently during the addition of the CNBr and is allowed 
to react for eight minutes on ice. 
The CNBr-activated Sepharose is then washed with five volumes of a solution 
containing 0.25 M NaHCO.sub.3 pH 9.0. 
The CNBr-activated Sepharose is then mixed with a solution containing a 
known quantity of poly(.gamma.-glutamylcysteinyl)glycine. This suspension 
is allowed to react overnight at room temperature. 
The above suspension is then poured into a glass or plastic column 
containing a glass frit in the bottom which allows passage of liquid but 
not the metal-binding matrix. As the liquid drips from the column (it can 
be removed using gravity flow or by pumping), the matrix is washed by 
addition of at least five volumes of solution containing 0.25 M 
NaHCO.sub.3, pH 9.0. This is followed by washing the column with five 
volumes of the same solution containing 1 M NaCl and five volumes of a 
solution containing 1 M ethanolamine, pH 9.0 (pH adjusted with HCl). The 
column can be stored in this latter solution, then washed again with 0.25 
M NaHCO.sub.3, followed by distilled water. 
The column is then washed with five volumes of a solution containing 0.1 M 
oxalic acid. This removes the cadmium which was bound by the polypeptides 
while the polypeptides were still within the cell. It also frees the 
binding sites of the polypeptides for binding more metal ion. 
A stock solution containing 5 .mu.M CdCl.sub.2 containing a small amount of 
radioactive .sup.109 Cd is passed through the column and fractions are 
collected from the bottom of the column and assayed for the presence of 
the radioactive cadmium. No radioactive cadmium can be detected until all 
of the metal-binding sites on the column are saturated. Knowledge of the 
specific gravity of the cadmium solution allows determination of the 
amount of cadmium bound by a specific batch of metal-binding matrix. 
Cadmium bound to the column is removed by again washing the column with a 
small volume of a solution containing 0.1 M oxalic acid. This wash 
releases the cadmium ion from the column in a concentrated solution and 
results in the regeneration of cadmium binding sites on the column. The 
end result is a significant concentration of the cadmium from the 
beginning aqueous solution. 
The polypeptides were first identified by their ability to bind radioactive 
cadmium. Extracts from Cd-tolerant Datura innoxia cells were passed 
through a Sephadex G-50 column which separates molecules based primarily 
on size. Fractions from the columns were assayed for cadmium and it was 
found that certain fractions contained greater than 90% of the cadmium 
known to be within the cells. These fractions were collected and further 
purified by affinity chromatography on Thiopropyl Sepharose 6B, which 
binds compounds which are rich in cystein (we knew from amino acid 
labeling experiments that cysteine was one of the major components of the 
cadmium binding fractions). It was found that the components of the 
fractions contained only three different amino acids (by amino acid 
analysis of the purified material); cysteine, glutamate and glycine in a 
ratio close to 3:3:1. We attempted to sequence the polypeptides using 
Edman degradation, but this did not result in the degradation of the 
polypeptides, suggesting that either the peptide bonds were blocked or 
were not linked to one another via the .alpha.-carboxyl group. We 
sequenced the polypeptides using a combination of enzymatic degradation of 
the polypeptides, starting from the carboxyl end. The presence of 
.gamma.-carboxamide linkages was first suggested by the stability of the 
polypeptides to Edman degradation and was confirmed by .sup.13 C NMR. 
Experimentation was conducted at ambient temperatures, but the polypeptides 
are stable over a temperature range of at least 4.degree. to 85.degree. C. 
Selectivity of the polypeptide may vary with temperature; this would be 
useful in designing a commercial process. The Sepharose used in the 
experiments had a hydrated size of from 60 to 140 microns. 
TABLE 
______________________________________ 
Cd concentration, 
Bound Cd, 
micromolar moles Cd/mole polypeptide 
______________________________________ 
1 0.2 
5 1.41 
20 1.66 
47.5 1.91 
100 2.01 
______________________________________ 
The Table shows the results of a series of experiments in which aqueous 
solutions having varying Cd concentrations were passed through a column 
2.5 cm I.D..times.20 cm long containing an estimated 12.45 mg of poly 
(.gamma.-glutamylcysteinyl)glycines on Sepharose. The column was 
regenerated after each of the 5 solutions was passed through it. A small 
amount of radioactive Cd was present in each solution in order to 
determine the total amount of Cd bound by the column. It can be seen that 
the Cd capacity of the column was dependent on the concentration of Cd in 
the solution.