at the heart of the present invention is chemical modification of a protein so as to first introduce carboxyl moieties into the n - butylamino side groups of lysine residues within the protein . the modified protein molecules are then crosslinked using a bifunctional crosslinking agent to yield a biodegradable , superabsorbent , protein hydrogel . as noted above , the protein starting material can be selected from any source , animal , vegetable , or microbial , without limitation . for instance , while soy protein is preferred due to its low cost , the protein hydrogel described herein can be manufactured from other oilseed proteins , leaf proteins ( e . g . alfalfa ), microbial proteins , animal proteins , and proteins recovered from food processing wastes . crude protein concentrates , as well as protein isolates will function equally well in the present invention . and , since the protein hydrogel is not generally intended for consumption , the starting material need not be of food grade . the preferred protein source is defatted soy beans , which are extracted with water to yield a soy protein isolate . generally , the soy beans are crushed to meal ( defatted soy flour can be purchased commercially ), and then extracted with copious quantities of water . extraction is accomplished by conventional means using any type of suitable equipment ( mixers , agitators , separatory funnels , etc .). extraction may be carried out in a continuous process , or batchwise . the extract is then treated to isolate the protein . this can be accomplished in any suitable fashion . for instance , this can be accomplished by treatment with acid to precipitate the proteins dissolved within the extract to yield soy protein isolate . the protein will normally precipitate from solution at about ph 4 . 5 the soy protein isolate ( spi ) may be optionally dialyzed or further purified ( e . g ., by recrystallization ) if desired . other isolation methods , such as evaporation of the solvent , or chromatography , can be used with equal success . while the present invention may be practiced with any type of protein , for brevity and clarity only , the remainder of the specification shall be limited to protein hydrogels made using the above - described spi . the spi is then modified with a carboxylic group - containing acylating agent . the acylating agent reacts with n - butylamino groups of lysine residues within the spi , and functions to introduce carboxyl moieties into the spi . preferably , the acylating agent is a polycarboxylic anhydride , a mono - anhydride , a dianhydride , or a combination thereof . as used herein , the term &# 34 ; anhydride &# 34 ; shall mean any of the preceding types of anhydrides . suitable dianhydrides which can be used in the present invention include , for example , benzenetetracarboxylic dianhydride , cyclobutane tetracarboxylic dianhydride , diethylene - triamine - pentaacetic dianhydride , and ethylenediaminetetraacetic acid dianhydride ( edtad ). edtad is the preferred acylating agent . again , for brevity , the description which follows will be limited to addition of edtad to the spi . this is for brevity and clarity only , and is understood not to limit the invention claimed herein in any fashion . introduction of the edtad into the spi is performed by step - wise addition of solid edtad to an aqueous solution of spi . it must be remembered , however , that edtad is a bifunctional reagent which is capable of cross - linking polypeptides either inter - or intramolecularly . two possible reaction pathways for the reaction of edtad with a protein are shown below : ## str1 ## wherein pro is the protein being modified . in reaction i , one molecule of edtad reacts with two lysyl residues to form a linkage . when the reaction of the protein with edtad proceeds by reaction i , the result is the incorporation of only one carboxyl moiety per lysyl residue . moreover , if reaction i occurs between subunits of a protein molecule , the intramolecular crosslinking may impair swelling of the modified protein . in reaction ii , one molecule of edtad reacts with one lysyl residue and one water molecule . in this reaction , three carboxyl moieties per lysyl residue are incorporated into the protein , and no linkages are formed . this greatly increases the net anionic charge of the modified protein , which aids in unfolding the protein structure . because no linkages are formed , the swellability of the modified protein is not impaired . in light of the bifunctionality of edtad , in order to form a protein hydrogel having maximum absorbency , edtad should be added to the spi under conditions which favor reaction ii over reaction i . conditions which favor reaction ii over reaction i are those conditions wherein the protein is present in dilute solution , and the individual protein molecules are partially dissociated and / or denatured , thereby lessening the possibility of edtad reacting with two protein molecules . the reaction can be carried out at a temperature range of from about 5 ° c . to about 100 ° c . it is preferred that the reaction be conducted at mildly elevated temperatures , from ambient to about 100 ° c ., under basic conditions , about ph 8 to ph 12 . however , the reaction conditions should not be so rigorous as to cause hydrolytic degradation of the protein chains . the preferred reaction protocol to optimize reaction of the protein by the mechanism of reaction ii is to first incubate the spi in a dilute aqueous solution of about ph 8 to ph 12 , at a mildly elevated temperature of about 65 ° c . most preferably , the ph of the solution should approach 12 . however , care must be taken not to exceed about ph 12 . if the alkalinity of the solution rises above approximately ph 12 , the protein may suffer extensive alkaline hydrolysis . the concentration of spi in the solution should be on the order of about 1 to 2 %. the spi should be incubated for approximately 30 minutes at 65 ° c . longer incubations times are acceptable so long as alkaline hydrolysis does not occur . the incubation period serves to dissociate and / or denature the protein molecules of the spi . the incubation may also be performed under acidic conditions , down to about ph 2 . however , since the preferred acylation reaction takes place in alkaline solution , it is preferred that the incubation is also done under alkaline conditions so as to minimize salt formation during the acylation reaction . after incubation , the solution is allowed to return to room temperature , and solid edtad is added slowly thereto , over a period of one to three hours . the reaction should be carried within a ph range of from about 8 to 12 . it is preferred that the ph of the reaction be held constant during acylation by the addition of base , preferably naoh . this can be done automatically using a commercially - available ph - stat apparatus ( fisher scientific ). under these conditions , little or no alkaline hydrolysis of the spi occurs . of course , the reaction conditions can be easily adjusted by one of skill in the art such that reaction i prevails . for instance , a shortened incubation period , or omission of the incubation step entirely will tend to favor reaction i , as will adding the edtad to a more concentrated spi solution . performing the acylation at an alkalinity closer to ph 9 tends to favor the reaction i pathway over the reaction ii pathway . adjusting the relative rates of the two reactions will change the characteristics of the final protein hydrogel . while optimization of reaction ii yields a protein hydrogel having superior swellability and greater overall anionic charge , optimization of reaction i yields a stiffer , less absorbent hydrogel , which is desirable in some applications . knowledge of the interplay between the two reactions allows the physical characteristics of the final gel product to be tailored to fit a wide variety of final applications . the ratio of reaction by the reaction i pathway versus the reaction ii pathway can be determined by electrometric titration of various modified and unmodified spi samples . the titration curves of the modified samples are then compared to unmodified samples subjected to the same reaction conditions . the number of carboxyl groups per 10 5 gmole of protein is calculated from the number of moles of h + ion dissociated ( or , by the number of moles of naoh consumed ) by the protein during titration from ph 2 . 0 to the isoionic point of the protein . titration curves for native spi , and spi subjected to ph 12 and 65 ° c . are essentially identical ( data not shown ), illustrating that heat treatment at ph 12 does not result in deamidation of the glutamine and asparagine residues of spi . knowing this , any increase in the carboxyl group content of spi modified under these conditions must be due to incorporation of edtad at the lysyl residues of the spi . an illustrative electrometric titrimetric plot is shown in fig2 . here , ▪ is the curve for 91 % edtad - modified spi , ▴ is the curve for 49 % edtad - modified spi , and  is the curve for unmodified spi . the hydrogels used in this plot were modified according to the above protocol . the perpendicular intersecting lines indicate the isoionic point for each curve . the extent of acylation can be varied so as to modulate the physical characteristics of the final gel product . this can be done quite easily by varying the ratio of protein to added edtad ( or other acylating agent ). the greater the amount of edtad added per unit protein , the greater the extent of modification . fig1 shows a plot of the percent of lysine residues within spi which are acylated at a given ratio of edtad to spi ( g / g ). for instance , at 0 . 5 grams of edtad per gram of spi , approximately 90 % of the lysine residues of spi have been modified . these ratios will , of course , vary depending on the nature of the starting protein . factors such as whether the protein is a single chain or formed from several sub - chains will have an effect on the extent of modification . extent of modification is expressed herein as the percentage of available lysine residues which have been acylated . it must also be remembered that the crosslinking step , described in full below , also utilizes lysine residues within the protein to crosslink the protein chains . therefore , it is preferred that the extent of modification not exceed 98 % of available lysine residues . this maximum extent of modification should also be decreased if the starting protein is particularly low in lysine residues . in order to obtain the advantages of increased carboxyl moiety content , it is preferred that a minimum of 50 % of the lysine residues of the starting protein be acyl modified . fig3 shows the relationship between the number of lysine residues and the carboxyl group content of edtad - modified spi as a function of the extent of modification for spi modified according to the above - described preferred protocol . here , native spi , shown in the left - hand plot , has about 146 carboxyl groups and 45 lysine residues per 10 5 gmoles ( approximately 870 total amino acid residues ) of spi . the 49 % edtad - modified spi ( middle plot ) is found to contain 212 carboxyl groups and 23 lysine residues , while the 91 % edtad - modified spi is found to contain about 295 carboxyl groups and 4 lysine residue per 10 5 gmole spi . fig3 shows that for each lysine residue modified as described above , an average of 2 . 3 carboxyl groups are incorporated into the protein . this confirms that , under the preferred reaction protocol , reaction of spi with edtad predominantly follows the reaction ii pathway . after acylation , the protein solution is exhaustively dialyzed against deionized water to remove salts ( in this case , primarily sodium edta ) formed in the reaction . the dialyzed modified protein may be optionally lyophilized to yield an acyl - modified protein . edtad is the preferred acylating agent because , inter alia , it is essentially non - toxic . the only reactive groups introduced into the protein by the addition of edtad are the carboxyl groups . when added to the spi according to the protocol described above , any unreacted edtad will readily react with water and naoh , to be converted into sodium ethylenediaminetetraacetic acid ( edta ). since sodium edta is a &# 34 ; generally regarded as safe &# 34 ; ( gras ) food additive , there is no concern in regard to the toxicity or environmental safety of any residual amount of sodium edta ( if any ) remaining in the modified protein . unlike poly ( acrylate ) or poly ( acrylamide )- based hydrogels , which may contain residual monomers which are toxic , the present protein hydrogel , if it contains any residual reagents , would only contain residual sodium edta . while not being limited to any particular mode of operation , it is believed that the edtad acylating agent , by reaction with the lysyl residues of the protein , causes extensive unfolding of the protein molecules via intramolecular electrostatic repulsion caused by the carboxylic acid substituents on the acylating agent . this is believed to convert the rigid , globular structure of soy globulins into a random - coil - type , polyanionic polymer . the substantial polyanionic character which the carboxylic acid moieties impart to the spi are believed to provide numerous sites for water binding . evidence of the unfolding of the soy globulins is provided by fluorescent spectroscopy . fig4 is an emission spectra of native spi (( 1 ), dotted line ), unmodified spi subjected to 65 ° c . and ph 12 ( 2 ), and three edtad - modified spi &# 39 ; s of increasing percent modification ( curves ( 3 ), ( 4 ), and ( 5 ), respectively ). the emission spectrum of native spi , shown by the dotted line , has a maximum fluorescence intensity at 340 . 8 nm . the maximum fluorescence intensity ( λ max ) of unmodified spi subjected to the reaction conditions of the preferred protocol ( 65 ° c ., ph 12 , curve ( 2 )) shifted to 343 . 8 nm , and showed a markedly increased intensity relative to the native spi . this indicates that denaturation / dissociation of the protein is caused by the incubation conditions , prior to addition of edtad . upon modification with edtad , the λ max of fluorescence emission shows a shift to longer wavelengths ( a red shift ) and a decrease in fluorescence intensity . the greater the extent of modification with edtad , the greater the red shift , and the greater the decrease in fluorescence intensity ( curves ( 3 ), ( 4 ), and ( 5 )). these changes in fluorescence intensity indicate that tryptophan residues within the spi are increasingly exposed to solvent due to unfolding of the protein upon modification with edtad . after acylation , the dialyzed and optionally lyophilized modified spi is crosslinked using a bifunctional crosslinking reagent . a wide variety of suitable bifunctional crosslinking agents are known in the art . dialdehydes , for instance , like dianhydrides , will also react with lysine residues to form crosslinks between polypeptide chains . bifunctional aldehydes are excellent crosslinking reagents . in the present invention , any type of dialdehyde , without limitation , can function as a crosslinking reagent . the preferred bifunctional crosslinking reagent is a bifunctional aldehyde having the formula wherein x is an integer of from 2 to 8 . the preferred bifunctional aldehyde from within this small group of homologs is glutaraldehyde ( x equals 3 ). crosslinking is preferably carried out in aqueous solution . here , in order to maximize crosslinking ( both intra and intermolecular linkages ), a relatively concentrated protein solution is used , and the ph maintained at about ph 7 to ph 10 . for instance , to a 15 % aqueous solution of acylated spi at ph 9 . 0 is added a suitable amount of a 25 % aqueous solution of glutaraldehyde . for example , about 150 μl of the 25 % glutaraldehyde solution would be added to 10 ml of the 15 % protein solution . the mixture is then thoroughly stirred , and cured overnight at room temperature . the cured gel is then air dried in an oven at 40 ° c . the residual amount of glutaraldehyde ( if any ) contained within the product protein hydrogel can be controlled by careful analysis of the available lysine residues in the modified spi . by optimizing the ratio of glutaraldehyde to available lysine residues , any residual amount of glutaraldehyde can be minimized , or the residual amount can be eliminated entirely . also , if a residual amount of glutaraldehyde is found to be present , the effects of the glutaraldehyde can be neutralized by treating the protein hydrogel with low molecular weight amino compounds such as ethanolamine or glycine . table 1 is a tabulation of the water uptake capacities of various edtad - modified protein hydrogels based upon the extent of edtad modification . the values for the amount of water retained in the first four gels represents the amount of water retained within gels made at ph 12 and 65 ° c . after centrifugation at 214 × g . as noted in the table , at 66 % modification , 1 gram of the protein hydrogel of the present invention is capable of absorbing over 100 grams of water . in other words , at 66 % modification , the present hydrogel is capable of absorbing over 10 , 000 % of its weight in water . table i______________________________________water uptake capacities of ethylenediaminetetraacetic dianhydride - modified soy protein hydrogel . sup . b % modification water uptake ( g water / g dry gel )* ______________________________________0 . sup . 6 . 3 + 0 . 4 . sup . a32 55 . 5 + 6 . 147 76 . 5 + 4 . 766 105 . 6 + 4 . 0______________________________________ * represents water retained by the swollen gel at 214 x g . . sup . a values represent mean ± standard error ( n = 3 ). . sup . b gels were made by crosslinking a 15 % edtadmodified spi with glutaraldehyde . several factors influence the ability of the present protein hydrogels to take up water . for example , fig5 depicts water uptake capacities as a function of protein concentration during crosslinking . the water uptake capacity of a 66 % edtad - modified hydrogel , crosslinked in an 8 . 5 % ( w / v ) solution is about 300 grams of water per gram of dry gel . in contrast , water absorption decreased to about 120 grams of water per gram of dry gel when the same 66 % edtad - modified soy protein was cross linked in a 15 % solution . this knowledge can be used to create hydrogels with varying water uptake capacities simply by manipulating the protein concentration in the crosslinking step . water uptake of the present hydrogels is also ph sensitive . fig6 shows that the higher the alkalinity of the water , the greater the water uptake . for spi - derived hydrogels , this was true for 32 %, 47 %, and 66 % edtad - modified gels . fig7 shows that the protein hydrogels of the present invention are also sensitive to the ionic strength of the water . fully reversible swelling and shrinkage of the present hydrogels occurs as the gel is cycled between deionized water and a 0 . 15 nacl solution . fig8 and 9 further show that the hydrogels of the present invention are capable of chelating divalent heavy metal cations . fig8 shows that , under equilibrium conditions , an edtad - modified , glutaraldehyde crosslinked hydrogel is capable of binding approximately 150 mg of pb ++ per gram of dry gel . fig9 is an identical plot for mercury . under equilibrium conditions , the same hydrogel is capable of binding approximately 200 mg of hg ++ per gram of dry gel . fig1 depicts a process flow chart of the preferred method of making the presently claimed protein hydrogel . briefly , a 1 - 2 % soy protein isolate solution is adjusted to ph 12 , and incubated at 65 ° c . for 30 minutes . the solution is then cooled to room temperature , and edtad is slowly added to the solution . the ph of the solution is maintained constant at 12 by addition of naoh . the solution is then filtered ( for instance , by dialysis ) and concentrated . glutaraldehyde is then added to the solution to crosslink the protein chains . the hydrogel is then cured overnight , and dried at 40 ° c . the following protocols are provided for illustrative purposes only to aid in a complete understanding of the claimed invention . it is understood that the examples do not limit the invention claimed herein in any manner . defatted soy flour is commercially available from many suppliers , including central soya , fort wayne , ind ., usa . glutaraldehyde and trinitrobenzenesulfonic acid ( tnbs ) can be obtained from sigma chemical co ., st . louis , mo ., usa . ethylenediaminetetraacetic dianhydride ( edtad ) can be obtained from aldrich chemical co ., milwaukee , wis ., usa . defatted soy flour was extracted with water at ph 8 . 0 at a flour to water ratio of 1 : 10 . the solution was centrifuged and the supernatant adjusted to ph 4 . 5 with 2m hcl to precipitate the proteins . the precipitate was redissolved in water at ph 8 , dialyzed against water ( ph 8 . 0 ) overnight , and lyophilized to yield soy protein isolate ( spi ). protein concentration was determined by the dry weight method . a weighted aliquot of protein stock solution in deionized water was dried to a constant weight at 105 ° c . in a vacuum oven . the protein content is then expressed as w / w %. acylation of the spi was performed by the step - wise addition of solid edtad to a 1 % aqueous solution of the spi at 65 ° c . the ph of the protein solution is kept constant during the acylation by adding 1 n naoh with a ph - stat ( fisher scientific ). the duration of the acylation is normally on the order of two to three hours . 150 μl of a 25 % aqueous solution of glutaraldehyde was added to 10 ml of a 15 % ( w / w ) solution of the acylated protein at ph 9 . 0 the mixture was stirred thoroughly with a magnetic stirrer , and the resultant gel cured overnight at room temperature . the cured gel was then air dried in an oven at 40 ° c . the extent of acylation is expressed as the percentage of the total number of lysyl residues modified . the lysine content of the unmodified spi , and the acylated spi was determined by the tnbs method described by hall et al . ( analyst , 1973 : 98 , 673 ), which is incorporated herein by reference for its teaching of protein modification analysis : to 1 ml of 4 % nahco 3 was added 0 . 8 ml of a solution containing less than 5 mg spi , followed by the addition of 0 . 2 ml of tnbs solution ( 12 . 5 mg / ml ). the mixture was incubated at 40 ° c . for 2 hours , and then 3 . 5 ml of concentrated hcl was added to the mixture . the tube was stoppered and kept at 110 ° c . for 3 hours , and then cooled . after cooling , the volume was made up to 10 ml with deionized water . the solution was extracted twice with anhydrous diethyl ether . the tube was unstoppered and heated to 40 ° c . to allow the residual ether to escape . the absorbance of the yellow ε - tnp lysine solution was then measured at 415 nm against a blank . the amount of lysyl residues in the acylated and unacylated spi was then determined from a standard curve constructed using lysine . the swelling properties of the subject protein hydrogels was analyzed in the following fashion : a known amount of dry gel is placed in a preweighed filter pouch similar in appearance to a tea bag . the pouch is then heat sealed . a control pouch of the same weight , without any gel , is also sealed . both pouches are then immersed in deionized water at ph 6 . 7 at room temperature for 24 hours . the pouches are then centrifuged at 214 × g for 5 minutes . after centrifugation , both pouches are weighed . the wet weight of the gel is determined by subtracting the weight of the control pouch from that of the sample pouch . the two pouches are then dried at 100 ° c . to a constant weight and re - weighed to calculate the dry weight of the gel . from the wet and dry weight of the gel sample , the grams of water absorbed per gram of dry gel is determined . it is understood that the present invention is not limited to the particular embodiment , reagents , steps , or methods described herein , but embraces all such forms thereof as come within the scope of the attached claims .
the present invention is a biodegradable , reversibly - swellable , polyvalent cation - binding , protein - based hydrogel which comprises an acyl - modified protein matrix in which the acyl - modified protein matrix is crosslinked with a bifunctional crosslinking reagent , and a method of making the same .