(49) Vol. 34, No. 9 (1978) T-405

(Received August 22, 1977)

SORPTION MECHANISM OF MERCURIC IONS BY KERATEINE OF THIOL-TYPE*

By Takeaki Miyamoto, Hiraku Ito, Masao Sugitani, and Hiroshi Inagaki

(Institute for Chemical Research, Kyoto University, Uji/Kyoto 611)

Abstract

Sorption behavior of Hg2+ by the kerateine of thiol-type, which was prepared from keratin, was investigated. The behavior was discussed in relation to (i) the amphoteric nature, (ii) the chemical modification of side-chain polar groups, and (iii) the heat denaturation of kerateine gels. It was found that the apparent isoelectric point of the kerateine gel was closely related to the pH at which the Hg 2+ uptake showed the minimum. The Hg 2+ uptakes by modified kerateine gels in acid media depended on their modification history. The results indicated that in acid media, the dominant binding modes of Hg 2+ consist in chemosorption by the thiol as well as amino groups in the kerateine gel and in chelation of Hg2+ through these groups. On the other hand, in neutral and alkaline media, very high Hg2+ uptakes were observed for all types of kerateine gels independent of their modifica tion history. Such high Hg" uptakes could be explained in terms of the cooperative interactions between electron negative atoms incorporated in the kerateine gel, namely, sulphur, nitrogen, and oxygen rather than the ionization effect of the acidic groups present. The swelling volume of the heat-denatured kerateine gels decreased considerably with increasing the treatment temperature, and a good relation was found between the rate of Hg2+ uptake and swelling volume of the gel: the rate of Hg2+ uptake decreased with decrease in the swelling volume.

In addition, it is well known that polypeptides INTRODUCTION such as wool kerateine are easy to undergo differ In a previous paper we have reported on the ent types of denaturation.4-6 Especially, heat chemical processings of waste wool to denaturation is accompanied by a decrease in the prepare solid gel-particles (kerateine gels), and solubility. Such solubility change of kerateine their basic characteristics as adsorbent for mercuric molecules7 will affect its sorption behavior of ions.1 The kerateine gels are prepared from soluble Hg2+ The sorption mechanism of Hg2+ to the proteins extracted from wool, and contain a large kerateine gel is a complex matter and has not yet proportion of active thiol groups, which may play been fully understood. an important role in the Hg2+ uptake, especially The present study was intended to investigate in acid media. following three effects on the sorption behavior of Wool kerateine consists of various polypeptides Hg2+ by the kerateine gel of thiol type: (i) Effect which contain not only thiol but also amino, of the amphoteric nature of kerateine gels, (ii) carboxyl, hydroxyl and disulphide groups, 2,3 and Effect of the chemical modification of side-chain the amphoteric nature of kerateine results in the polar groups, and (iii) Effect of the physical different sorption mechanism of Hg` for different modification such as "heat denaturation" pH-region.1 This means that contributions of these EXPERIMENTAL side-chain groups will be involved more or less in the Hg2+ binding, depending on the pH of solution. Materials * Interaction of Wool Kerateine and its Derivatives The kerateine gels of thiol-type were prepared with Heavy Metal Ions (Part II) in the same way as reported previously.1 In Table 1 Part I, this Journal, 34, T-16 (1978) are listed some characteristics of the sample gels T-406 SEN-1 GAKKAI SHI(報 文) (50)

Table 1. Characteristics of Kerateine Gels out using a Hitachi Automatic Amino Acid Ana

lyser Type KLA-5 (Hitachi Seisakusho Co., Ltd.,

Tokyo). Table 2 shows the amino acid composi

tions of the kerateine gel of thiol-type, WG-T and

together with that of native wool.

Determination of thiol content a) Each sample (50 mg) was shaken for 1 hr. The thiol content (SH-content) was determined in HgCI2 aqueous solution (25 ml) having by the method of Friedman and Noma.8 The pH 3.5 and 200 ppm of Hg2+ details of experimental conditions has been de used for the present study. Collagen (Bovine scribed before.1

Achilles ) and polyglycine, purchased from Analysis of mercuric ion uptake

Sigma Chemical Co., St. Louis, Mo., U.S.A., were The uptake of Hg2+ exhibited by the kerateine used as model polypeptides to be compared with gel was determined with a Shimadzu atomic kerateine gels. absorption spectrometer Model AA-61OS (Shima

All chemicals were of laboratory grade quality, dzu Seisakusho Co., Ltd., Kyoto). The details of and they were used without further purification. the determination procedure has been described

Standard solution of HgCl2 with a known concen before.1 Experimental conditions specific to the tration of 1000 ppm was obtained from Nakarai present study were as follows: 50 mg of the gel

Chemical Co., Ltd., Kyoto. The solutions with was shaken in 75 ml of a buffered aqueous solution lower concentrations were prepared by diluting of HgCl2 with a concentration of 200 ppm at 20•Ž

the standard solution appropriately with distilled for lhr. For this purpose Clark-Lubs' buffer water or Clark-Lubs' buffer solution. solutions were used. Unless otherwise stated, the

Amino acid analysis values of the Hg2+ uptakes given in this paper are

The polypeptides were hydrolysed in 6N HC1 those found under the aforementioned condition.

under vacuum for 24 hr at 110•Ž. The HCI was Chemical modification of side-chain polar groups

removed by freeze-drying. Analyses were carried Acetylation was carried out by immersing the

Table 2. Amino Acid Compositions of Kerateine Gel, Native Wool and Collagen.a

a) Values given in moles per 100 moles of amino acids . (51) Vol. 34, No. 9 (1978) T-407

kerateine gel in acetic anhydride and glacial acetic

acid with trace quantities of sulphuric acid and

dimethylaniline.9 Esterification and deamination

were carried out by treating the kerateine gel with

methanolie-HCI and with sodium nitrate and glacial

acetic acid, respectively.10 The modified kerateine

gels were then washed with water and ethanol, and dried under vacuum. Heat treatment of kerateine gels

A known weight of kerateine gel was immersed

in O2-free water at different temperature for 1 hr.

The gels were then filtered, and dried under

vacuum and reweighed. No weight decrease was

found for the kerateine gels thus denatured by Fig. 1 pH variations of solution medium with this treatment. time for kerateine gel (WG-T2) in the

Determination of swelling volume and water-regain absence of HgC12.

1g of the gel was placed in a 2.0 ml cylinder

(readable to 0.01 ml) to which I ml of water was added, and then the cylinder was gently tapped.

The volume of swollen gel was thus measured at

different time according to a procedure employed

by Marhol and Cheng.11 The water-regain of the

gels was determined as follows: 1 g of the gel was allowed to stand in water at 20•Ž for 1 hr. The gel

was then filtered and lightly pressed between

filter papers to remove surface moisture. The

swollen gel was weighed, and dried under vacuum

and reweighed.

RESULTS AND DISCUSSION Fig. 2 pH variations of solution medium with Effects of amphoteric nature of kerateine gels time for kerateine gel (WG-T2) in the

Hg2+ sorption behavior of the kerateine gel in presence of HgCl2. acid media is very complex, as mentioned in our

previous paper.' This problem will be dealt with higher than about 3.6 showed a steep decrease in this section on the basis of the amphoteric similar to that in the absence of HgC12. However,

nature of kerateine gels . the decrease was followed by a slower decrease The pH of aqueous HgC12 solution medium was which continued up to about 60 min., as seen in changed with time by adding the gel , unless Figure 2. When the initial pH was below 3.6, the buffered.1 Such pH variation will be conditioned pH variation exhibited a maximum. It is obvious by two factors, namely the initial pH of solution that the slowly decreasing region appeared due to medium and the presence of HgCl2 . Figure 1 the uptake process of Hg2+ by the kerateine gel. shows typical pH variations with time for a kera The time dependence of pH variation in this region teine gel, WG-T2 , in the absence of HgC12. The pH is in parallel to that of the Hg 2+ uptake by the values either decreased or increased depending on kerateine gel; the equilibrium uptake was attained the initial pH value (due to the amphoteric nature after 60 min., although a slight increase in the of kerateine gels) uptake was further recognized.1 , but approached their proper equilibrium values after 20 min . In Figure-3 are shown plots of the initial and On the other hand, the pH variation in the equilibrium pH observed for WG-T2 gel in the

presence of HgCI2 within an initial pH range absence and presence of HgC12. In the absence of T-408 SEN-I GAKKAISHI(報 文) (52)

with increase in the number of protons present in the medium. Effects of side-chain polar groups on Hg 2+ sorption behavior It has been pointed out that active thiol groups contained in the kerateine gel play an important role in the Hg2+ uptake, particularly in acid media.1 However, the kerateine gel contains not only thiol but also amino, carboxyl, hydroxyl and disulphide groups, and it may be expected that these polar groups will contribute more or less to the Hg2+ uptake. Such contributions may be evaluated by blocking these groups. Table 3 shows the Hg2+ uptakes by new types of gels which were derived from WG-TI by its chemical modification in different manner. Esteri-

Fig. 3 Plots between the initial and equilibrium fication and acetylation prevent carboxyl and

pH values of solution medium observed for amino groups from ionizing, respectively. Deami kerateine gel (WG-T2) in the absence (•›) nation converts mainly primary amino groups to and presence (•œ) of HgC12 . hydroxyl groups, although the reaction is not entirely specific to amino groups.10 The result HgC12, the difference between initial and equilib indicates that in acid media, esterification of the rium pH vanished around at pH 3.6, and this pH carboxyl groups induced no significant effect upon corresponds to the apparent isoelectric point of the uptake, suggesting that the Hg2+ uptake in the the gel studied here (this point was different for lower pH region is not directly concerned with the different sample gel but within a range from 3.5 carboxyl groups. Data on the esterified gel above to 4.0). These values are distinctly lower than pH 7.0 were not available, because the risk of those reported for native wool and S-carboxy- hydrolysis of ester groups will come up in alkaline methylated wool kerateine.2 However the reason media. On the other hand, the blockings of amino is, at present, unknown for us. One thing to be groups gave an appreciable decrease in the uptake mentioned here is that the pH at which the Hg2+ in acid media. This implies that the Hg2+ uptake uptake by our gel showed the minimum1 was by the kerateine gel is concerned not only with almost identified with the apparent isoelectric the active thiol groups but also much with the point. primary amino groups, and further that chemo From the figure it is also recognized that the sorption of Hg2+ to the primary amino group equilibrium pH was considerably lowered by the and/or chelation of Hg2+ through this group are presence of HgCl2. This means that the uptake one of the important binding modes of Hg2+, process of Hg2+ by the kerateine gel is associated particularly in acid media. It is also pointed out

Table 3. Effect of Protein Group Modification on Hg2+ Uptake

2) Each sample (20 mg) was shaken for 1 hr in HgC1 2 aqueous solution (30 ml) with a concentration of 200 ppm. (53) Vol.34, No.9(1978) T-409 that the acetyl and hydroxyl groups resulted from with that by kerateine gel. The investigation on the above modifications are less important in the polyglycine will provide information on coopera Hg2+uptake than the primary amino group. These tive actions of main-chain peptide bonds or glycine results are consistent with those reported recently residues upon the Hg2+ binding; whereas that on by Friedman and Masri.12 From results on the collagen will provide information of effects of Hg2+ uptake by synthetic polypeptides, they cysteine and cystine upon the Hg2+ binding, found that amino groups of lysine residues are because collagen contains no such groups but very effective for the Hg2+ uptake in acid media comparable amount of acidic groups (13 moles/ in comparison with glutamic acid and tyrosine 100 moles of amino acids) to kerateine gel, as seen residues. in Table 2. Furthermore, it may be expected that In relation to the sorption mechanism in acid the difference between collagen and polyglycine media, some preliminary experiments on the de in the Hg2+ uptake partly reflects the contribution sorption of Hg2+ bound to the kerateine gel were of the acidic groups. The result showed that the carried out by using 0.01M ethylenediaminetetra Hg2+ uptakes by polyglycine and collagen were acetic acid (EDTA) as mercury binding agent. 13 distinctly lower than that by the kerateine gel, It was found that the recovery of Hg2+ bound to although the pH dependences of these three types the kerateine gel in acid media was only 30% by of polypeptides were similar to each other. It is the treatment of 0.01M EDTA. This suggests that also shown that the Hg2+ uptake by collagen, there are, at least, two modes of the Hg2+ binding, which contains relatively a large amount of acidic such as chelation of Hg2+ through the polar groups groups, is not so much different from that by present, and chemosorption. In fact, it is known polyglycine, even in alkaline media. This suggests that HgCI2 reacts with the thiol groups in kerateine that the higher Hg2+ uptake exhibited by the to form products of types W-S-HgC1or W-S-Hg-S-W, kerateine gel cannot be interpreted merely in where W- represents kerateine.14,15 The primary terms of the ionization effect of the acidic groups. amino group also has ability to form product of a On the other hand, it is recognized that mercuric type W-NH-HgCl.12 On the basis of these results ions possess high affinity to sulphur atoms incor we may conclude that the dominant mechanism porated in cysteine and cystine residues not only of Hg2+ uptake in acid media consists in chemo in acidic but also in alkaline media.16 This fact sorption by the thiol as well as amino groups in may explain our observation that the Hg2+ uptakes the kerateine gel. exhibited by the kerateine gel were greatly higher It is seen in Table 3 that prominent increases in than those by collagen. Contribution of main-chain the Hg2+ uptake were observed for all types of peptide bond or glycine residue seems to be kerateine gels independent of their modification moderately involved in the Hg2+ binding inalkaline history, when the pH of medium goes from neutral media. In addition, a preliminary experiment to alkaline. This implies that the sorption mecha showed that the Hg2+ uptakes by different kera- nism of Hg2+ in the alkaline region is quite different from that in the acidic. Such a feature of the Hg2+ sorption has already been pointed out in our previous paper.' For the higher Hg2+ uptakes in alkaline media, aspartic and glutamic acids (ca. 20 moles/ 100 moles of amino acids) will contribute much to the Hg2+ binding , in a conse quence that the number of ionized carboxyl groups increases with increasing pH of the medium . How ever, it is impossible to attribute the increases in the Hg2+ uptake only to the ionization effect of these acidic groups , as mentioned below. Figure 4 shows the pH dependences of Hg2+ Fig. 4 pH dependences of Hg 2+ uptakes by uptakes by polyglycine and collagen in comparison polyglycine, collagen, and kerateine gel. T-410 SEN-IGAKKAISHI(報 文) (54) teine gels were little influenced by their SH- molecules partly due to the rearrangement of the contents in alkaline media. This suggests that in side-chain polar groups.4 Such conformational alkaline media, the disulphide bond may contribute change will affect the swelling property of the to the Hg2+ binding as similarly as the thiol kerateine gel. This allows us to expect that heat groups does. denaturation of the kerateine gel produces changes As stated in the above, the sorption of Hg2+ in in the rate of metal ion uptakes, since the ability alkaline media is effected by different mechanisms of a gel to remove metal ions from aqueous brought about by different functional groups of solution depends on the amount of water within kerateine. In addition to such complexities, one the gel phase. Table 4 shows swelling volumes of should take, at least, two things into consideration. kerateine gels denatured at different temperature One is that kerateine molecules tend to assume a and rates of Hg2+ uptakes observed at pH 3.0. for randomly coiled conformation in alkaline region, the treated gels. As expected, the swelling volume as revealed by Bradbury et al.17 with nmr. This of the denatured kerateine gels decreases consider conformational change might be related to the ably with increasing the treatment temperature, Hg2+ sorption phenomenon. The other is a change and a good relation is found between the rate of in the ionic state of mercury with the pH of Hg 2+ uptake and swelling volume of the denatured medium, which has generally been known. Thus sample gel: the rate of Hg2+ uptake decreases with we are, at present, not in a position to propose decreasing swelling volume. However, it should be any definitive sorption mechanism of Hg2+ What noted that each sample gel exhibits almost the we may point out on this stage is that the Hg2+ same Hg2+ uptakes after 24 hr, independent of sorption will be effected by cooperative inter denaturation condition. actions between electron negative atoms incor In connection to the above findings, we deter porated in kerateine, namely, sulphur, nitrogen mined SH-contents for the denatured sample gels. and oxygen. The result is summarized in Table 5, in which the Effects of heat denaturation water-regains and Hg2+ uptakes observed at pH 7.0 Last part of this paper concerns effects of heat are also given. One can see that the SH-content denaturation of kerateine gels on the sorption remains almost constant independent of their behavior of Hg2+ It is known that heat denatura treatment temperature, while the water-regain and tion of soluble keratin fractions, such as S Hg2 uptake of sample gels strongly depend on carboxylmethyl kerateine and Cc-keratose, makes the treatment temperature. From this result we them insoluble." This solubility change has been may conclude that the rate of Hg2+ uptake is assigned to conformational change of keratin dominantly decided by the swelling property. This conclusion permits us to point out a superior Table 4. Rate of Hg2+ Uptakes by Denatured performance of our kerateine gels. The gel is Kerateine Gels and Their Swelling Volumes ready, as it stands, to remove Hg2+ efficiently from aqueous solution without such a pre-condi tioning as one must usually do for other synthetic

Table 5. Characterization of Denatured Kerateine Gels

a) Each sample (20 mg) was shaken for the required a) Each sample (20 mg) was shaken for 1 hr . in time in HgCI2 aqueous solution having pH 3.0 HgCI2 aqueous solution (30 ml) having pH 7.0 and 200 ppm of Hg2+ and 200 ppm of Hg2+ (55) Vol. 34 No. 9 (1978) T-411 adsorbents. 6. F. Macritchie, J. Polymer Sci., Symp., No. 55, 139 (1976). This work was presented at the Annual Meeting 7. See, e.g., J.R. Parrish and R. Stevenson, Anal. Chim. Acta, 70, 189 (1974). of the Society of Fiber Science and Technology, 8. M. Friedman and A. T. Noma, Text. Res. J., Japan, held in Tokyo in June, 1976. 40, 349 (1970). 9. I. C. Watt and J. D. Leeder, Trans. Fraday Acknowledgement Soc., 60, 1335 (1964). It is a pleasure to acknowledge a financial 10. J. D. Leeder and I. C. Watt, J. Phys. Chem., 69, 3280 (1965). support for this work under a research grant from 11. M. Marhol and K. L. Cheng, Talanta, 21, 751 the International Wool Secretariat. (1974). 12. M. Friedman and M. S. Masri, J. Appl. REFERENCES Polymer Sci., 17, 2183 (1973). 1. T. Miyamoto, M. Sugitani, H. Ito, T. Kondo, 13. M. Friedman and A. C. Waiss, Jr., Environ. and H. Inagaki, Sen-i Gakkaishi (J. Soc. Fiber Sci. Technol., 6, 457 (1972). Sci. Tech., Japan), 34, T-16 (1978). 14. P. R. Brown and J. O. Edwards, Biochem., 2. W.G. Crewther, R.D.B. Fraser, F.G. Lennox, 8, 1200 (1969). and H. Lindley, Advan. Protein Chem., 20, 15. See, e.g., E. H. Hinton, Jr., Text. Res. J., 44, 191 (1965). 233 (1974). 3. J. H. Bradbury, ibid., 27, 111 (1973). 16. D.F.S. Natusch and L.J. Porter, J. Chem. Soc. 4. J. D. Leeder and M. Lipson, J. Appl. Polymer (A), 252" (1971). Sci., 7, 2053 (1963). 17. J. H. Bradbury and D. E. Peter, Int. J. Peptide 5. R. S. Asquith and M. S. Otterburn, Appl. Protein Res., 7, 191 (1975). Polymer Symp., 18, 277 (1971).