Environ. Sci. Technol. 1998, 32, 516-522
Rates of Reduction of N-Chlorinated Peptides by Sulfite: Relevance to Incomplete Dechlorination of Wastewaters JAMES S. JENSEN† AND GEORGE R. HELZ* Chemistry and Biochemistry and Water Resources Research Center, University of Maryland, College Park, Maryland 20742
Biologically induced fragmentation of proteins during wastewater treatment produces peptides, which form longlasting organic chloramines when the water is disinfected with Cl2. To protect aquatic wildlife from residual chlorine, including chloramines, wastewaters are often treated with sulfur dioxide or sulfite salts. This strategy incompletely eliminates residual chlorine species. Here we report that dechlorination rate constants of N-chloropeptides are 1-2 orders of magnitude smaller than those for NH2Cl and some aliphatic organic chloramines. Slow rates explain the prevalence of N-chloropeptides in dechlorinated wastewaters after faster reacting chlorine species have been eliminated. Dechlorination is subject to general acid catalysis. For N-chlorinated leucylalanine, the rate law above pH 6 in phosphate buffer at 25 °C and I ≈ 0.1 M is as follows: rate ) (9.92 ( 0.41 × 103[H2PO4-] + 5.70 ( 0.52 × 108[H3O+] + 5.3 ( 0.2)[SO32-][Cl-Leu-Ala] (concentrations in M, time in s). Rate constants for other peptides appear to be of similar magnitude; variations in the acid-catalyzed terms among different hydrophobic peptides correlate with solvation energies of side chains. The kinetic data suggest that reducing N-chloropeptides in wastewaters by 75% or more will require reaction times generally >0.5 h at environmentally acceptable SIV doses and pH values.
Introduction Chlorine-disinfected municipal wastewaters contain toxic agents, many of which are organic and inorganic Nchloramines (1, 2). To protect aquatic wildlife, especially fish, increasing numbers of wastewater treatment plants now include a dechlorination step after disinfection, just prior to discharge. Most often, SO2 or sulfite salts are applied for this purpose (3, 4). Here, these dechlorinating agents will collectively be called SIV compounds because they contain sulfur in the +IV oxidation state. The goal of dechlorination is to reduce all oxidative forms of residual chlorine to Cl-, including that in N-chloramines. However, field and laboratory evidence shows that dechlorination with SIV is incomplete on a kHSO3- > kSO32-, an order opposite to that observed in other systems in which SIV is a reducing agent (19, 23). As a general matter, protonation is expected to diminish the facility with which a reducing agent will surrender electrons to an oxidizing agent. Figure 3 shows that a reasonable fit can be obtained to the data in ref 21 with a model similar to eq 5. However, to fit the data below pH 6, it is necessary to propose an additional reaction: VOL. 32, NO. 4, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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H3O+ + Cl-triAla + HSO3- + 3H2O f 3H3O+ + H-triAla + SO42- + Cl-
-
δ[Cl-triAla] ) k4[H3O+][Cl-triAla][HSO3-] δt
k4 (6) (7)
The following constants were used to calculate the curve in Figure 3: k1 ) 1.3 × 104 M-2 s-1, k2 ) 1.2 × 109 M-2 s-1, k3 ) 9.9 M-1 s-1 and k4 ) 2.1 × 107 M-2 s-1. The values of k1-k3 are similar in magnitude to those in Table 3 for Cl-Ala-Ala and Cl-Leu-Ala. The rate constant for the H3O+-catalyzed reaction involving HSO3- as reductant is less than 2% of the rate constant involving SO32- as reductant (i.e., the triAla analogue of reaction 3). Roughly the same relationship between the analogous constants for NH2Cl has been reported (19). In view of the similar kinetic behavior during dechlorination of all five monochloramines in Table 3, it seems reasonable that the reactions all operate by a similar mechanism. The mechanism of Yiin et al. (19) for NH2Cl dechlorination also accounts for the new information here on N-chloropeptides. In this mechanism, a ternary intermediate is formed in which SO32- binds to Cl on the chloramine and a proton donor (H2PO4-, H3O+, H2O) binds to the lone pair on N. This intermediate then dissociates, releasing chlorosulfate, which hydrolyzes rapidly to SO42- + Cl- (24). Proton donors of greater acidity speed the reaction by withdrawing electron density to a greater extent from the N-Cl bond. Hence the rate constant for H3O+ is much larger than the rate constant for H2PO4-. Rate constants for dechlorination of NH2Cl in the presence of a series of proton donors obey the Bronsted relationship (19). While the range of observed rate constants encountered among the different dipeptides in Table 1 is not large, it is significant. All the dipeptides have an identical core structure and differ only in the side chains of the N- and C-terminal amino acids. It is reasonable to look to inductive and steric effects caused by these side chains to explain the variations. The inductive effect would influence rates by affecting the basicity of the amino N. However, the inductive effect should have only a small influence among the peptides in Table 1. Except for glycine, the differences in the side chains occur only after the second C atom from the amine. As expected, when the pKa values of the unchlorinated N-terminal amino acid in each dipeptide were compared to the rate constants in Table 1, the correlation was poor. The same was true when pKa values of the amino N in the dipeptides, themselves were compared. The steric effect influences rates through restriction of access of reactants to the reactive site on a molecule. It is plausible that steric effects could be particularly important in the Yiin et al. (19) mechanism, which requires formation of a three-component intermediate. However, comparisons of the rate constants in Table 1 with molecular volumes of the side chains (25) produced insignificant correlations; this was true whether the comparison was made to the volume of the N-terminal side chain or to the total volume of the Nand C-terminal side chains. On the other hand, there appears to be a relationship between the observed rate constants (Table 1) and hydrophobicity of the side chains in the case of dipeptides composed of amino acids with poorly solvated side chains (Figure 4). As a measure of hydrophobicity, the free energy of transfer of the side chain components between water and a nonpolar solvent (26) was used. Figure 4 shows that the dipeptides with the most hydrophobic side chains possess the slowest rate constants. Serylleucine and phenylalanylglycine, which have side chains that participate in H-bonding, react more rapidly than anticipated from this relationship. 520
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FIGURE 4. Relationship between kobs for dipeptides (Table 1) and hydrophobicity of the side chains of dipeptides. Free energy of transfer is the free energy reduction caused by transferring the side chain of an amino acid from water to an organic solvent; the scale is based on glycine which is assigned ∆G ) 0. Values on the horizontal axis are sums for both side chains in each dipeptide. Aspartylglycine is not plotted because no ∆G value is available. The implication of Figure 4 is that the extent of a peptide’s solvation, which will affect its molecular conformation, is an important factor in its reactivity with SIV. This may be of practical importance in view of Helz and Nweke’s (5) evidence that much of the SIV-refractory residual chlorine in wastewater can be extracted into octanol. While the inductive effect apparently does not account for differences in reaction rate among the dipeptides in Table 1, it probably does account for the broader differences among the compounds in Table 3. For example, the pKa for NH2Cl is 1.44 and for N-chloroglycylglycine is -0.67 (11). If the value for N-chloroglycylglycine is representative of peptides, then the ∼2 order of magnitude pKa difference between NH2Cl and N-chloropeptides nicely explains the similar differences in the k1 and k2 values in Table 3. Prospects for Improving Dechlorination Treatment. The observation that SIV-refractory residual chlorine in wastewaters includes a prominent contribution from Nchloropeptides (6) can be understood from the comparison in Table 3. In ordinary wastewaters, which have a nearneutral pH and total P usually below 10-4 M, the H3O+catalyzed term will dominate. Under these conditions, peptides can compete much less effectively than inorganic and aliphatic monochloramines for SIV, due to their much lower rate constants. Furthermore, as Helz and Nweke (5) have noted, other inorganic forms of residual chlorine (HOCl, OCl-, NHCl2, NCl3) will be reduced much faster than any of the monochloramines. Thus, N-chloropeptides will be survivors of the dechlorination process and after several minutes will become prevalent constituents of total residual chlorine, as is observed. As shown in Table 3, rate constants for the peptides studied here are all of similar magnitude. Because of this and because SIV-refractory residual chlorine at wastewater treatment plants includes a prominent contribution from moderately hydrophobic N-chloropeptides (6), it is reasonable to explore ways of enhancing the efficiency of dechlorination treatment by considering peptide dechlorination rates. We will use ClLeu-Ala as a model for peptides generally, because it is the one for which we have the most accurate data above pH 6. Two simplifying assumptions will be made. First, we assume that dechlorination of individual peptides in wastewater is a pseudo-first-order process. This will often be the
FIGURE 5. Time needed to achieve 50% reduction of N-chloroleucylalanine at 25 °C as a function of pH and SxIV. (SxIV is the HSO3+ SO32- remaining after rapidly reduced residual chlorine has been destroyed.) Time needed to achieve 97% reduction (5 half-lives) would be 5-fold larger for the same pH and SxIV conditions. Calculations assume no catalysis by acids other than H3O+ and N-chloroleucylalanine , SxIV. case in treatment situations, where it is common practice to add a SIV dose that is about 50% greater on a molar basis than the total residual chlorine in the water to be dechlorinated. More than 90% of the residual chlorine reacts within 1 min or so (5). After this initial phase, the ratio of excess SIV (henceforth, SxIV) to the remaining residual chlorine will be >5, and subsequent changes in [SxIV] due to dechlorination reactions will be small. Second, we will neglect general acid catalysts, which in general are much less important in natural waters and wastewaters than they are in laboratory experiments involving high buffer concentrations (27). For example, the maximum influence of H2PO4- on the rate of dechlorination of Cl-Leu-Ala, according to eq 5, occurs near pH 7. In order for the H2PO4--catalyzed term to exceed the H3O+-catalyzed term at pH 7, H2PO4- must exceed 6 mM (200 ppm as P). Such a concentration would be unusual, even in a wastewater. On the basis of these two assumptions, the concentration of SxIV needed to produce 50% reduction in Cl-Leu-Ala within a given time interval at a given pH and 25 °C can be calculated from the following equation. Here, SxIV is in ppm as SO2, time is in minutes, pKa is for HSO3- (i.e., 7.2 at 25 °C), and the k values are as given in Table 3; the constant, 741, reconciles the various units of measurement:
SxIV )
741[1 + 10(pKa-pH)] [k210-pH + k3]t1/2
(8)
Figure 5 is a plot of eq 8 and shows that achieving high degrees of N-chloropeptide reduction is unlikely without holdup tanks to provide reaction time. Such tanks are not currently included in most dechlorination systems. Figure
5 shows that dechlorination is favored by acidic conditions. However even at pH 6, 50% reduction of Cl-Leu-Ala can be accomplished in 1 min only with SxIV of approximately 20 ppm; 97% reduction (5 half-lives) would require either waiting 5 min at 20 ppm SxIV or raising SxIV to 100 ppm and waiting 1 min. In either case, such high SxIV concentrations would have environmentally unacceptable consequences (28). At higher pH, longer reaction times or higher SxIV concentrations are necessary. For example at pH 8, dechlorination of ClLeu-Ala requires approximately 4 times longer than at pH 6. To achieve 50% reduction of Cl-Leu-Ala with a SxIV concentration of 1 ppm, reaction times of 0.3-2 h (depending on pH) are needed. Figure 5 does not take into account temperature variations. Evidence in ref 21 suggests that k3 may depend strongly on temperature whereas the other constants may be less sensitive. If this preliminary indication is supported in future work, then cold weather would lengthen dechlorination times at high pH but have less effect near neutrality. It is of interest that the observed 26-min half-life for reduction of dechlorination-resistant residual chlorine at a wastewater treatment plant (5) is predicted reasonably well by Figure 5. This wastewater had a pH of ∼6.5 and a SxIV of ∼1 ppm. The residual chlorine in real wastewaters consists of a mixture of compounds and cannot be expected to conform exactly to Figure 5. Nonetheless, because the best information now available (6) suggests that hydrophobic N-chloropeptides comprise a major fraction of dechlorinationresistant residual chorine, Figure 5 may serve as a useful guide to how real wastewaters behave during dechlorination.
Acknowledgments U.S. Geological Survey support to the Maryland Water Resources Research Center made this work possible.
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(25) Creighton, T. E. Proteins: Structures and Molecular Properties; W. H. Freeman: New York, 1993; 507 pp. (26) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246, 2211-2217. (27) Perdue, E. M.; Wolfe, N. L. Environ. Sci. Technol. 1983, 17, 635642. (28) Kijak, P. J.; Helz, G. R. Environ. Sci. Technol. 1988, 22, 11711177.
Received for review August 18, 1997. Revised manuscript received November 25, 1997. Accepted November 28, 1997. ES9707365
