Environmental Health PerspectivesVol. 72, pp. 5-11, 1987

Biochemistry of Protein-IsocyanateInteractions: A Comparison of the Effectsof Aryl vs. Alkyl Isocyanatesby William E. Brown,* Allen H. Green,*t Thomas E. Cedel,*and Jennifer Cairns*

In addition to their use in the polyurethane and pesticide industries, isocyanates have proven to beuseful probes for the exploration of protein structure. This paper focuses on three aspects of isocyanates:their broad reactivity, their reversible interaction with cholinesterases, and the relative hydrolysis ratesof alkyl and aryl isocyanates. The broad reactivity of isocyanates as well as the demonstrated affinitylabeling of serine and sulfhydryl esterases are discussed. Extension of the affinity labeling studies toinclude the analysis of the inhibition of cholinesterases by methyl isocyanate shows that methyl isocyanateis not an effective inhibitor of any of the cholinesterases. The inhibition of cholinesterases by alkylisocyanates shows a pattern of decreased specificity with decreased alkyl chain length. The inhibition ofcholinesterases by isocyanates is shown to be reversible, with a maximum rate of reversal seen at phys-iological pH. This reversal is characteristic of the reaction of an isocyanate with a sulfhydryl group.Finally, the affinity labeling of proteins must compete successfully with the hydrolysis of isocyanates inaqueous solution. The hydrolysis of alkyl isocyanates is shown to be significantly slower than that of thearyl isocyanates.

The application of alkyl and aryl isocyanates to thepesticide and polyurethane industries is of major com-mercial interest worldwide. In addition, the isocyanateshave proven to be valuable tools to protein chemistsmaking correlations between protein structure andfunction. It is the information from these latter studiesthat may prove valuable to our understanding of howisocyanates affect human and animal populations. As areagent used for the study of protein structure and func-tion, isocyanates have two major characteristics thatmake them attractive. First, they are highly reactivewith a variety of functional groups found on biologicalmacromolecules, and second, they have a finite lifetimethat enables them to react with selected functionalgroups but not to exist long enough to cause significant,nonspecific modification of the macromolecule understudy. It is the purpose of this paper to explore both ofthese advantages of the isocyanates as protein modifi-cation reagents, to summarize information already inthe literature, and to present new data relevant to eachpoint. Of particular interest to this symposium will bethe comparison of the biochemical results for the arylisocyanates and the alkyl isocyanates. In the formercase, the emphasis will be on toluene diisocyanate (TDI)

*Department of Biological Sciences, Carnegie-Mellon University,4400 Fifth Ave., Pittsburgh, PA 15213.

tPresent address: Family Practice Center, Norristown, PA 19401.

for which there is extensive toxicological information(1), and in the latter case, the emphasis will be on methylisocyanate (MIC), which is the focus of this symposium.The first point of discussion will be the reactivity of

the isocyanates. Table 1 shows the typical reactions ofisocyanates with different functional groups that occuron proteins and other biological macromolecules. Vir-tually every functional group gives a defined productwith an isocyanate under some defined set of conditions.For the purpose of this paper the discussion will centeronly on those products that readily form under phys-iological conditions and thus are potential sites of re-action under exposure conditions. This narrows the can-didates to the hydroxyl group (reaction 2), thesulfhydryl group (reaction 3), and the imidazole group(reaction 5). Although the other reactions are possible,they are less probable because significantly reduced lev-els of the appropriate reactive form of the functionalgroup exist under physiological conditions. For exam-ple, the amine reaction with the isocyanate is optimalwith a nonprotonated amine, and under physiologicalconditions less than 5% of the protein amines are non-protonated. As will be discussed later, the reaction ofthe isocyanates with the hydroxyl group of serine hasbeen demonstrated to be the result of the modificationof the serine esterases (7). The modification of the ty-rosyl-hydroxyl has been demonstrated with model com-pounds (8) but has not been observed in proteins. In

BROWN ET AL.

Table 1. Reactions of isocyanates (R-N=C=O) with biologicallyrelevant functional groups.

Reactive functional group Product Reference0

H2N-R' 11 (2)R-NH-C-NH-R'

0HO-R' 1I (3)

R-NH-C-0-R'0

HS-R' II (4)R-NH-C-S-R'

0 0011 11 11 (5)

HO-C-R' R-NH-C-0-C-R'0

R-NHr--R' + CO2o c

C = C -R' R-NH-C-N N

N NRcN +_ _

H20 R-NH2 + C02

addition, the reaction with hydroxyl, R'OH, shown inTable 1 is analogous to the hydrolysis reaction of theisocyanate that results in the formation of carbon diox-ide and the corresponding amine (Table 1). It is impor-tant to note that any site-specific reaction that one de-scribes is occurring in successful competition with thishydrolysis reaction. This is an important considerationin the case of the imidazole nitrogen reaction since al-though it is a good nucleophile at physiological pH, thereaction of secondary amines is slow relative to the rateof isocyanate hydrolysis.Of particular interest is the reaction with the sulfhy-

dryl group that can occur at physiological pH, whoseproduct is stable under acidic conditions, but which isfully reversible under slightly alkaline pH conditions (8).It is worth noting that the reversal of this reactionproceeds through formation of the isocyanate (4). Theimplication of this observation will be discussed later.Finally, the imidazole group is a potential site of mod-ification at physiological pH, and, although model com-

pounds have been synthesized, this specific modificationhas not been observed in proteins to date. All of theaforementioned reactions take place with both the alkyland aryl isocyantes.The diverse reactivity of the isocyanates make them

attractive as potential molecular "rulers" to measurestructural features ofproteins. To this end, studies wereperformed to measure the intramolecular distance be-tween amino acids on a protein by crosslinking aminoacid side chains with bifunctional isocyanates of defineddimensions (9). This analysis was extended to mono-

functional isocyanates in the case ofthe serine proteases(chymotrypsin, trypsin, and elastase), and it was foundthat not only did the isocyanates discriminate betweendifferent esterases but they also demonstrated a chain-length dependence for inhibition of a single esterase (7).Subsequent biochemical analysis showed that the re-action was irreversible even in the presence of strongnucleophilic reagents, the modification was on the ac-tive-site serine 195 in the case of chymotrypsin (10), andthat the alkyl chain was buried in the hydrophobic sub-strate-binding pocket of the enzyme (11). Thus the alkylisocyanates had been shown to be excellent active-sitedirected affinity probes for the serine esterases. It isworth noting that although these were in vitro exper-iments, they were carried out at pH 7.5 and representstoichiometric inhibition of the enzymes in aqueous me-dium.Subsequent to this work, in vitro titrations of other

enzymes were performed; noteworthy among thesewere papain and alcohol dehydrogenase (8), which wereboth inhibited by butyl isocyanate. The major differ-ences between these studies and those previously citedfor the serine esterases are that the inhibition of bothof these enzymes results from modification of their ac-tive site sulfhydryl group and that the inhibition by theisocyanates was reversible under slightly alkaline pH(pH : 7.0).

Finally, it has been shown that in in vitro titrationsofthe cholinesterases with a variety ofisocyanates, bothalkyl and aryl, there is both inhibitory discriminationof a single isocyanate for different cholinesterases andof different isocyanates for a single cholinesterase (12).This effect is similar to the same effect seen with otheranticholinesterases such as the organophosphates andcarbamates and has been discussed elsewhere (13). Thepurpose of this paper is to extend the characterizationof the cholinesterase-isocyanate interaction to includemethyl isocyanate and to demonstrate that MIC is notan effective inhibitor of the cholinesterases. Further-more, it will be shown that the inhibition of the cholin-esterases by other isocyanates is fully reversible underphysiological conditions.The second point to be made in this paper is that

these site-specific stoichiometric inhibitions of esterasesby isocyanates and indeed any site-directed affinity la-beling of a protein by any isocyanate must competesuccessfully with the simultaneous hydrolysis of the iso-cyanates by water. Though substantial information isavailable on the respiratory effects of the aryl isocya-nates (1) and much has been presented at this meetingon the effects of methyl isocyanate, little informationhas been presented on the lifetime of the different is-ocyanates in aqueous media. The results presented inthis paper will show that the alkyl isocyanates are sig-nificantly more stable than the ayl isocyanates againsthydrolysis. This extended lifetime of MIC may accountfor the significant differences seen in the inhalation ir-ritation between TDI and MIC.

6

CHEMISTRY OF PROTEIN-MIC INTERACTIONS

Methods

MaterialsHorse serum cholinesterase (12.9 units/mg) and eel

cholinesterase (355 units/mg) were purchased fromSigma Chemical Company and used without further pu-rification. Human serum cholinesterase was purifiedfrom plasma obtained from the Central Blood Bank ofPittsburgh, PA. Purification to a specific activity of 150units/mg was achieved by a combined purificationscheme of Main et al. (14) and Lockridge et al. (15).Hexyl, butyl, o-tolyl, and p-tolyl monoisocyanates were

purchased from Eastman Organic Chemicals and havea purity of 98% or greater. Methyl isocyanate and hex-amethylene diisocyanate were purchased from AldrichChemical Company.

Enzyme AssaysHorse and human serum cholinesterases were as-

sayed according to the procedure of Ellman et al. (16)with butyrylthiocholine (Sigma) as substrate. Eel cho-linesterase was assayed by the same procedure, ace-

tylthiocholine (Sigma) being used as substrate. One unitof activity was defined as the hydrolysis of 1 ,umole ofbutyrylthiocholine (or acetylthiocholine) per minute at250C.

Cholinesterase Titration with IsocyanatesThe titration of the cholinesterases was carried out

exactly as described previously (12). Initial protein con-

centrations were 1 mg/mL in 20 mM phosphate buffer,pH 6.9, and the isocyanates were added as aliquots fromstock solutions of isocyanates prepared in acetone. Theisocyanates were dissolved in acetone to facilitate theirsolubility and dissolution in the aqueous reaction mix-ture. The acetone never exceeded 10% of the final re-action volume and was found to have no effect on cho-linesterase activity in parallel experiments. The resultsare expressed as percent of the initial activity of cho-linesterase.

Reversal of Isocyanate InactivationHuman serum cholinesterase (1 mg/mL) was titrated

with hexamethylene diisocyanate (HDI) as described inthe preceding section to 28% of its initial activity. Thefinal reaction mixture was adjusted to a pH of 7.5 andincubated at room temperature. Aliquots of the ad-justed reaction mixture were removed over a 26-hr timeperiod and immediately assayed for cholinesterase ac-

tivity. The results are expressed as the percent of thesample's pretitration activity as a function of time. Sam-ples titrated to lower initial remaining activity showedrecovery to a lower final level (data not shown).

pH Dependence of ReversibilityHuman serum cholinesterase was titrated to 28% of

its initial activity with HDI at pH 6.9 as previouslydescribed. Aliquots of the final reaction mixture wereincubated at different pH values following titration witheither acid (HCI) or base (NaOH) to the desired pH.Aliquots from each of these incubations were removedat prescribed time intervals, diluted into the assaybuffer, and immediately assayed in the standard assayprotocol. This procedure generated curves similar tothat in Figure 2 for each pH under study. From eachcurve, the initial velocity for the recovery of activity ata defined pH is measured. The final results are thenexpressed as the rate of reactivation as a function ofpH.

Measurement of Hydrolysis RatesThe rates of hydrolysis of hexamethylene monoiso-

cyanate (HMI) and of o- and p-toluene monoisocyanate(TMI) are being determined using proton nuclear mag-netic resonance (NMR). All results reported here wereperformed on the Bruker WH300 NMR instrument inDr. Chien Ho's laboratory at Carnegie-Mellon Univer-sity. Utilization of NMR for the kinetic studies made itpossible to accurately measure the quantity of waterpresent in the hydrolysis experiment, the quantity ofisocyanate present, the rate of disappearance of theisocyanate, and the type of and rate of appearance ofall products and intermediates. This paper reports onlythe rate of isocyanate disappearance while a future pub-lication will provide a complete analysis of the hydrol-ysis ofthese monoisocyanates and certain diisocyanates.All hydrolysis experiments were performed under sim-ilar conditions, and thus a typical protocol will be given.Experiments were performed in both deuterated di-methyl sulfoxide (DMSO) and deuterated acetone.These solvents were chosen because the solubility ofboth water and the isocyanates in them provide a uni-form miscible system. Acetone was the preferred sol-vent in later experiments due to the observed catalyticeffect DMSO had on the hydrolysis reaction. A NMRtube was filled with 1.0 mL of solvent, and the spectrumwas measured to determine the amount ofwater presentin the sample. A measured quantity of water was thenadded to the solvent such that the concentration wasbetween 0.11 and 3.33 M for these experiments. A spec-trum was taken to verify the water concentration. Atzero time, a measured quantity of isocyanate was addedto the water-solvent mixture, and spectra were takenat defined intervals. The first data point was collectedwithin 1.5 min of introduction and mixing of the iso-cyanate, and data collection was computer-controlled,with the most rapid sequence of time points being 30-sec intervals. For each isocyanate, defined resonanceswere identified that represented the unhydrolyzed is-ocyanate-for example, the protons on the alpha carbonfor HMI. The second-order rate constants were derived

7

BROWN ET AL.

from the slopes of the linear second-order plots for thedisappearance of the isocyanate in each case. The re-ported rate constants represent the average of at leastthree experiments performed at concentrations of iso-cyanate ranging from 0.019 to 1.08M and concentrationsofwater ranging from 0.11 to 3.33 M with ratios ofwaterto isocyanate ranging from 0.4 to 10.

Results and DiscussionIn Vitro Effect of MIC on Cholinesterases

In experiments performed under conditions similarto those previously used (12), eel acetyl cholinesteraseand equine serum butyryl cholinesterase were titratedwith methyl isocyanate. The results of these titrationsare illustrated in Figure 1. It can be seen that althoughthere is some discrimination between the two cholin-esterases for methyl isocyanate, neither of the enzymesare effectively inhibited by MIC. For comparison thetitration of serum cholinesterase by HDI is shown onthe graph to illustrate the effect of a stoichiometric in-hibitor. Compiled in Table 2 is a comparison of the effectof different alkyl and aryl mono- and diisocyanates onthe two cholinesterases. The diisocyanates generally ap-pear to be the more potent inhibitors of the cholines-terases, as discussed previously (12), while the alkylmonoisocyanates show a marked dependence on chainlength, with the shorter chain isocyanates being thepoorer inhibitors. This is similar to the effect observedwith the inhibition of chymotrypsin by the alkyl iso-cyanates and may reflect a decrease in the specific af-

4

4..

c

0)u

L.0)

(L

a

A

A

0

0

20 40 60Molar Ratio Isocyanate

0-

80 100 120to Enzyme

FIGURE 1. Titration of cholinesterases with methyl isocyanate:horse serum cholinesterase in 20 mM phosphate buffer titratedwith () methyl isocyanate and (O) hexamethylene dilsocyanate;(A) eel acetyl cholinesterase, under the same conditions titratedwith methyl isocyanate.

Table 2. Inhibition of cholinesterases by isocyanates at variousmolar ratios of isocyanate/protein for 50% enzyme inhibition.

Isocyanate/protein ratio for50% inhibitiona

Horse serumIsocyanate enzyme Eel enzyme

Methyl isocyanate 51 560Butyl isocyanate 10 109Hexyl isocyanate 4.0 55Hexamethylene diisocyanateb 3.2 1.82,6-Toluene diisocyanateb 0.8 2022,4-Toluene diisocyanateb 68 700

a Calculations of molar ratios assumed that all protein was cholin-esterase.bData taken from a previous study (12) for comparison. Experi-

ments were performed under identical conditions.

finity making the protein reaction less competitive withthe hydrolysis reaction.

Reversible Nature of Isocyanate Inhibitionof CholinesterasesEven though MIC is not an effective inhibitor of cho-

linesterase, it is relevant to examine the characteristicsof the isocyanate-cholinesterase conjugates formed byother isocyanates. In light of the reversible nature ofthe BIC-papain and BIC-alcohol dehydrogenase modi-fications (8), studies were conducted on the reversibilityof the isocyanate-cholinesterase conjugates. In findingsreported earlier (17,18), it was found that upon incu-bation of isocyanate-cholinesterase conjugates at pH7.5, there is almost complete recovery of the enzymaticactivity. Reported herein are the previously unpub-lished details of the experiments performed by ourgroup. Figure 2 shows the rate of the recovery of en-zymatic activity at pH 7.5 for a sample of human serumcholinesterase which had been initially inhibited by HDIto 28% of control activity. Two characteristics of therecovery can be seen in this plot. The first is the timefor reversal, this process appears to be complete within7 hr. The second characteristic is that the reversal isessentially complete (89% of initial activity). This latterobservation should be qualified by the fact that the cho-linesterase was not completely inhibited (i.e., the initialreaction product of the isocyanate with the cholinester-ase retained 28% of its pre-inhibition activity). This in-itial value for the reversibility experiments was chosenbecause, as seen previously (12), the inhibition titrationcurve is linear to this point, indicating a minimum ofnonspecific modification of the enzyme. Beyond thispoint, nonspecific inhibition begins to appear that mayresult in loss of activity by second site modification. Lossof complete reversibility below this point may reflecteither involvement of a second irreversible modificationthat may also result in enzyme inhibition or irreversibledenaturation of the enzyme as a result of the more ex-tensive modification.

8

CHEMISTRY OF PROTEIN-MIC INTERACTIONS

24-

z0 20

u 16

L 120

< 8

>4

-JwiLI

4 5 6 7 8 9 10

pH

FIGURE 3. pH dependence of the recovery of cholinesterase activityfollowing inhibition by isocyanate. Human serum cholinesterase,inhibited to 28% of its original activity by HDI, is incubated atroom temperature under a variety of pH conditions. The initialrate of reactivation at each pH was measured from plots similarto those in Fig. 2.

2 4 6TIME (HOURS)

FIGURE 2. Rate of recovery of cholinesterase activity following in-hibition by HDI. Human serum cholinesterase, inhibited to 28%of its original activity, is incubated at room temperature in 20 mMphosphate at pH 7.5.

pH Dependence of the ReversibilityThe reversibility shows a marked pH dependence as

illustrated in Figure 3. The low pH stability and alkalinepH reversal are similar to those seen with the modifi-cation of the active site sulfhydryl in papain and alcoholdehydrogenase (3). Unlike papain and alcohol dehydro-genase, however, this loss of reactivation at high pH isnot due to irreversible denaturation of the enzyme atthese pH values since control experiments show reten-tion of activity of unmodified enzyme up to pH 10. Thisloss of reactivation may be due either to induced insta-bility in the enzyme as a result of the initial modificationby the isocyanate or to shift of the isocyanate from areversibly modified active site amino acid (i.e., a sulfhy-dryl group) to an irreversibly modified active-site aminoacid (e.g., a hydroxyl or an amino group). As mentionedabove, the modification of a sulfhydryl by an isocyanateyields a product that is reversible under slightly alkalinepH and proceeds through the isocyanate functional

group before either being hydrolyzed or reacting withanother strong nucleophile, such as the amino groupabove its pKa. This suggests that a sulfhydryl containingprotein or peptide may act as a carrier of the isocyanategroup for possible reaction upon its release at a latertime and/or place. Although no direct evidence existsfor this mechanism under physiological conditions, it iscertainly a hypothesis that can be tested with the useof labeled isocyanates.

Relative Hydrolysis of Alkyl and ArylIsocyanatesBecause of the vast amount of information available

on the respiratory effects ofTDI (1) and the most recentobservations of the respiratory effects of MIC (19), itwas thought that the finding of pulmonary sensitizationobserved with MIC and not with TDI under similarconditions might be explained by the difference in theirlifetime in aqueous environments. From previously pub-lished data (7) it was found that the rate of disappear-ance of the alkyl isocyanates, BIC and HMI, was sec-ond-order as expected and was the same for the twoalkyl isocyanates within experimental error. This wouldsuggest that there may be no significant difference inthe rate of hydrolysis of the alkyl monoisocyanates asa function of the alkyl chain length. Thus MIC wouldhave a half-life of approximately 2 min in an aqueousphosphate-buffered solution at pH 7.5 (the conditionschosen for the in vitro inhibition studies of the serine

zz

E 70wcc

> 60

U 50

zw 40crwCL

301

9

PC)

10 BROWN ET AL.

esterases). As mentioned previously, the finite lifetimeof a reagent is a characteristic that is beneficial in invitro protein chemical modification studies since the re-agent would be stable enough for site-specific modifi-cations but would hydrolyze before extensive nonspe-cific modifications would take place. However, under invivo physiological conditions, where a variety of differ-ent proteins exist simultaneously at very high concen-trations, the potential for nonspecific modification isgreatly increased. Therefore, the longer a reagent's re-active lifetime, the more potential a reagent has forextensive modification of biological macromolecules. Tounderstand the differences in the pulmonary effects ofMIC and TDI, it thus became important to determinethe relative difference in hydrolysis rates between thealkyl and aryl isocyanates. Table 3 lists the second-orderrate constants determined for the hydrolysis of HMIand o- and p-toluene monoisocyanates (o-TMI, p-TMI).Although performed at stoichiometric rather than sat-urating conditions of water concentration, the relativehydrolysis rates of the alkyl and aryl isocyanates wouldindicate a 5- to 1400-fold faster rate for the hydrolysisof the aryl monoisocyanates over the alkyl monoiso-cyanate, depending on the isomer of aryl isocyanate andthe solvent chosen for the study. It is known that DMSOenhances certain organic reactions and thus the datacollected in acetone may be the most significant; how-ever, emphasis should be placed on the relative com-parison of the rates in each solvent rather than betweensolvents. These results would support the speculationthat the greater pulmonary sensitization seen with theMIC relative to TDI (19) may in part be due to thelonger lifetime ofMIC in the airways due to a relativelyslower rate of hydroysis. These rates are meant to il-lustrate relative rates and at this point in our investi-gation do not represent absolute values for the aqueoushydrolysis rates.

In summary, the data thus far would indicate thatMIC has a relatively long lifetime, is highly reactive,and is not an anticholinesterase reagent. In addition,the lack of efficient inhibition of cholinesterase by MICand the reversibility of the inhibition by other isocyan-ates in the in vitro experiments might explain the in-ability of in vivo exposure experiments to show anyeffect on serum cholinesterase (20). Thus the destruc-tive nature ofMIC may be due to indiscriminant labelingof biological macromolecules. At this point it is worthmentioning the recent presentation of data from theexposure of guinea pigs to radioactively labeled TDI

Table 3. Second-order rate constants for the hydrolysisof isocyanates under controlled aqueous conditions.

Second-order rateconstant/M-min

Isocyanate In DMSO In acetoneHexamethylene monoisocyanate 5.4 x 1O-3 4.1 X 10-4o-Tolyl monoisocyanate 1.8 x 10-1 1.9 x 10-3p-Tolyl monoisocyanate 7.68 ND

(21). The results of these studies indicate that TDI istaken up in the blood of the animals in a dose-responsemanner with no significant difference between animalspreviously sensitized to TDI and nonsensitized animals.In addition, all of the radioactivity in the blood of theexposed animals appears irreversibly attached to a sin-gle protein species of 71,000 molecular weight. Theseresults would indicate a very specific mechanism for theinteraction of TDI with the physiological system. Al-though the data reported here would indicate real dif-ferences between the alkyl and aryl isocyanates in theirreaction specificity and hydrolysis rates, it is intriguingto speculate as to whether there is a similarity in theuptake of the MIC and TDI physiologically. Again, thisis a question that can be resolved with the aid of alabeled MIC probe.

This investigation was supported in part by a grant from the Win-ters Foundation to Dr. Brown.

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3. Staab, H. A., and Benz, W. Synthese und Eigenschaften von N-Carbonsaureamiden der Azole eine neue Isocyanat-synthese.Ann. Chem. 648: 72-82 (1961).

4. Sidgwick, N. V. The Organic Chemistry of Nitrogen, 3rd ed. (I.T. Millar and H. D. Springall, Eds.), Oxford University Press,London, 1966.

5. Adams, P., and Baron, F. A. Esters of carbamic acid. Chem.Rev. 65: 567-602 (1965).

6. Naegeli, C., and Tyabji, A. Uber dem Umsatz aromatischer Is-ocyansaure-ester mit organischen Sauren. I. Theorie und An-wendung der Reaktion fur die preparative Darstellung von Saure-anhydriden. Helv. Chim. Acta 17: 931-957 (1934).

7. Brown, W. E., and Wold, F. Alkyl isocyanates as active sitespecific reagents for seine proteases. Reaction properties. Bio-chemistry 12: 828-835 (1973).

8. Twu, J.-S., and Wold, F. Butyl isocyanate, an active site specificreagent for yeast alcohol dehydrogenase. Biochemistry 12: 381-386 (1973).

9. Fasold, H. Zur chemischen untersuchung der Tertiarstruktur vonProteinen. I. Quervernetzung des Myoglobin sowie Prufungseines Molekulargewichtes und nativen Zustandes. Biochem. Z.342: 288-294 (1965).

10. Brown, W. E., and Wold, F. Alkyl isocyanates as active sitespecific reagents for serine proteases. Identification of the active-site seine as the site ofreaction. Biochemistry 12:835-851 (1973).

11. Brown, W. E. Alkyl isocyanates as active site specific reagentsfor seine proteases. Location of alkyl binding site in chymotryp-sin by x-ray diffraction. Biochemistry 14: 5079-5084 (1975).

12. Brown, W. E., Green, A. H., Karol, M. H., and Alarie, Y. C.E. Inhibition of cholinesterase activity by isocyanates. Toxicol.Apple. Pharmacol. 63: 45-52 (1982).

13. Brown, W. E. The chemistry and biochemistry of isocyanates: anoverview. In: Current Topics in Pulmonary Pharmacology andToxicology (M. A. Hollinger, Ed.), Elsevier Science PublishingCo., New York, 1986, pp. 200-225.

14. Main, A. R., Soucie, W. G., Buxton, I. L., and Arinc, E. Thepurification of cholinesterase from horse serum. Biochem. J. 143:733-744 (1974).

15. Lockridge, O., Eckerson, H. W., and LaDu, B. N. Interchaindisulfide bonds and subunit organization in human serum cholin-esterase. J. Biol. Chem. 254: 8324-8330 (1979).

CHEMISTRY OF PROTEIN-MIC INTERACTIONS 11

16. Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Feather-stone, R. M. A new and rapid calorimetric determination of ace-tycholinesterase activity. Biochem. Pharmacol. 7: 88-95 (1961).

17. Brown, W. E., Green, A. H., Karol, M. H., and Alarie, Y. Re-versible active-site specific inhibition of cholinesterase by iso-cyanates. Toxicologist 2: 164-165 (1982).

18. Dewair, M., Baur, X., and Mauermayer, R. Inhibition of acetyl-cholinesterase by diisocyanate and its spontaneous reactivation.Int. Arch. Occup. Environ. Health 52: 257-261 (1983).

19. Ferguson, J. S., Schaper, M., Stock, M. F., Weyel, D. A., andAlarie, Y. Sensory and pulmonary irritation with exposure tomethyl isocyanate. Toxicol. Apple. Pharmacol. 82: 329-335 (1986).

20. Karol, M. H., Hansen, G. A., and Brown, W. E. Effects of inhaledhexamethylene diisocyanate (HDI) on guinea pig cholinesterase.Fundam. Appl. Toxicol. 4: 284-287 (1984).

21. Hill, B. L., Karol, M. H., and Brown, W. E. The fate of inhaled14C-toluene diisocyanate in sensitized guinea pigs. Toxicologist 6:15 (1986).