A portion of a MALDI MS spectrum taken from a complete peptic digest of CBDP-modified human serum albumin. An aliquot of the complete digest was diluted 10-fold with 50% acetonitrile plus 0.1% trifluoroacetic acid, and 1 μl was applied to a MALDI target plate. The values shown indicate the monoisotopic masses for VRYTKKVPQVSTPTL at 1717 amu, LVRYTKKVPQVSTPTL at 1830.1 amu, VRYTKKVPQVSTPTL with an added mass of 170 on Tyr411 to give 1887 amu, and LVRYTKKVPQVSTPTL with an added mass of 170 on Tyr411 to give 2000.1 amu. The structure is for the 2000.1 amu peptide which carries o-cresyl phosphate attached to Tyr411. The accession number for human albumin in the NCBI database is gi:122920512.

MS spectrum from a mixture of tyrosine and CBDP, after 15.5 hours of reaction. Peaks marked by masses represent the reactants and the reaction products. The inserted structures are consistent with the indicated masses. Masses found in a mass spectrum of the buffer/matrix alone are marked by an asterisk (*). Three prominent, unassigned masses (at 288.0, 348.0 and 394.0 amu) are marked by a pound sign (#).

A portion of a MALDI MS spectrum taken on a complete tryptic digest of CBDP-inhibited human BChE. BChE was reacted with a 40-fold excess of CBDP for 30 seconds, and the reaction was stopped by addition of an equal volume of acetonitrile. The sample was digested with trypsin and the complete digest was diluted 10-fold in 50% acetonitrile plus 0.1% trifluoroacetic acid before applying 1 μl to the MALDI target plate. The values shown indicate the monoisotopic masses. The active site peptide SVTLFGES₁₉₈AGAASVSLHLLSPGSHSLFTR has a mass of 2928.4. Five adducts on serine 198 (serine 8 in the peptide) were found. They have masses of 3008.3, 3036.5, 3084.5, 3098.3, and 3114.3. Adduct structures are associated with the indicated masses. The accession number for human BChE in the NCBI database is gi:116353.

A low-energy, collision induced fragmentation spectrum of the active site peptide from BChE, labeled with o-cresyl phosphate (monoisotopic mass of 3098.3 amu). The tryptic digest of CBDP-modified BChE was separated offline by HPLC and the fraction containing the 3098 amu mass was infused into the QTRAP 4000 mass spectrometer, using static infusion. The spectrum is the sum of 500 scans. Annotated masses include a singly-charged b-ion series (b2-b4), a singly-charged y-ion series (y3-y18) and a doubly-charged y-ion series (y18-y25). The symbol Δ indicates ions that have dehydroalanine in place of serine due to beta-elimination of the OP (mass of OP plus mass of a molecule of water). All other, unannotated, major peaks could be accounted for as b- or y-ions minus water or minus amine, as a-ions, or as internal fragments.

The 170 amu added mass indicates that the cyclic saligenin moiety was displaced from CBDP during this reaction. See Scheme 2. Note that the masses in Scheme 2 are for the neutral species. If the o-cresyl moiety had been eliminated from CBDP, rather than the saligenin, the added mass would have been 168 amu.

To further test the reaction of CBDP with tyrosine and to re-examine the issue of the chemical nature of the adducts formed, 1 mM CBDP was reacted with 1 mM free tyrosine, at pH 7.8, and samples were withdrawn at intervals. Simple MALDI mass spectra of the samples showed prominent masses consistent with the presence of the protonated forms of tyrosine ([M+H]⁺¹ = 181.9 amu), CBDP ([M+H]⁺¹ = 276.9 amu), a tyrosine-CBDP ring-opened adduct ([M+H]⁺¹ = 458.0 amu), and o-cresyl phosphotyrosine ([M+H]⁺¹ = 352.0 amu), see Scheme 4 Note that the masses given in Scheme 4 are for the neutral charge state. No phosphorylated tyrosine with an added mass of 80 from HPO₃ was seen. This is noted because the +80 adduct is the major form found in the reaction of CBDP with the active site serine of butyrylcholinesterase.

Formation of the phospho-, o-cresyl phospho-, and ring-opened saligenin minus cresyl-adducts can be rationalized by a logical sequence of reactions, as shown in Scheme 6. Initial reaction of CBDP with BChE would be expected to yield a ring-opened derivative, by analogy with the tyrosine-CBDP reaction (see Scheme 4). Subsequent hydrolysis of the ring-opened species could remove either the cresyl moiety or the saligenin moiety. Loss of the cresyl would yield the +186 amu species, and loss of the saligenin would yield the +170 amu species. The existence of both of these species strongly implies the transient existence of the ring-opened derivative. Failure to observe the ring-opened species argues that it is hydrolyzed more rapidly than it is formed. Finally, hydrolysis of either the +170 or the +186 amu adduct would yield the phosphoserine adduct (+80 amu).

MALDI post source decay fragmentation spectrum of the CBDP-labeled human serum albumin peptide LVRYTKKVPQVSTPTL ([M+H]⁺¹ = 2000.2 amu). The spectrum was for an HPLC purified fraction of a peptic digest. The masses are centered over the peaks to which they apply. The y-axis is expanded 6.7-fold for peaks between 0 and 1800 m/z. The 306.0 amu mass, enclosed in a box, indicates the Tyr immonium ion derived from o-cresyl phosphate, whose structure is shown. The 1830.2 mass is the parent ion minus the OP.

Time course for the reaction of CBDP with tyrosine at pH 7.8. The circles indicate CBDP, the squares a tyrosine-CBDP ring-opened adduct, and the triangles o-cresyl phosphotyrosine.

MALDI post-source decay fragmentation spectrum of the human BChE active site peptide labeled at Ser198 with phosphate (monoisotopic mass of 3008.7 amu). The spectrum was taken from an offline HPLC purified fraction of the tryptic digest. The masses are centered over the peaks to which they apply. The y-axis is expanded 6.7-fold for peaks between 0 and 2500 m/z. The symbol Δ indicates ions that contain dehydroalanine in place of serine 198 due to loss of phosphate plus a molecule of water. The y22 ion at 2275.1 amu retains the phosphate on serine 198.

We chose to work with CBDP rather than with TOCP because it is accepted that TOCP is activated to CBDP in vivo [16; 17]. Activation involves oxidation of TOCP to di-o-cresyl-o-(alpha-hydroxy)tolyl phosphate, in the liver [16] followed by cyclization, with the aid of albumin, to yield CBDP in the serum (Scheme 1). Cyclization will occur spontaneously, but at 1/10 the rate of that catalyzed by albumin [18].

For saligenin to have been eliminated, two phosphorus-oxygen bonds must have been broken. To accomplish this, sequential reactions would be expected with an intermediate in which only one of the cyclic saligenin bonds to the phosphorus was hydrolyzed (see Scheme 3). Note that the ring-open structure depicted is only one of two possible forms. Choice of this depiction is for illustrative purposes and is not intended to indicate the actual chemical mechanism. Also note that the masses in Scheme 3 are for the neutral forms. The added mass from CBDP for such an intermediate would be 276 amu. Peptides VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL with an added mass of 276 amu would appear at 1993 amu (1717.0 + 276 amu) and 2106 amu (1830.1 + 276 amu), respectively. Careful examination of the MALDI spectrum revealed peaks at these masses, but their intensities were barely 2-fold above background. More convincing evidence for this ring-opened intermediate was found when CBDP was reacted with free tyrosine (see the section entitled “Adducts formed from the reaction of CBDP with free tyrosine”).

Casida and co-workers have shown that reaction of CBDP with trypsin and chymotrypsin also resulted in the displacement of saligenin. They also argued that the two bonds were broken in sequential steps, the first being displaced upon initial reaction with the nucleophilic active-site serine, and the second being hydrolyzed in a rapid “aging” reaction [16; 17; 19] (see Scheme 5). Aging is a well established phenomenon exhibited by serine hydrolases. It is defined as loss of the alcohol group from the organophosphorylated-serine adduct. It is catalyzed by groups in the active site of the OP-inhibited enzyme [23]. Aging is generally considered to be a unique function of serine hydrolases. It is noteworthy to mention here that OP-albumin does not age. Organophosphorus adducts which age rapidly on butyrylcholinesterase or acetylcholinesterase are stable on albumin [24]. Since displacement of saligenin occurs upon reaction of CBDP with Tyr411 on serum albumin, we suggest that the second step does not require enzymatic intervention such as occurs in aging, and therefore should be called dealkylation rather than aging. This conclusion is supported by the results from the reaction of CBDP with free tyrosine, where release of saligenin also occurs. That reaction can be seen to proceed via the expected two-step mechanism without any apparent assistance for the second step.