Cysteine ionization

As the four microscopic constants cannot be determined by a titration curve, spectrophotometric analysis (UV absorption of R-S ) was necessary. The pX (8.65) of cysteine betaine (ionization of a thiol in the presence of a positive nitrogen) and the pK (8.75) of S-methyl cysteine (ionization of an amino group in the presence of neutral sulfur) closely mimic the and /c2 dissociation pathways and suggest that these values should be close to each other... 

As a more sensitive detection method, MS can be very useful in amino acid determinations. For example, S-carboxymethyl-(R) cysteine or SCMC, is a mucolytic agent used in the treatment of respiratory diseases. The development of a method utilizing high performance IEC and atmospheric pressure ionization (API) mass spectrometry to quantify SCMC in plasma has been described.66 This method is simple (no derivatization needed), rapid (inn time 16 min.), sensitive (limit of quantification 200 ng/mL in human plasma), and has an overall throughput of more than 60 analyses per day. API-MS was used successfully with IEC to determine other sulfur-containing amino acids and their cyclic compounds in human urine.67 IEC has also been used as a cleanup step for amino acids prior to their derivatization and analysis by gas chromatography (GC), either alone or in conjunction with MS.68 69... 

Anacardio, R., Cantalini, M.G., De Angelis, F., and Gentile, M., Quantification of S-carboxymethyl-(R)-cysteine in human plasma by high-performance ion-exchange liquid chromatography/atmospheric pressure ionization mass spectrometry, /. Mass Spectrom., 32, 388, 1997. 

S., Simultaneous determination of urinary cystathionine, lanthionine, S-(2-aminoethyl)-L-cysteine and their cyclic compounds using liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization, /. Chromatogr. B, 698, 301, 1997. 

The most significant amino acids for modification and conjugation purposes are the ones containing ionizable side chains aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, and tyrosine (Figure 1.6). In their unprotonated state, each of these side chains can be potent nucleophiles to engage in addition reactions (see the discussion on nucleophilicity below). 

The mechanism of zinc deprivation by 3-nitrosobenzamide was elucidated most recently. When the reconstituted nucleocapsid protein p7 of HIV-1 (15 i-M) was incubated with 3-nitrosobenzamide (300 iM) at pH 7.5, three disulfide bonds per protein molecule were formed while 3-nitrosobenzamide was reduced to the hydroxylamine. Molecular masses of p7 adducts augmented by one or two 3-nitrosobenzamide residues were observed by electrospray ionization MS, consistent with covalent bond formation between cysteine sulfur and the nitroso nitrogen atom127. 

S. D. Lewis, F. A. Johnson, J. A. Shafer, Effect of Cysteine-25 on the Ionization of His-tidine-159 in Papain as Determined by Proton Nuclear Magnetic Resonance Spectroscopy. Evidence for a Hisl59-Cys25 Ion Pair and Its Possible Role in Catalysis , Biochemistry 1981, 20, 48-51. 

Example Peptides often contain sulfur from cysteine. Provided there are at least two cysteines in the peptide molecule, the sulfur can be incorporated as thiol group (SH, reduced) or sulfur bridge (S-S, oxidized). Often, both forms are contained in the same sample. At ultrahigh-resolution, the contributions of these compositions to the same nominal m/z can be distinguished. The ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) FT-ICR mass spectrum of native and reduced [D-Pen jenkephalin gives an example of such a separation (Fig. 3.25). [39] The left expanded view shows fully resolved peaks due to and C2 isotopomers of the native and the all- C peak of the reduced compound at m/z 648. The right expansion reveals the peak of the native plus the... 

Bartels, M.J. Quantitation of the Tetra-chloroethylene Metabolite A-Acetyl-5-(trichlorovinyl)cysteine in Rat Urine Via Negative Ion Chemical Ionization Gas Chromatography/Tandem Mass Spectrometry. Biol. Mass Spectrom. 1994, 23, 689-694. 

Modification of an introduced cysteine is both time- and concentration-dependent. It is therefore possible to measure the rate of sulfhydryl modification by applying low concentrations of MTS over short periods of time. The rate of reaction depends on the following factors (1) the permeability of the access pathway to the substituted cysteine, (2) electrostatic potentials, (3) the degree of ionization of the thiol, and (4) local steric constraints (Karlin and Akabas, 1998). The rate constant provides important information about the physicochemical environment of the introduced cysteine residue, relative to other accessible residues within... 

The crystal structure (Strop et al. 2001) reveals a homodimer with the zinc atom ligated by the sulfur atoms of two cysteines (Cys 32 and Cys 90) and the nitrogen atom of a histidine (His 87), as is the case for the plant-type enzyme (Fig. 11.3). The active site contains an HEPES buffer molecule in a position that implicates involvement of Asp 34 in the transport of protons after ionization of the zinc-bound water. 

There are at least three possibile ways in which the inhibitor can bind to the active site (1) formation of a sulfide bond to a cysteine residue, with elimination of hydrogen bromide [Eq. (10)], (2) formation of a thiol ester bond with a cysteine residue at the active site [Eq. (11)], and (3) formation of a salt between the carboxyl group of the inhibitor and some basic side chain of the enzyme [Eq. (12)]. To distinguish between these three possibilities, the mass numbers of the enzyme and enzyme-inhibitor complex were measured with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI). The mass number of the native AMDase was observed as 24766, which is in good agreement with the calculated value, 24734. An aqueous solution of a-bromo-phenylacetic acid was added to the enzyme solution, and the mass spectrum of the complex was measured after 10 minutes. The peak is observed at mass number 24967. If the inhibitor and the enzyme bind to form a sulfide with elimination of HBr, the mass number should be 24868, which is smaller by about one... 

Some amino acids have additional ionizable groups in their side-chains. These may be acidic or potentially acidic (aspartic acid, glutamic acid, tyrosine, cysteine), or basic (lysine, arginine, histidine). We use the term potentially acidic to describe the phenol and thiol groups of tyrosine and cysteine respectively under physiological conditions, these groups are unlikely to be ionized. It is relatively easy to calculate the amount of ionization at a particular pH, and to justify that latter statement. 

Similar calculations as above for the basic side-chain groups of arginine pK 12.48) and lysine pK 10.52), and the acidic side-chains of aspartic acid (pATa 3.65) and glutamic acid (pAfa 4.25) show essentially complete ionization at pH 7.0. However, for cysteine (pATa of the thiol group 10.29) and for tyrosine (pAfa of the phenol group 10.06) there will be negligible ionization at pH 7.0. 

We have shown the cysteine thiol group as uncharged. The pAfa for this group in cysteine is about 10.3, and application of the Henderson-Hasselbach equation (see Section 4.9) indicates there will be negligible ionization at pH 7. Nevertheless, under the influence of a suitable basic group, e.g. arginine pATa 12.5, ionization to thiolate may be possible. In such an environment, thiolate may act as the nucleophile in the mechanism. 

Other than water, protein is the major constituent of meat averaging nearly 21% in heef or chicken meat, with fat varying fiom 4.6 to 11.0% in beef and fiom 2.7 to 12.6% in chickoi. The principal radiolytic reactions of aqueous solutions of aliphatic amino acids are reductive deamination and decarboxylation. Alanine yields NH3, pyruvic add, acetaldehyde, propionic acid, CO2, H2, and ethylamine (6). Sulfur-containing amino adds are espedally sensitive to ionizing radiation. Cysteine can be oxidized to cystine by the hydroxyl radical or it can react with the hydrated electron and produce... 

Ultraviolet difference spectra have frequently been used to measure the ionization of the phenolic hydroxyl of tyrosines. The sulfydryls of cysteines and the imidazoles of histidines are also amenable to difference spectroscopy. 

An additional point should be noted from table 3.3. Whereas the amino acid side chains (R groups) that are normally charged at physiological pH are restricted to five amino acids (aspartic acid, glutamic acid, lysine, arginine, and sometimes histidine), a number of potentially ionizable R groups are part of other amino acids. These include cysteine, serine, threonine, and tyrosine. The ionization reac-... 

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