Modification of human serum albumin with trifluoromethyl-substituted aryl halides and sulfonates

Gerig, J. T.; Reinheimer, John D.

doi:10.1021/ja00834a029

Cited by 38 publications

(18 citation statements)

References 10 publications

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“…[25] In general, the existence of preferential sites for N-homocysteinylation seems a priori paradoxical, given that the rates of homocysteinylation measured for various proteins correlate with their content of lysine and with the rate of aminolysis measured for free lysine in solution. [3] However, as demonstrated previously, [27] significant variations of pK a value concern only a small fraction of protein lysine residues. Consequently, if bulk rate constants are measured, the high reactivity of a few activated lysines is largely masked by the vast majority that possess a normal pK a value.…”

Section: Discussionmentioning

confidence: 55%

“…[26] A decrease of several units in the pK a value is not uncommon for protein side chains: an unusually reactive lysine residue with a pK a value of 5.9 has been identified in acetoacetate decarboxylase, [27] and a pK a value of 7.9 was reported for Lys199 in HSA. [25] Generally speaking, pK a depression of a lysine can occur if it is buried in a hydrophobic environment [28,29] or if it neighbours positively charged amino acids (such as histidine, arginine or another lysine [29] ).…”

Section: Discussionmentioning

confidence: 99%

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Mechanism of Hydrolysis and Aminolysis of Homocysteine Thiolactone

Garel

Tawfik

2006

Chemistry A European J

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Homocysteine thiolactone (tHcy) is deemed a risk factor for cardiovascular diseases and strokes, presumably because it acylates the side chain of protein lysine residues ("N-homocysteinylation"), thereby causing protein damage and autoimmune responses. We analysed the kinetics of hydrolysis and aminolysis of tHcy and two related thiolactones (gamma-thiobutyrolactone and N-trimethyl-tHcy), and we have thereby described the first detailed mechanism of thiolactone aminolysis. As opposed to the previously studied (thio and oxo)esters and (oxo)lactones, aminolysis of thiolactones was found to be first order with respect to amine concentration. Anchimeric assistance by the alpha-amino group of tHcy (through general acid/base catalysis) could not be detected, and the Brønsted plot (nucleophilicity versus pK(a)) for aminolysis yielded a slope (beta(nuc)) value of 0.66. These data support a mechanism of aminolysis where the rate-determining step is the formation of a zwitterionic tetrahedral intermediate. The beta(nuc) value and steric factors dictate a regime whereby, at physiological pH values (pH 7.4), maximal reactivity of tHcy is exhibited with primary amine groups with a pK(a) value of 7.7; this allows the reactivity of various protein amino groups towards N-homocysteinylation to be predicted.

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Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Mechanism of Hydrolysis and Aminolysis of Homocysteine Thiolactone

Garel

Tawfik

2006

Chemistry A European J

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“…Lysine 199, which is known to bind hydrophobic anions such as aspirin and benzyl penicillin, was also determined to be a predominant binding site for both TDI and MDI. Gerig and Reinheimer [93] determined that the pK a of the aspirin binding site (later determined to be Lys199) of albumin was 7.9. These authors hypothesized based on the reactivity of human serum albumin with dinitrofluorobenzene that there exist two lysines on HSA that have a pK a as low as 7.9.…”

Section: Protein-selective Haptenation Targets: the Diisocyanate-amentioning

confidence: 99%

Haptenation: Chemical Reactivity and Protein Binding

2011

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Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

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“…Trifluoromethyl-dinitrobenzene sulfonate (CF;-DN BS) was made according to Gerig and Reinheimer [ 11] and Gerig et al [12], Proteins (600 mg) and sulfonates (from 0.1 to 20 m M) were let to react: 20 h, 4°C, pH 7.4 in phosphate buffer (20 ml). Several stoi chiometric conditions have been used.…”

Section: Haptenizaiion and Handling O F Soluble Proteinsmentioning

confidence: 99%

Cell Surface Modifications with Trifluoromethyl Dinitrophenyl-Soluble Protein Conjugates: Immunogenic Role of Noncovalently Bound Hapten

Bischoff

Maugras

Poignant

et al. 1984

Int Arch Allergy Immunol

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Bovine serum albumin (BSA) was derivatized, under mild conditions, with trifluoromethyl-dinitrophenyl (CF3-DNP), a haptenic group cross-reacting with trinitrophenyl (TNP). High-field nuclear magnetic resonance of fluorine (¹⁹F-NMR) permitted to calculate the number of covalently and noncovalently bound haptenic groups per BSA molecule. Further dialysis against paratoluene sulfonic acid permitted to obtain CF₃-DNP-BSA conjugates from which noncovalently bound hapten had been removed. Soluble conjugates containing 4 covalenty bound plus 1 noncovalently bound hapten groups, or only 4 covalently bound groups, were added to splenocytes in culture. These splenocytes, after such treatments, were added to effector lymphocytes in a 5-day culture aimed at the generation of cytotoxic T lymphocytes (CTL) against the CF₃-DNP-induced cell surface modification antigens. It was found that only the BSA conjugates that contained noncovalently bound haptens were able to generate CTL against target cells whose surface had been directly modified with CF₃-DNP, whereas BSA bearing only covalently bound hapten groups were not. Replacing BSA by human serum albumin, or CF₃-DNP by TNP, gave comparable results. Thus, under the conditions used, haptenic groups covalently bound to their soluble carrier protein did not generate hapten-dependent CTL, but noncovalently bound or free haptenic groups at very low concentration were able to do so.

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Modification of human serum albumin with trifluoromethyl-substituted aryl halides and sulfonates

Cited by 38 publications

References 10 publications

Mechanism of Hydrolysis and Aminolysis of Homocysteine Thiolactone

Mechanism of Hydrolysis and Aminolysis of Homocysteine Thiolactone

Haptenation: Chemical Reactivity and Protein Binding

Cell Surface Modifications with Trifluoromethyl Dinitrophenyl-Soluble Protein Conjugates: Immunogenic Role of Noncovalently Bound Hapten

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