New Insect Juvenile Hormone Mimics: Thiolcarbamates

A prodecure for the quantitative determination of lysinoalanine (LAL) by gas chro-matography-selected ion monitoring was developed. α,ε-Diaminopimelic acid was chosen as the internal standard, and the content of LAL in commercial foods was analyzed. Of the samples analyzed, the content was highest in pidan. Wheat flour-based products (Chinese noodles, pretzels and crackers) and milk products (condensed milk and lactic acid beverages) also contained a significant amount of LAL. Ordinary milk, soybean (soy milk and soy protein isolate) and meat products (ham and Hamburg steak) contained a low amount of LAL. It was confirmed that the possibility of low-level LAL formation existed in foods cooked at home without any alkaline treatment.     

Aristolochic acid-induced accumulation of methylglyoxal and Nε-(carboxymethyl)lysine: an important and novel pathway in the pathogenic mechanism for aristolochic acid nephropathy

Aristolochic acid, found in the Aristolochia species, causes aristolochic acid nephropathy (AAN) and can develop into renal failure. Methylglyoxal (MGO) is a highly cytotoxic compound generated from the metabolic process of glucose or fatty acids. It binds to proteins and forms N(ε)-(carboxymethyl)lysine (CML), which contributes to aging and diabetes mellitus complications. However, no relevant literature explores the relationship of MGO and CML with AAN. By injecting AA (10mg/kg BW) into C3H/He mice for 5 consecutive days, we successfully developed an AAN model and observed tubular atrophy with decreased renal function. Creatinine clearance also decreased from 10.32 ± 0.79 ml/min/kg to 2.19 ± 0.29 ml/min/kg (p<0.01). The concentration of MGO in kidney homogenates increased 12 × compared to the control group (from 18.23 ± 8.05 μg/mg of protein to 231.16 ± 17.57 μg/mg of protein, p<0.01), and CML was observed in the renal tubules of the mice by immunohistochemistry. Furthermore, compared to the control group, GSH levels decreased by 0.32 × (from 2.46 ± 0.41 μM/μg of protein to 0.78 ± 0.15 μM/μg of protein, p<0.01), whereas intra-renal antioxidant capacity decreased by 0.54×(from 6.82 ± 0.97 U to 3.71 ± 0.25 U; unit is equivalent to μM Trolox/mg of protein, p<0.01). In this study, we found that serious kidney damage induced by AA is related to an increase and accumulation of MGO and CML.     

Determination of <Emphasis Type="Italic">N</Emphasis><Superscript>ɛ</Superscript>-(carboxymethyl)lysine in food systems by ultra performance liquid chromatography-mass spectrometry

We report the use of ultra pressure liquid chromatography (UPLC), coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode to determine the advanced glycation endproduct, N ^ɛ-(carboxymethyl)lysine (CML). The procedure was applied to acid hydrolyzates of protein isolated from a range of foods (milks processed at different temperatures, butter, cheese, infant formulae, bread, raw and cooked minced beef and olive oil). Highest levels of CML were determined in white bread crust (15.2 ± 0.63 mmol/mol Lys), wholemeal bread crust (13.1 ± 0.61 mmol/mol Lys) and evaporated full-fat milk (4.86 ± 0.77 mmol/mol Lys). Lowest levels of CML were measured in raw minced beef beef (0.03 ± 002 mmol/mol Lys), raw full-fat cow’s milk (0.08 ± 0.03 mmol/mol Lys) and pasteurized skimmed cow’s milk (0.09 ± 0.002 mmol/mol Lys). CML could not be detected in olive oil.     

Chemical composition of eggs of the Olive Ridley Lepidochelys olivacea (Testudines: Cheloniidae) and it's potential as a food source

The Olive Ridley is a worldwide distributed species with high nesting production per season, and in La Escobilla Oaxaca, México, there is a 70% of non-hatched eggs that are lost. In order to evaluate their potential use as a source for human and animal food products, their chemical composition was analyzed. Lyophilized egg samples from 25 turtles were obtained and were analyzed following the analytical methods for fatty acids, protein, fat, ash, moisture, amino acids, vitamins, cholesterol and microbiological agents. The analytical composition obtained was (g/100g): moisture (4.7), ash (3.8), protein (53.7), and fat (47.4). The essential amino acid (g aa/100g protein) content was: Ile (4.4), Lys (6.6), Leu (7.4), Met+Cys (8.8), Phe+Tyr (10.8). The vitamin content was: retinol (340 microg/100g), cholecalciferol (5.91 microg/100g) and 8.6 mg/100 tocopherol, 0.3 mg/100g thiamine and 1.1 mg/100g riboflavin. The total lipid content (TL), fatty acids (FA), and cholesterol (Chol) were divided into three groups based on the weight of the turtle: (TL) (44.3-48.7-49.1g/100g) and (Chol) (518.4-522.5 mg/100g-728.7). A total of 17 Saturated FA (SFA), 8 Monounsaturated FA (MUFA) and 11 Polyunsaturated FA (PUFA) were identified. The most abundant SFA (mg/100g) were: C14:0 (445-772), C16:0 (485-1263); MUFA: C16:1 (456-716), C18:1n-9c (904-1754), and PUFA: C20:4n-6 (105-217); two n-3 fatty acids were identified EPA (48-103) and DHA (97-189).There were significant differences (Fisher, p < 0.05) for: (Chol), total FA, SFA, MUFA, PUFAs and n-3 (EPA + DHA) FA. It was not detected any microbiological agent. In conclusion, lyophilized L. olivacea eggs are an option for its inclusion in the development of food products as they can be used as a high quality biological protein and n-3 fatty acid source for fortification and enrichment.     

Determination of isometamidium residues in animal-derived foods by liquid chromatography-tandem mass spectrometry

In this paper, a new determination method for isometamidium residues in animal-derived foods was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Isometamidium residues in bovine tissues and milk were extracted with the mixed solution of acetonitrile and 0.25 mol/L of ammonium formate-methanol (v/v, 1:1), concentrated and degreased, and determined by LC-MS/MS with quantification by external standard method. The results showed that the peak area of chromatogram was linearly related to the concentration of isometamidium in the range of 1-100 μg/L, and the limits of detection (LOD) and quantification (LOQ) were 0.05 μg/kg and 5 μg/kg, respectively. The average recoveries of spiked samples were in the range of 73.8-93.9% with relative standard deviations ranged from 2.3% to 7.5%. This method is simple, accurate and suitable for the identification and quantification for isometamidium in animal-derived foods.     

Cross-linking of wheat gluten proteins during production of hard pretzels

The impact of the hot alkaline dip, prior to pretzel-baking, on the types and levels of cross-links between wheat proteins was studied. Protein extractability of pretzel dough in sodium dodecyl sulfate containing buffer decreased during alkaline dipping [45?s, 1.0% (w/v) NaOH, 90°C], and even more during baking (3?min at 250°C) and drying (10?min at 135°C). Reducing agent increased the extractability partly, indicating that both reducible (disulfide, SS) and non-reducible (non-SS) protein cross-links had been formed. The decrease in cystine levels suggested β-elimination of cystine releasing Cys and dehydroalanine (DHA). Subsequent reaction of DHA with Lys and Cys, induced the unusual and potentially cross-linking amino acids lysinoalanine (LAL) and lanthionine (LAN), respectively, in alkaline dipped dough (7?μmol LAN/g protein) and in the end product (9?μmol LAL and 50?μmol LAN/g protein). The baking/drying step increased sample redness, decreased Lys levels more than expected based on LAL formation (57?μmol/g protein), and induced a loss of reducing sugars (99?μmol/g protein), which suggested the potential contribution of Maillard-derived cross-links to the observed extractability loss. However, levels of Maillard products which possibly cross-link proteins, are small compared to DHA-derived cross-links. Higher dipping temperatures, longer dipping times, and higher NaOH concentrations increased protein extractability losses and redness, as well as LAL and LAN levels in the end product. No indications for Maillard-derived cross-links or LAL in pretzel dough immediately after dipping were found, even when severe dipping conditions were used.     

Oxidation of glycated proteins: age-dependent accumulation of N epsilon-(carboxymethyl)lysine in lens proteins

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of fructoselysine (FL) in glycated (nonenzymatically glycosylated) proteins in vitro and has also been detected in human tissues and urine [Ahmed et al. (1986) J. Biol. Chem. 261, 4889-4894]. In this study, we compare the amounts of CML and FL in normal human lens proteins, aged 0-79 years, using specific and sensitive assays based on selected ion monitoring gas chromatography-mass spectrometry. Our results indicate that the lens content of FL increases significantly between infancy and about age 5 but that there is only a slight, statistically insignificant increase in FL between age 5 and 80 (mean +/- SD = 1.4 +/- 0.4 mmol of FL/mol of Lys). In contrast, the lens content of the oxidation product, CML, increased linearly with age, ranging from trace levels at infancy up to 8 mmol of CML/mol of lysine at age 79. The ratio of CML to FL also increased linearly from 0.5 to 5 mol of CML/mol of FL between age 1 and 79, respectively. These results indicate that CML, rather than FL, is the major product of glycation detectable in adult human lens protein. The age-dependent accumulation of CML in lens protein indicates that products of both glycation and oxidation accumulate in the lens with age, while the constant rate of accumulation of CML in lens with age argues against an age-dependent decline in free radical defense mechanisms in this tissue.     

Conversion of lysine to N(epsilon)-(carboxymethyl)lysine increases susceptibility of proteins to metal-catalyzed oxidation.

Metal-catalyzed oxidation (MCO) of proteins leads to the conversion of some amino acid residues to carbonyl derivatives, and may result in loss of protein function. It is well documented that reactions with oxidation products of sugars, lipids, and amino acids can lead to the conversion of some lysine residues of proteins to N(epsilon)-(carboxymethyl)lysine (CML) derivatives, and that this increases their metal binding capacity. Because post-translational modifications that enhance their metal binding capacity should also increase their susceptibility to MCO, we have investigated the effect of lysine carboxymethylation on the oxidation of bovine serum albumin (BSA) by the Fe(3+)/ascorbate system. Introduction of approximately 10 or more mol CML/mol BSA led to increased formation of carbonyls and of the specific oxidation products glutamic and adipic semialdehydes. These results support the view that the generation of CML derivatives on proteins may contribute to the oxidative damage that is associated with aging and a number of age-related diseases.     

High-performance liquid chromatographic determination of N-epsilon-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate.

A recently reported methodology for amino acid analysis by HPLC has been adapted for quantification of N-epsilon-(2-propenal)lysine (a modified lysine by reaction with malondialdehyde that has been found in enzymatic digests of foods and in urine) in biological samples. We describe its use for investigating the in vitro degradation of N-epsilon-(2-propenal)lysine using rat tissue homogenates. Lysine dipeptide, used as a control in the incubation mixtures, and the lysine released by the hydrolytic action of the homogenates in the in vitro incubations are quantified in the same way. The samples are subjected to a cleanup prederivatization step using PD-10 disposable columns (Pharmacia). This allows precolumn derivatization with diethylethoxymethylenemalonate (50 min, 50 degrees C) and resolution of the derivatives of the compounds of interest by reversed-phase HPLC (binary gradient, 45 min) with quantification based on the uv absorption of the derivatives at 280 nm (detection limits below 1 pmol). The entire analysis takes 110 min. This method can be of general use for the determination of N-epsilon-(2-propenal)lysine in the context of research dealing with protein deterioration by reaction with malondialdehyde in biological systems and in foods. A method for the synthesis of N-epsilon-(2-propenal)lysine, used as external standard for the HPLC analysis, is described.     

Interaction of lysinoalanine with the protein synthesizing apparatus

Lysinoalanine [N epsilon-(DL-2-amino-2-carboxyethyl)-L-lysine; LAL], a nephrotoxic lysine analog, inhibits the lysyl-tRNA-synthetase (EC 6.1.1.6) of prokaryotic and eukaryotic cells competitively at micromolar concentrations. Incorporation of [14C]lysine into protein by a cell-free eukaryotic protein-synthesizing system was inhibited by LAL. Inhibition was 69.7% and 18.4% at LAL concentrations of 1.0 mM and 0.1 mM, respectively. LAL was incorporated into protein as well as being an inhibitor as indicated by the incorporation of [14C]LAL into protein by the cell-free eukaryote protein-synthesizing system. The proteins labeled with [14C]LAL co-electrophoresed with those labeled with [14C]lysine. These results indicate that LAL is an inhibitor of both prokaryote and eukaryote lysyl-tRNA-synthetase. Furthermore, it is incorporated into protein. Both of these actions can be factors in the nephrotoxicity of this common food contaminant. Possible mechanisms for the toxicity of lysinoalanine are discussed.     

Structure-sweetness relationship in thaumatin: importance of lysine residues

To clarify the structural basis for the sweetness of thaumatin I, lysine-modified derivatives and carboxyl-group-modified derivatives were prepared by chemical modification followed by chromatographic purification. The sweetness of derivatives was evaluated by sensory analysis. Phosphopyridoxylation of lysine residues Lys78, Lys97, Lys106, Lys137 and Lys187 markedly reduced sweetness. The intensity of sweetness was returned to that of native thaumatin by dephosphorylation of these phosphopyridoxylated lysine residues except Lys106. Pyridoxamine modification of the carboxyl group of Asp21, Glu42, Asp60, Asp129 or Ala207 (C-terminal) did not markedly change sweetness. Analysis by far-UV circular dichroism spectroscopy indicated that the secondary structure of all derivatives remained unchanged, suggesting that the loss of sweetness was not a result of major disruption in protein structure. The five lysine residues, modification of which affected sweetness, are separate and spread over a broad surface region on one side of the thaumatin I molecule. These lysine residues exist in thaumatin, but not in non-sweet thaumatin-like proteins, suggesting that these lysine residues are required for sweetness. These lysine residues may play an important role in sweetness through a multipoint interaction with a putative thaumatin receptor.     

Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen

N epsilon-(Carboxymethyl)lysine (CML) is formed on oxidative cleavage of carbohydrate adducts to lysine residues in glycated proteins in vitro [Ahmed et al. (1988) J. Biol. Chem. 263, 8816-8821; Dunn et al. (1990) Biochemistry 29, 10964-10970]. We have shown that, in human lens proteins in vivo, the concentration of fructose-lysine (FL), the Amadori adduct of glucose to lysine, is constant with age, while the concentration of the oxidation product, CML, increases significantly with age [Dunn et al. (1989) Biochemistry 28, 9464-9468]. In this work we extend our studies to the analysis of human skin collagen. The extent of glycation of insoluble skin collagen was greater than that of lens proteins (4-6 mmol of FL/mol of lysine in collagen versus 1-2 mmol of FL/mol of lysine in lens proteins), consistent with the lower concentration of glucose in lens, compared to plasma. In contrast to lens, there was a slight but significant age-dependent increase in glycation of skin collagen, 33% between ages 20 and 80. As in lens protein, CML, present at only trace levels in neonatal collagen, increased significantly with age, although the amount of CML in collagen at 80 years of age, approximately 1.5 mmol of CML/mol of lysine, was less than that found in lens protein, approximately 7 mmol of CML/mol of lysine. The concentration of N epsilon-(carboxymethyl)hydroxylysine (CMhL), the product of oxidation of glycated hydroxylysine, also increased with age in collagen, in parallel with the increase in CML, from trace levels at infancy to approximately 5 mmol of CMhL/mol of hydroxylysine at age 80.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57+/-14 years, HbA1c 8.0+/-1.8%) to 42 non-diabetic controls (C) (age 45+/-17 years). The urinary excretion of total CML in diabetic patients was increased by approximately 30% (DM: 0.58+/-0.21; C: 0.45+/-0.14 microM/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36+/-0.17; C: 0.27+/-0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).     

Hamster zonae pellucidae cannot induce physiological acrosome reactions in chemically capacitated hamster spermatozoa in the absence of albumin

Cauda epididymal hamster spermatozoa were capacitated with D-penicillamine in a chemically defined (protein-free) medium (= "chemical" capacitation). Hamster zonae pellucidae were incapable of inducing functional acrosome reactions in chemically capacitated hamster sperm in a protein-free medium during sperm-egg coincubation. The culture medium used throughout incubation was a modified Tyrode's solution containing 10 mM sodium lactate, 100 microM sodium pyruvate and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm motility was maintained in all media with PHE (20 microM penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). Additional D-penicillamine (125 or 500 microM) or 3 mg/ml bovine serum albumin (high control: TALP-PVA) was used to capacitate sperm during preincubation at 1-2 X 10(6) sperm/ml for 4.0 h at 37 degrees C in 5% CO2 in air. Sperm were then coincubated (2 X 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA or TLP-PVA +/- additional D-penicillamine (total: 500 or 125 microM) for 1.5 or 6.0 h. Percent egg penetration was used as the definitive index of sperm capacitation and functional acrosome reactions. Chemically capacitated sperm did not penetrate eggs (0.0 +/- 0.0%) in the absence of albumin during 1.5 h of sperm-egg coincubation. When sperm were chemically capacitated with 125 microM or 500 microM D-penicillamine, then coincubated with eggs for 6.0 h in the absence of albumin, only 18.8 +/- 28.6% and 23.7 +/- 29.7%, respectively, of eggs were penetrated. Significantly (p less than 0.05) more eggs (67.7 +/- 22.4%) were penetrated when coincubated with chemically capacitated sperm for 1.5 h in medium containing albumin. These results demonstrate that zonae pellucidae of hamster eggs require the presence of albumin to efficiently induce functional acrosome reactions in sperm that are chemically capacitated with D-penicillamine.     

Lysine maintenance requirement and efficiency of its utilisation in young pigs as estimated by comparative slaughter technique

An experiment was carried out on weaner pigs (initial BW 10.8 kg) to estimate the maintenance requirement for lysine (Lys) and its marginal efficiency of utilisation using a comparative slaughter technique. Three groups of six pigs each were fed purified diets for 21 days supplying Lys at 19.5, 78 or 195 mg/kg W0.75, which corresponded to 50, 200 or 500% of the assumed maintenance requirement. All other essential amino acids were given at 50% excess. At the end of the experiment, pigs were killed for whole-body nitrogen (N) and amino acid analysis. A representative group of six pigs was analysed at the beginning of the experiment. Based on regression equations, relating Lys or N retention to Lys intake, Lys requirement for zero Lys retention was estimated to be 121 mg/kg W0.75, while Lys requirement corresponding to zero N retention was 41.7 mg/kg W0.75. At N equilibrium, the pigs lost 65 mg of Lys per kg W0.75 daily while at zero Lys retention, the daily N retention was 156 mg/kg W0.75 . The marginal efficiency of lysine utilisation was 0.91. It is concluded that zero lysine retention is a better criterion of lysine maintenance requirement than zero N retention.     

Reaction of ascorbate with lysine and protein under autoxidizing conditions: formation of N epsilon-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of glucose adducts to protein in vitro and has been detected in human tissue proteins and urine [Ahmed, M. U., Thorpe, S. R., & Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894; Dunn, J. A., Patrick, J. S., Thorpe, S. R., & Baynes, J. W. (1989) Biochemistry 28, 9464-9468]. In the present study we show that CML is also formed in reactions between ascorbate and lysine residues in model compounds and protein in vitro. The formation of CML from ascorbate and lysine proceeds spontaneously at physiological pH and temperature under air. Kinetic studies indicate that oxidation of ascorbic acid to dehydroascorbate is required. Threose and N epsilon-threuloselysine, the Amadori adduct of threose to lysine, were identified in the ascorbate reaction mixtures, suggesting that CML was formed by oxidative cleavage of N epsilon-threuloselysine. Support for this mechanism was obtained by identifying CML as a product of reaction between threose and lysine and by analysis of the relative rates of formation of threuloselysine and CML in reactions of ascorbate or threose with lysine. The detection of CML as a product of reaction of ascorbate and threose with lysine suggests that other sugars, in addition to glucose, may be sources of CML in proteins in vivo. The proposed mechanism for formation of CML from ascorbate is an example of autoxidative glycosylation of protein and suggests that CML may also be an indicator of autoxidative glycosylation of proteins in vivo.     

Purine contents of soybean-derived foods and selected Japanese vegetables and mushrooms

Purine contents of soybean-derived food and various other Japanese foods were quantitatively determined by high-performance liquid chromatography (HPLC). Purine contents were as follows: soybean-derived foods, 21.9-172.5 mg/100 g or 100 mL; Japanese vegetables, 2.3-171.8 mg/100 g; Japanese mushrooms, 9.5-142.3 mg/100 g. Since purine levels in these foods did not exceed 200 mg/100 g, we recommend that eating of them should be adopted and good dietary habits followed.     

Molecular size and immunoreactivity of ethanol extracted soybean protein concentrate in comparison with other products

Soy protein products are widely used as high nutrient sources in many food industries. However, allergenic proteins make it as one of the “big 8” foods. The objective was to analyze the molecular size, changes in protein structure and immunoreactivity of ethanol extracted soy protein concentrate in comparison with other major commercial soybean protein products extracted at different pH, and allergen stability after pepsin hydrolysis. The results showed that immunoreactivity of defatted soy white flakes (SPC2) was 227.7 mg IgE/kg protein or 102.4 mg IgE/kg product based on enzyme-linked immunosorbent assay (ELISA), the highest compared with other soy products; soy protein isolate (SPI) was the lowest (76.7 mg IgE/kg protein). Solubility of most allergenic proteins was higher at pH 12 than at pH 8.2, and the lowest at pH 2, especially no ß-conglycinin was extracted at pH 2. These results contribute to the understanding of the mechanism of immunoreactivity reduction in Soycomil and demonstrate competitive advantages compared to other soy protein products.     

Lysine maintenance requirement and efficiency of its utilisation in young pigs as estimated by comparative slaughter technique

An experiment was carried out on weaner pigs (initial BW 10.8 kg) to estimate the maintenance requirement for lysine (Lys) and its marginal efficiency of utilisation using a comparative slaughter technique. Three groups of six pigs each were fed purified diets for 21 days supplying Lys at 19.5, 78 or 195 mg/kg W^0.75, which corresponded to 50, 200 or 500% of the assumed maintenance requirement. All other essential amino acids were given at 50% excess. At the end of the experiment, pigs were killed for whole-body nitrogen (N) and amino acid analysis. A representative group of six pigs was analysed at the beginning of the experiment. Based on regression equations, relating Lys or N retention to Lys intake, Lys requirement for zero Lys retention was estimated to be 121 mg/kg W^0.75, while Lys requirement corresponding to zero N retention was 41.7 mg/kg W^0.75. At N equilibrium, the pigs lost 65 mg of Lys per kg W^0.75 daily while at zero Lys retention, the daily N retention was 156 mg/kg W^0.75. The marginal efficiency of lysine utilisation was 0.91. It is concluded that zero lysine retention is a better criterion of lysine maintenance requirement than zero N retention.     

Modification by homocysteine thiolactone affects redox status of cytochrome C

Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.