Synthesis and characterization of fluorinated homocysteine derivatives as potential molecular probes for ¹⁹F magnetic resonance spectroscopy and imaging

A N-trifluoroacetyl-protected amino acid containing a thioester function, 2,2,2-trifluoro-N-(2-oxo-tetrahydrothiophen-3-yl)acetamide (TFA-tHcy), has been synthesized and characterized. It was then used to prepare a fluorine-labeled N-homocysteinylated protein, ¹⁹F-Hcy-εN-Lys-albumin, that was characterized by SDS-PAGE, MALDI-TOF-MS, UV-vis and ¹⁹F NMR spectroscopy. On average, four N-trifluoroacetylhomocysteine residues were covalently conjugated to human serum albumin through the N-substituted homocysteine thiolactone. The in situ homocysteinylation of human plasma proteins with TFA-tHcy has also been performed and has led to the formation of N-homocysteinylated proteins, with albumin modification accounting for ca. 75% of all fluorine-labeled human plasma proteins. The synthesized fluorinated molecular probes can be potentially used as informative molecular probes for in vivo ¹⁹F magnetic resonance spectroscopy and imaging.     

Brain infarction correlates more closely with acrolein than with reactive oxygen species

Although it is thought that the major factor responsible for cell damage is reactive oxygen species (ROS), our recent studies have shown that acrolein is more toxic than ROS. Thus, the relative importance of acrolein and ROS in cell damage during brain infarction was compared using photochemically induced thrombosis model mice. The levels of acrolein-conjugated albumin, and of 4-hydroxynonenal (HNE)-conjugated albumin and 8-OHdG were evaluated as indicators of damage produced by acrolein and ROS, respectively. The increase in acrolein-conjugated albumin was much greater than the increase in HNE-conjugated albumin or 8-OHdG, suggesting that acrolein is more strongly involved in cell damage than ROS during brain infarction. It was also shown that infarction led more readily to RNA damage than to DNA or phospholipid damage. As a consequence, polyamines were released from RNA, and acrolein was produced from polyamines, especially from spermine by spermine oxidase. Production of acrolein from spermine by spermine oxidase was clarified using spermine synthase-deficient Gy mice and transglutaminase 2-knockout mice, in which spermine content is negligible or spermidine/spermine N¹-acetyltransferase activity is elevated.     

Fluorescein-conjugated bovine albumin; physical and biological properties

Fluorescein-bovine albumin conjugates have been prepared and found not to differ appreciably in size, shape, and homogeneity from the precursor, bovine serum albumin. Fluorescein has also been conjugated to rat plasma proteins. Their disappearance rates from the circulation of rats correspond with those obtained from the use of isotope labeling. Their sites of localization in rat tissues were shown to be in the cytoplasm but not in the nuclei of Kupffer cells, fixed macrophages, granulocytes, and proximal renal tubules. Adsorption to endothelium was a characteristic finding. Extracellular localizations were predominantly in the lumina of blood vessels and proximal renal tubules (but never in the lumina of collecting tubules), and the interstitial fluid of skeletal and cardiac muscle (but not that of glandular organs such as the adrenals, liver, and spleen). BAC absorption from the skin of rabbits requires days whereas sodium fluorescein absorption is measured in hours, attesting to the persistence of the colloidal state of BAC in vivo. Fluorescein conjugates have been used to visualize the transcapillary passage of circulating proteins in the mesenteric circulation of frogs and rats by direct microscopic observation and found to diffuse slowly in the manner predicted for plasma proteins. The normal cutaneous vessels of the rat are impermeable in the gross to the labeled proteins; second degree burn promptly increases the permeability of these vessels rendering the presence of the label detectable in the gross in the skin. The process of labeling does not render guinea pig albumin antigenic, although slight antigenicity results from labeling whole plasma protein. It is believed that sufficient biological evidence is presented to support the conclusion that fluorescein-conjugated plasma proteins, particularly albumin, behave in vivo like their native precursors.     

Polyamines in renal failure

Summary. The levels of polyamines (putrescine, spermidine and spermine) and polyamine oxidase in plasma of patients with chronic renal failure were determined. The level of putrescine was increased but the level of spermine was decreased in the plasma of these patients. The patients also had increased plasma polyamine oxidase activity leading to increased degradation of spermine. As acrolein was a major toxic compound produced from spermine by polyamine oxidase, the levels of free and protein-conjugated acrolein in plasma were also measured. Acrolein levels were enhanced in plasma of patients with chronic renal failure. The accumulated acrolein found as protein conjugates was equivalent to 170 μM, which was about 5-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by spermine oxidase in plasma. An increase in putrescine, spermine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. After patients with chronic renal failure had undergone hemodialysis, their levels of plasma polyamines, spermine oxidase and acrolein returned towards normal. It is likely that acrolein produced from spermine accumulates in the blood due to decreased excretion into urine and may function as a uremic “toxin”.     

Studies on the inhibition of human thrombin: effects of plasma and plasma constituents

The effects of blood plasma and some plasma constituents on several types of thrombin inhibitors were quite varied. Two active esters were rapidly destroyed by serum albumin; one of these reacted initially with Lys-199, the residue that is also acylated by aspirin. Of two sulfonyl fluorides one was unaffected by albumin, and the other bound reversibly to albumin; this binding was greater with albumin acetylated at Tyr-411 near the binding site for medium-chain fatty acids. The effects of a chloromethyl ketone were inhibited, apparently reversibly, by albumin but were practically abolished by glutathione. Of two potent reversible inhibitors one was unaffected by plasma constituents, while the other was over 10-fold less potent in plasma than in fibrinogen. The effect of plasma could be partially explained by binding to albumin and lipoproteins.     

Acrolein impairs ATP binding cassette transporter A1-dependent cholesterol export from cells through site-specific modification of apolipoprotein A-I

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde, but the factors that control its reactions with nucleophilic groups on proteins remain poorly understood. Lipid peroxidation and threonine oxidation by myeloperoxidase are potential sources of acrolein during inflammation. Because both pathways are implicated in atherogenesis and high density lipoprotein (HDL) is anti-atherogenic, we investigated the possibility that acrolein might target the major protein of HDL, apolipoprotein A-I (apoA-I), for modification. Tandem mass spectrometric analysis demonstrated that lysine 226, located near the center of helix 10 in apoA-I, was the major site modified by acrolein. Importantly, this region plays a critical role in the cellular interactions and ability of apoA-I to transport lipid. Indeed, we found that conversion of Lys-226 to N(epsilon)-(3-methylpyridinium)lysine by acrolein associated quantitatively with decreased cholesterol efflux from cells via the ATP-binding cassette transporter A1 pathway. In the crystal structure of truncated apoA-I, Glu-234 lies adjacent to Lys-226, suggesting that negatively charged residues might direct the modification of specific lysine residues in proteins. Finally, immunohistochemical studies with a monoclonal antibody revealed co-localization of apoA-I with acrolein adducts in human atherosclerotic lesions. Our observations suggest that acrolein might interfere with normal reverse cholesterol transport by HDL by modifying specific sites in apoA-I. Thus, acrolein might contribute to atherogenesis by impairing cholesterol removal from the artery wall.     

Increase in putrescine,amine oxidase,and acrolein in plasma of renal failure patients

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 microM, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.     

Activation of MMP-9 activity by acrolein in saliva from patients with primary Sjögren’s syndrome and its mechanism

We have recently reported that the altered recognition patterns of immunoglobulins due to acrolein conjugation are at least partially responsible for autoimmune diseases in patients with primary Sjögren’s syndrome (pSS). In the current study, it was found that the specific activity (activity/ng protein) of metalloproteinase-9 (MMP-9) in saliva was elevated about 2.4-fold in pSS patients. Accordingly, it was examined whether MMP-9 is activated by acrolein. It was found that the MMP-9 with 92 kDa molecular weight was activated by acrolein. Under the conditions studied, Cys99, located in the propeptide, was conjugated with acrolein together with Cys230, 244, 302, 314, 329, 347, 361, 373, 388 and 516, which are located in fibronectin repeats and glycosyl domains, but not on the active site of MMP-9. In addition, 82 and 68 kDa constructs of MMP-9s, lacking the NH₂-terminal domain that contains Cys99, were not activated by acrolein. The results suggest that acrolein conjugation at Cys99 caused the active site of MMP-9 to be exposed. Activation of MMP-9 by acrolein was inhibited by cysteine, and slightly by lysine, because these amino acids inhibited acrolein conjugation with MMP-9. Conversely, MMP-9 activity in the presence of 50 μM acrolein was enhanced by 100 μM histidine. This was due to the inhibition of acrolein conjugation with His405 and 411 located at the Zn²⁺ binding site of MMP-9. These results suggest that activation of 92 kDa MMP-9 by acrolein is involved in tissue damage in pSS patients and is regulated by cysteine and histidine, and slightly by lysine. Activated 82 and 68 kDa MMP-9 s were not detected in saliva of pSS patients by Western blotting.     

Acrolein produced from polyamines as one of the uraemic toxins

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.     

Probing Calcium Ion-Induced Conformational Changes of Taiwan Cobra Phospholipase A2 by Trinitrophenylation of Lysine Residues

Phospholipase A₂ (PLA₂) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca²⁺. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca²⁺. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca²⁺ when Ca²⁺ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca²⁺. Alternatively, the nonessentiality of Lys-65 for PLA₂ activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca²⁺, suggesting that Ca²⁺ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca²⁺ are well consistent with their functional roles in the catalytic mechanism of PLA₂, and suggest that the occurrence of conformational changes with PLA₂ could be explored by chemical modification studies.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

γ-Glutamyl semialdehyde and 2-amino-adipic semialdehyde: biomarkers of oxidative damage to proteins

Reactive oxygen species are formed in the body by several natural processes and by induced oxidative stress. The reactive oxygen species may react with the various biomolecules of the body, including proteins. In order to assess the impact of oxidative damage to proteins, we have tried to identify oxidized amino acids in blood proteins which might serve as biomarkers of oxidative damage. When oxidative damage is induced into bovine serum albumin by metal-catalysed oxidation systems, the aldehyde groups formed can be derivatized by fluoresceinamine (FINH₂). Following acid hydrolysis of FINH₂-derivatized protein, two major oxidation products, γ-glutamyl semialdehyde (GGS) and 2-amino-adipic semialdehyde (AAS), were found and identified by HPLC and MS. Isolation and identification of oxidized amino acids from homopolymers (poly-Arg,-Pro,-Lys,-Trp or -Leu) confirmed that GGS can originate from Arg or Pro, while AAS is an oxidation product of Lys. When oxidative stress was induced in rats by treatments with t-butyl hydroperoxide or acrolein, rat plasma protein levels of GGS and AAS were found to be significantly higher compared with control rats. The AAS-content in serum albumin or in total plasma proteins collected from eight different mammalian species was found to be inversely proportional to their maximum lifespan potential. The content of AAS in plasma proteins of untreated adult rats showed a positive correlation with the age of the rat. In young rats a negative correlation with age was found for both GGS and AAS. We conclude that GGS or AAS may be useful novel biomarkers of oxidative damage to proteins in vivo.     

The heterogeneity of uridine diphosphate glucuronyltransferase from rat liver

1. The glucuronide conjugation of p-nitrophenol, phenolphthalein, o-aminophenol and 4-methylumbelliferone by rat liver microsomes has been studied. The detergent Triton X-100 activated UDP-glucuronyltransferase activity towards all these substrates, therefore the optimum activating concentration was added in all experiments. 2. Mg²⁺ enhanced the conjugation of the substrates. 3. With phenolphthalein substrate inhibition occurred but this could be relieved by adding albumin, which binds excess of phenolphthalein. 4. Kinetic constants of the substrates and UDP-glucuronate have been determined. Mutual inhibition was found with the substrates p-nitrophenol, 4-methylumbelliferone and phenolphthalein. p-Nitrophenol conjugation was inhibited competitively by phenolphthalein and 4-methylumbelliferone. 5. o-Aminophenol did not inhibit the conjugation of the other three substrates because these are conjugated preferentially to o-aminophenol. 6. It is concluded that the four substrates are conjugated by one enzyme at the same active site.     

Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry

Elevated levels of homocysteine (Hcy) are associated with cardiovascular and neurodegenerative diseases in humans. Hcy becomes a component of human proteins as a result of N-homocysteinylation of protein lysine residues by Hcy-thiolactone, which affects the protein's structure and function, and contributes to Hcy-related pathology. Albumin is the major target for N-homocysteinylation in human blood in vivo. Previous work has identified Lys-525 as a predominant site of N-homocysteinylation in vitro and in vivo. Here we show that Lys-4, Lys-12, Lys-137, Lys-159, Lys-205, and Lys-212 of human albumin are susceptible to N-homocysteinylation in vitro and provide evidence that two of those residues, Lys-137 and Lys-212, in addition to Lys-525, are N-homocysteinylated in vivo in human plasma.     

The hypoalbuminaemia of ovine fascioliasis: the influence of protein intake on the albumin metabolism of infected and of pair-fed control sheep.

Studies using ¹²⁵I-albumin and ⁵¹Cr-labelled plasma proteins showed that the hypoalbuminaemia which developed in sheep during the migratory stage of Fasciola hepatica infections was brought about by a combination of reduced albumin synthesis and plasma volume expansion. It was suggested that these changes were a reflection of the attendant liver damage and possibly of preferential utilisation of amino acids for immunoglobulin production. During the biliary stage of the disease, when the animals developed even more marked hypoalbuminaemia, increased albumin degradation arising from excessive plasma leakage into the gut were the outstanding features. The severity of these changes was closely linked to the state of the albumin pools which in turn was related to such factors as the plane of nutrition, appetite and fluke burden of the host. More albumin was catabolised by sheep with low fluke burdens, and in animals with the same level of infection, greater rates of catabolism were associated with a high protein intake. Sheep which catabolised most albumin became the least hypoalbuminaemic and survived longest. These animals also synthesised most albumin. It was shown that by impairing albumin synthesis, inappetence was an important additional factor in the hypoalbuminaemia of heavy infections, particularly if superimposed on a low protein diet. Nevertheless, irrespective of the size of their adult fluke burden, chronically-infected sheep were able to synthesise more albumin than pair-fed controls.     

Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Phospholipase A₂ (PLA₂) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA₂ and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA₂ and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA₂. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA₂ and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA₂ activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca²⁺ binding and antigenicity of Lys-6-modified PLA₂ were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA₂ to 32% of that of PLA₂. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA₂ activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA₂ is almost the same as that of native PLA₂. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA₂, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA₂.     

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

Radioimmunoassay of N'-nitrosonornicotine.

Antibodies specific for the precarcinogen N′-nitrosonornicotine (NNN) were obtained in rabbits immunized with a N′-nitroso-5′-carboxynornicotine-human serum albumin conjugate. The NNN derivative was synthesized by reduction and nitrosation of 2′,3′-dehydronornicotine and covalently conjugated to the macromolecule with the use of a carbodiimide. A radioimmunoassay was developed which could detect as little as 0.2 ng of NNN. Analyses of plasma supplemented with NNN indicated that it was possible to detect 2 ng NNN/ml sample. Nicotine, cotinine, pyridine, and pyrrolidine derivatives are ineffective inhibitors of the antigen-antibody reaction. N′-Nitrosoanabasine inhibits effectively, but this derivative has been reported not to occur in tobacco. The radioimmunoassay has been used to monitor nitrosation of nornicotine and anabasine under chemical conditions and to determine the rate constants for the reactions.