Differential modification of hemoglobin chains by acetaldehyde

Acetaldehyde-hemoglobin adducts have been suggested as potential markers for alcohol consumption. These adducts were formed in vitro with [14C]acetaldehyde and separated into hemoglobin subunits by cation-exchange chromatography to examine the relative modification of the alpha- and beta-chains. The effect of varying concentrations of acetaldehyde on the relative amounts of polypeptide adducts and on the specific radioactivities of undissociated hemoglobin (Hb) following reaction with hydroxymercurybenzoate (HMB) was also studied. There were linear relationships (P less than 0.05) between increasing levels of [14C]acetaldehyde (0.0, 0.1, 0.2, 0.5 mM) and the radioactivities of the alpha- and one of the two beta-chain adducts (22, 25, 53 dpm/mg Hb and 151, 272, 626 dpm/mg Hb, respectively). Increases in radioactivities of a minor unidentified hemoglobin adduct fraction were also observed. The ratios of specific radioactivities of beta-to alpha-chain (8.8 +/- 1.2 SEM) did not vary with the concentrations of acetaldehyde. Although the amounts of undissociated hemoglobin following reaction with HMB did not increase with increasing concentrations of acetaldehyde, the significant increase of specific radioactivities of this fraction (152, 1967, and 6562 dpm/mg Hb for 0.1, 0.2, and 0.5 mM acetaldehyde, respectively) suggested possible crosslinks within the tetramer or dimer. The amino acid analysis of alpha- and beta-subunit adducts formed with 0.1 and 0.5 mM acetaldehyde showed that unreacted cysteine residues were more often detected at the higher acetaldehyde concentration consistent with the formation of cysteine adducts labile to acid hydrolysis or the shielding of cysteine residues in acetaldehyde-modified Hb against the subunit separation by HMB treatment. Thus acetaldehyde reacts differentially with the alpha- and beta-hemoglobin subunits and with the undissociated hemoglobin molecule.     

Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain

The covalent binding of [14C]acetaldehyde to purified beef brain tubulin was characterized. As we have found for several other proteins, tubulin bound acetaldehyde to form both stable and unstable adducts. Unstable adducts (Schiff bases) were stabilized, and rendered detectable, by treating incubated reaction mixtures with the reducing agent sodium borohydride. In short-term incubations, the majority of the adducts formed were unstable, but the percentage of total adducts that were stable gradually increased with time. Stable adduct formation was greatly increased by the inclusion of sodium cyanoborohydride in reaction mixtures (reductive ethylation). When reaction mixtures were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the alpha- and beta-chains of the heterodimeric tubulin molecule, the alpha-chain of free tubulin, but not intact microtubules, was the preferential site of stable adduct formation under both reductive and nonreductive conditions. Denaturation studies showed that the native tubulin conformation was necessary for the alpha-chain to show enhanced reactivity toward acetaldehyde. Competition binding studies showed that alpha-tubulin could effectively compete with beta-tubulin and bovine serum albumin for a limited amount of acetaldehyde. Unstable acetaldehyde adducts with free tubulin or microtubules did not exhibit alpha-chain selectivity. Analysis of reaction mixtures indicates that lysine residues are the major group of the protein participating in adduct formation. These data indicate that the alpha-chain of free tubulin is the preferential site of stable acetaldehyde-tubulin adduct formation. Further, these data raise the possibility that alpha-tubulin may be a selective target for acetaldehyde adduct formation in cellular systems.     

Enrichment of cysteinyl adducts of human serum albumin

We report a method to enrich cysteinyl adducts of human serum albumin (HSA), representing biomarkers of exposure to systemic electrophiles. Because the major site of HSA adduction is the single free sulfhydryl group at Cys34, we used thiol-affinity resins to remove mercaptalbumin (i.e., unadducted HSA) from the cysteinyl adducts. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment of HSA. Differences in adduct content were detected across samples of freshly isolated, archived, and commercial HSA. Cysteinylated and glycosylated adducts were present in all samples, with abundances decreasing in the following order: commercial HSA > archived HSA > fresh HSA. After enrichment of HSA, mercaptalbumin was no longer observed in mass spectra. The ratios of HSA adducts post-/preenrichment, quantified via the Bradford assay and gel electrophoresis, were 0.029 mg adducts/mg HSA in fresh HSA and 0.323 mg adducts/mg HSA in archived HSA. The apparent elevation of adduct levels in archived samples could be due to differences in specimen preparation and storage rather than to differences in circulating HSA adducts. We conclude that thiol-affinity resins can efficiently remove mercaptalbumin from HSA samples prior to characterization and quantitation of protein adducts of reactive systemic electrophiles.     

Binding and photochemistry of enantiomeric 2-(3-benzoylphenyl)propionic acid (ketoprofen) in the human serum albumin environment.

Global analysis of circular dichroism multiwavelength data and time resolved fluorescence was applied to investigate the interaction of R(-)- and S(+)-ketoprofen (KP) with human serum albumin (HSA) in buffer solution at neutral pH. The most stable drug:protein adducts of 1 : 1 and 2 : 1 stoichiometry were characterized as regards the stability constants and the absolute circular dichroism spectra. The spectra of the diastereomeric 1 : 1 conjugates are negative with minima at ca. 350 nm for R(-)-KP and 330 nm for S(+)-KP, those of the 2 : 1 complexes are both negative with minimum at 340 nm and quite similar in shape to each other, thereby showing that the protein loses chiral recognition capability upon multiple binding. HSA intrinsic time resolved fluorescence data obtained exciting at 295 nm point to Trp 214 being located in the secondary binding site for both KP enantiomers. The photodegradation of the S(+)- and R(-)-KP:HSA complexes was studied by steady state photolysis using lambda(irr) > 320 nm. No decrease of the photodegradation quantum yields was observed in 1 : 1 complexes. An induction time for the photodegradation course in 2 : 1 complexes was observed. Transient absorption spectroscopy at lambda(exc) = 355 nm showed that triplet KP species were formed with stereo-differentiated lifetimes and high quantum yields (0.7-0.9). Secondary transients were consistent with the occurrence of photodecarboxylation and/or photoreduction within the protein matrix.     

Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions

The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins to form covalent adducts, and this damage has been implicated in pathologies associated with oxidative stress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that may serve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE with HSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues. The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNE Michael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selected reaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constants ranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 > H247 approximately K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity in subdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kinetics together with HSA structure and target residue pK(a) values suggest that location in the hydrophobic binding cavity and low predicted pK(a) of H242 account for its high reactivity toward HNE. H242 adducts may be preferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers for oxidative stress.     

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer's disease and aging. The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2 nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV-visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.     

Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography-mass spectrometry

A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 degrees C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine-cysteine-proline-phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 micromol/L of modified HSA could be obtained by applying 10 microL of NAPQI-HSA sample.     

Covalent binding of acetaldehyde to type III collagen

Incubation of neutral salt soluble type III pN-collagen with [14C]acetaldehyde in vitro resulted in the formation of spontaneously stable acetaldehyde-protein adducts. This reaction occurred primarily at lysine residues and it was not affected by 0.2-2 mM concentrations of ascorbate but addition of sodiumcyanoborohydride increased the stable adducts by 3-5-fold. When confluent cultures of human skin fibroblasts were incubated with physiologically relevant concentrations of acetaldehyde, it became covalently bound to type III procollagen secreted into the medium. We propose that acetaldehyde binding to collagen fibrils occurs in vivo following chronic alcohol consumption.     

Acetaldehyde adducts with proteins: Binding of [14C]acetaldehyde to serum albumin

Acetaldehyde, the immediate oxidation product of ethanol metabolism, was assessed for its ability to bind covalently to a purified protein in solution. Bovine serum albumin (BSA)² was used as the model protein incubated in the presence of 0.2 mm [¹⁴C]acetaldehyde at pH 7.4 and at 37 °C. Acetaldehyde formed both stable and unstable adducts with serum albumin. Unstable adducts were identified following stabilization with the reducing agent sodium borohydride. We examined both types of binding using trichloroacetic acid precipitation, gel filtration, and dialysis as means to separate bound from free acetaldehyde. All three methods of analysis gave comparable results except that the number of stable acetaldehyde adducts with albumin were significantly lower following separation by dialysis. The effects of l-cysteine, l-lysine, and reduced glutathione were assessed for their abilities as competitive reagents to decrease binding of [¹⁴C]acetaldehyde to BSA. Addition of cysteine caused a rather dramatic concentration-dependent reduction in [¹⁴C]acetaldehyde binding to BSA when compared to that caused by lysine which displayed a relatively mild effect on covalent binding. The presence of glutathione caused a concentration-dependent decrease in protein-bound radioactivity that was stronger than that by lysine but not as effective as cysteine. The ability of each reagent to reverse the formation of preformed acetaldehyde adducts with BSA was also examined. Only l-cysteine effectively decreased the number of unstable acetaldehyde adducts with BSA while stable binding of acetaldehyde remained essentially unaffected by any of the three reagents. These results indicate that acetaldehyde can covalently bind to protein and form unstable as well as stable adducts.     

Enhancement of acetaldehyde-protein adduct formation by L-ascorbate

The effect of L-ascorbate on the binding of [14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 mM) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 microM. An ascorbate concentration as low as 0.5 mM was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly-L-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.     

Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.     

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.     

Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide

A convenient method for postcolumn carbohydrate labeling has been developed. Eluates of borate complex anion-exchange columns are mixed with a reagent solution prepared from an aqueous solution of 2-cyanoacetamide and a borate buffer (pH 10.5), and the mixture is heated in a 10-m reaction coil at 100°C. Measurement of the absorbance of the product at 276 nm permits high reproducibility determination of 5 to 500 nmol of aldoses. Some carbonyl compounds are positive to this reaction, but most do not interfere with the analysis because their peaks do not appear in the aldose region. Ascorbate gives a small peak between those of mannose and fucose, but interference is negligible for equimolar amounts of ascorbate and these aldoses. This method is applied to and gives satisfactory results in the analysis of monosaccharides from various types of glycoconjugates.     

Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins

Acetaldehyde was found to form adducts with rat serum lipoproteins. The binding of [14C]acetaldehyde to lipoproteins was studied at low concentrations which are known to exist during ethanol oxidation. The amount of lipoprotein adducts was a linear function of acetaldehyde concentration up to 250 microM. Incubation of rat plasma low-density lipoproteins (LDL) with 200 microM acetaldehyde increased the disappearance rate of the 3H-label from the cholesterol ester moiety of LDL injected into normal rats. The data show that even low concentrations of acetaldehyde are capable of affecting LDL metabolism. These findings may provide an explanation for the low concentrations of serum LDL in alcoholics.     

The effect of acetaldehyde concentrations on the relative rates of formation of acetaldehyde-modified hemoglobins

The effect of various concentrations of acetaldehyde (0, 0.05, 0.1, 0.25, 0.5, 1.0, and 5.0 mM) on the relative rates of formation of hemoglobin acetaldehyde adducts detected in fractions eluted from cation exchange high-pressure liquid chromatography (HPLC) was investigated. When the hemoglobin and acetaldehyde mixtures were incubated at 37 degrees C for various time intervals up to 24 hr, increased amounts of HbA1c could be observed after 2 hr incubation with 1 mM or greater concentrations of acetaldehyde, or after 4 hr incubation with at least 0.5 mM acetaldehyde. An increase in the HbA1a + b fraction was not observed with 4 hr incubation time until the acetaldehyde level reached 1 mM. The HPLC method detected no difference in minor hemoglobins from alcoholic and normal subjects. Incubation of red blood cells at 37 degrees C for 1 hr with six consecutive pulses of 0.05 mM [14C]acetaldehyde showed no differences in the amounts of minor hemoglobins determined chromatographically at various pulse intervals. However, the measure of the 14C-label incorporation into hemoglobin showed that adducts eluting in the HbA1a+b fraction were formed at a faster rate than those eluting in the HbA1c or HbA0 fraction, respectively. The specific activities of the HbA1a+b fractions at 2, 4, and 6 pulses were 34, 128, and 949 cpm/mg hemoglobin; those of the HbA1c fraction were 15, 58, and 174 cpm/mg hemoglobin. This evidence of modification of hemoglobin by physiological levels of acetaldehyde from 14C-label incorporation suggests that an assay more sensitive than chromatographic separation of adducts might be clinically useful in detecting alcoholism or monitoring alcohol detoxification programs.     

Reaction of acetaldehyde with hemoglobin

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.     

Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch

A monomorphic albumin-like protein (CfSA) has been purified to homogeneity from the serum of African air-breathing catfish Clarias gariepinus Bloch. It has a molecular mass of approximately =70 kD and shows a lesser electrophoretic mobility than human serum albumin (HSA) in native gels. The protein exhibits cross-reactivity against rabbit anti-HSA serum and shows considerable similarity with HSA in secondary structure, however, with some differences, as indicated by a slight shift in the peaks around 267 nm and 278 nm and the absence of shoulders at 276 and 283. A certain degree of similarity also exists between their tertiary structures with respect to aromatic asymmetric environment as indicated by far-UV CD spectra and the visible range CD spectra of bilirubin complexes. CfSA-bilirubin complex is mainly characterized by bisignate CD Cotton effects (CDCEs), having minima and maxima wavelengths at 406 and 486 nm, respectively and unlike HSA, it shows prominent additional maxima around 426 nm. Based on the number of sulfhydryls, CfSA is in the rank of advanced teleosts. The occurrence of albumin in C. gariepinus in relation to the evolutionary dichotomy of albumin and other members of its multigene family in class Pisces has been discussed.     

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

PEGylation of human serum albumin: reaction of PEG-phenyl-isothiocyanate with protein

Successful and cost-effective PEGylation protocols require pure functionalized PEG reagents, which can be synthesized by simple and efficient procedures, exhibit high stability against hydrolysis, and maintain a level of reactivity with protein functional groups under mild reaction conditions. PEG-phenyl-isothiocyanate (PIT-PEG) is a new functionalized PEG having these characteristics, and has been synthesized by condensation of the bifunctional reagent 4-isothiocyanato phenyl isocyanate with monomethoxy PEG (mPEG). The data of (1)H NMR and colormetric analysis of the new PEG reagent establish that the mPEG has been quantitatively functionalized. The t 1/4 values for the hydrolysis of PIT-PEG5K in 100 mM phosphate solution at pH 6.5 and 9.2 are about 95 and 40 h, respectively. Incubation of human serum albumin (HSA, 0.5 mM) with a 10-fold molar excess of PIT-PEG (3K or 5K) at pH 6.5 and 9.2 generated PEG-HSA conjugates with average of 3.5 and 6.0 PEG chains per HSA molecule, respectively. The circular dichroism spectra of the conjugates showed that PEGylation of HSA has little influence on the secondary structure of HSA. The hexaPEGylated HSA, (TCP-PEG5K) 6-HSA, exhibited very high hydrodynamic volume, and the molecular radius of HSA increased from 3.95 to 6.57 nm on hexaPEGylation. The hexaPEGylation also increased the viscosity of 4% HSA from 1.05 to 2.10 cP, and the colloid osmotic pressure from 15.2 to 48.0 mmHg. The large increase in the hydrodynamic volume and the solution properties of (TCP-PEG5K) 6-HSA suggest that it could be a potential candidate as a plasma volume expander. PIT-PEG is a useful addition to the spectrum of functionalized PEG reagents available for surface decoration of proteins with PEG.     

Reactions of malonaldehyde and acetaldehyde with calf thymus DNA: formation of conjugate adducts

Our previous work has shown that treatment of nucleosides with malonaldehyde simultaneously with acetaldehyde affords stable conjugate adducts. In the present study we demonstrate that conjugate adducts are also formed in calf thymus DNA when incubated with the aldehydes. The adducts were identified in the DNA hydrolysates by their positive ion electrospray MS/MS spectra, by coelution with the 2'-deoxynucleoside standards, and, in the case of adducts exhibiting fluorescent properties, also by LC using a fluorescence detector. In the hydrolysates of double-stranded DNA (ds DNA), two deoxyguanosine and two deoxyadenosine conjugate adducts were detected and in single-stranded DNA (ss DNA) also, the deoxycytidine conjugate adduct was observed. The guanine base was the major target for the malonaldehyde-acetaldehyde conjugates and 2'-deoxyguanosine adducts were produced in ds DNA at levels of 100-500 adducts/10(5) nucleotides (0.7-3 nmol/mg DNA).