HCl Hydrolysis Research Articles

Fluorescent Labeling and Characterization of Cellulose Nanocrystals with Varying Charge Contents

Cotton-source cellulose nanocrystals (CNCs) with a range of surface charge densities were fluorescently labeled with 5-(4, 6-dichlorotriazinyl) aminofluorescein (DTAF) in a facile, one-pot reaction under alkaline conditions. Three CNC samples were labeled: (I) anionic CNCs prepared by sulfuric acid hydrolysis with a sulfur content of 0.47 wt %, (II) a partially desulfated, sulfuric acid-hydrolyzed CNC sample, which was less anionic with an intermediate sulfur content of 0.21 wt %, and (III) uncharged CNCs prepared by HCl hydrolysis. The DTAF-labeled CNCs were characterized by dynamic light scattering, atomic force microscopy, fluorescence spectroscopy and microscopy, and polarized light microscopy. Fluorescent CNCs exhibited similar colloidal stability to the starting CNCs, with the exception of the HCl-hydrolyzed sample, which became less agglomerated after the labeling reaction. The degree of labeling depended on the sulfur content of the CNCs, indicating that the presence of sulfate half-ester groups on the CNC surfaces hindered labeling. The labeling reaction produced CNCs that had detectable fluorescence, without compromising the overall surface chemistry or behavior of the materials, an aspect relevant to studies that require a fluorescent cellulose substrate with intact native properties. The DTAF-labeled CNCs were proposed as optical markers for the dispersion quality of CNC-loaded polymer composites. Electrospun polyvinyl alcohol fibers loaded with DTAF-labeled CNCs appeared uniformly fluorescent by fluorescence microscopy, suggesting that the nanoparticles were well dispersed within the polymer matrix.

The use of microorganisms in increasing the protein yield of cassava (Manihot esculenta Crantz) peel wastes

Growth and microbial protein production on hydrolyzed cassava peel waste by Trichoderma viride and Lactobacillus delbrueckii NRRL B-763 were investigated. Trichoderma viride was selected based on its high cellulase activity on filter paper (2.91 mg glucose/mL), cotton wool (3.08 mg glucose/mL) and carboxymethylcellulose (3.46 mg glucose/ mL) while Lactobacillus delbrueckii NRRL B-763 produced 5.84 mg protein/g in cassava peel after 72 h. Samples of cassava peel were hydrolyzed with the solutions of HCl, H2SO4 and NaOH at 0.5% concentration. The hydrolysate was neutralized to pH 6.5 and supplemented with KH2PO4 (5% w/v), urea (2.7% w/v) and (NH4)2SO4 (9% w/v). The hydrolysates produced by the solutions of HCl contained higher reducing sugar and soluble sugar content than H2SO4 and NaOH hydrolysates. The culture of Trichoderma viride was used in single culture fermentation of hydrolyzed cassava peels or in mixed culture fermentation with Lactobacillus delbrueckii NRRL B-763. Protein yield produced in 0.5% HCl hydrolysates was significantly (p ≤ 0.01) higher than that in H2SO4. The unhydrolyzed control samples produced the lowest protein. This study demonstrated the potential of cassava peel waste as a substrate for a recycling process and by- product recovery.

Principle component analysis combined with matrix-assisted laser desorption ionization mass spectrometry for rapid diagnosing the sera of patients with major depression

BackgroundPreviously, we demonstrated that 6M HCl hydrolysis followed by matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry is a useful technique to detect potential protein biomarkers in the sera collected from major depression (MD) patients and from healthy controls. MethodsIn this study, the effects of various organic acids in hydrolyzing proteins in serum were first examined. The organic matrixes commonly used in MALDI analysis were also examined for characterizing the hydrolyzed peptides. Finally, principle component analysis (PCA) was used to analyze the MALDI mass spectra of acid-hydrolyzed serum samples. ResultsIt was found that 20% TFA and sinapinic acid were the optimal reagents for hydrolysis and MALDI matrix. Samples collected from MD patients and healthy controls were readily classified through PCA analysis. A receiver operating characteristic (ROC) curve based on the ratio of the intensities of the two fragment ions (m/z 8606 and 9287) indicated by PCA plot was also constructed. The area under the curve was 0.845; the sensitivity and specificity were both 80%. ConclusionsAn analytical platform employing trifluoroacetic acid to hydrolyze serum proteins followed by MALDI-TOF/MS and PCA analysis was developed to rapidly differentiate the sera between MD patients and healthy controls.

Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Effect of acid hydrolysis in the microwave-assisted extraction of phenolic compounds from Geranium sibiricum Linne with the guidance of antibacterial activity

The feasibility and the effect of acid or alkali hydrolysis in microwave-assisted extraction as well as the antibacterial activity of the resulting extracts were studied to select appropriate phenols extraction method. The results demonstrated that acid hydrolysis was suitable for the extraction of phenols from Geranium sibiricum and hydrochloric acid (HCl) hydrolysis do not only performed the highest effectiveness, but also enhanced its antibacterial capacity. After the process was optimized, the total phenols content, yields of corilagin and geraniin were increased by 28.60, 53.80 and 35.65%, respectively, as compared to only 20% in microwave-assisted extraction (MAE). Scanning electron microscopy (SEM) was employed to verify the destructive effects of the methods, the results represented that HCl hydrolysis in MAE method had dramatic destructiveness on cell wall. Thus, HCl hydrolysis in MAE provides an efficient and rapid approach for the natural products extraction and the research also provides a valuable nature resource for healthy food industry. Key words: Phenols, Hydrolysis method, Microwave-assisted extraction, Geranium sibiricum Linne, Antibacterial activity.

A facile synthesis of deuterium labeled 2,2‐dimethyl‐[2H6]‐succinic acid and its anhydride

Deuterium labeled 2,2-dimethyl-[(2)H(6)]-succinic anhydride by a sequence of reactions involving Knoevenagel condensation of [(2)H(6)]-acetone with ethyl cyanoacetate in the presence of piperidine, Michael addition of cyanide, HCl hydrolysis, simultaneous decarboxylation, and subsequent dehydration using acetic anhydride in an overall yield of 34.23% based on [(2)H(6)]-acetone utilized in the reaction is reported. The title compounds were characterized and confirmed spectroscopically by Fourier transform infrared, (1) H-NMR, and Mass. The chemical purity as determined by HPLC was 99%. To the best of our knowledge, the synthesis of these specifically deuterium labeled compounds has not been reported so far.

Determination of chitosan with a modified acid hydrolysis and HPLC method

Acid hydrolysis and subsequent quantification of glucosamine (GlcN) are widely used for chitosan quantification. Degradation of GlcN during chitosan hydrolysis was the main reason for the decrease of recovery, which made the method improper for the quantification of chitosan. Ten milligram of chitosan hydrolyzed with 10mL mixed acid solution of HCl–H3PO4 (75:25 in molar ratio) showed the highest recovery, significantly higher than HCl hydrolysis. Further study revealed that the optimum conditions involved the hydrolysis with HCl–H3PO4 (4.5:1.5M) for 24h at 110°C. The hydrolysate was neutralized and derived with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) before HPLC quantification. The optimum ratio of FMOC-Cl:GlcN was 53:1, with excess FMOC-Cl induced by the high ionic strength of the solution. This quantification procedure was then validated and proved to be specific, with good linearity, accuracy, and precision, making it well-suited for the determination of chitosan.

A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate

The bicyclic diterpene (-)-sclareol is accumulated in glandular trichomes in Salvia sclarea (Schmiderer et al., 2008), and is a major terpenoid component of this plant species. It is used as the starting material for Ambrox synthesis, a synthetic ambergris analog used in the flavor and fragrance industry. In order to investigate the formation of sclareol, cDNA prepared from secretory cells of glandular trichomes from S. sclarea inflorescence were randomly sequenced. A putative copalyl diphosphate synthase encoding EST, SscTPS1, was functionally expressed in Escherichia coli. Whereas reaction of geranylgeranyl diphosphate with the putative copalyl diphosphate synthase followed by hydrolysis with alkaline phosphatase yielded a diastereomeric mixture of (13R)- and (13S)-manoyl oxide, HCl hydrolysis yielded (-)-sclareol (1) and 13-epi-sclareol as products. The product of the reaction of SscTPS1 with geranylgeranyl diphosphate was subjected to analysis by LC-negative ion ESI-MS/MS without prior hydrolysis. EPI scans were consistent with copalyl diphosphate to which 18 mass units had added (m/z 467 [M+H](-)). The enzymatic reaction was also carried out in the presence of 60% H2(18)O. LC-negative ion ESI-MS/MS analysis established an additional reaction product consistent with the incorporation of (18)O. Incubation in the presence of 60% (2)H2O resulted in the incorporation of one deuterium atom. These results suggest water capture of the carbocation intermediate, which is known to occur in reactions catalyzed by monoterpene synthases, but has been described only several times for diterpene synthases.

Chemical and bioassay characterisation of nitrogen availability in biochar produced from dairy manure and biosolids

Biochar is charcoal made from waste biomass and intended to be added to soil to improve soil function and reduce emissions from the biomass caused by natural degradation to CO2. Nitrogen (N) forms in biochar can be complex and their lability likely to be influenced by pyrolysis temperature which, together with the nature of carbon (C), will influence N mineralisation or immobilisation. These complex relationships are poorly understood, yet impact strongly on the potential agronomic value of biochar. In this study, N in different biochar samples produced from human and animal waste streams (biosolids and cow manure; each mixed with eucalyptus wood chips in a 1:1 dry wt. ratio) at different pyrolysis conditions (highest heating temperature 250, 350, 450 and 550°C) was extracted with 6M HCl. The acid hydrolysable, extractable N (THN) was fractionated into ammonia N (AN), amino acid N (AAN), amino sugar N (ASN) and uncharacterisable hydrolysable N (UHN). Biochar samples were also treated with 0.167M K2Cr2O7 acid to determine N potentially available in the long term. An incubation study of the different biochar samples mixed with acid washed sand was conducted at 32°C for 81 days to study both C and N turnover. During incubation, the CO2 released was trapped in NaOH and quantified. Hydrolysable N decreased as pyrolysis temperature increased from 250 to 550°C. Fractionation into AN, AAN, ASN and UHN revealed progressive structural rearrangement of N with pyrolysis temperature. Based on HCl hydrolysis and dichromate oxidation results, C and N in biochar became more stable as pyrolysis temperature increased. The ratio of volatile C to THN was a useful indicator of whether net N mineralisation or immobilisation of N in biochar occurred. THN thus seems a sound estimate of the labile N fraction in biochar in the short term; however, dichromate-oxidisable N is probably more meaningful in the long run. Further studies using different types of biochar need to be conducted under more realistic conditions to obtain more information on N availability in biochar once in soil.

Prebiotic organic microstructures.

Micro- and sub-micrometer spheres, tubules and fiber-filament soft structures have been synthesized in our experiments conducted with 3 MeV proton irradiations of a mixture of simple inorganic constituents, CO, N2 and H2O. We analysed the irradiation products, with scanning electron microscopy (SEM) and atomic force microscopy (AFM). These laboratory organic structures produced a wide variety of proteinaceous and non-proteinaceous amino acids after HCl hydrolysis. The enantiomer analysis for D,L-alanine confirmed that the amino acids were abiotically synthesized during the laboratory experiment. We discuss the presence of CO2 and the production of H2 during exothermic processes of serpentinization and consequently we discuss the production of hydrothermal CO in a ferromagnesian silicate mineral environment. We also discuss the low intensity of the Earth’s magnetic field during the Paleoarchaean Era and consequently we conclude that excitation sources arising from cosmic radiation were much more abundant during this Era. We then show that our laboratory prebiotic microstructures might be synthesized during the Archaean Eon, as a product of the serpentinization process of the rocks and of their mineral contents.

Electrophoretic and chromatographic evaluation of transgenic barley expressing a bacterial dihydrodipicolinate synthase

Nutritional quality of human and animal foodstuffs is determined by the content of essential amino acids. Barley is the fourth most important cereal of the world and the second most important cereal grown in the Czech Republic. Cereal grains such as barley contain insufficient levels of some essential amino acids, especially lysine. Dihydrodipicolinate synthase is the key enzyme involved in the regulatory step for lysine biosynthesis. Two constructs pBract214::sTPdapA and pBract214::mdapA containing the dapA gene from Escherichia coli coding for the bacterial dihydrodipicolinate synthase were used for transformation of barley. An Agrobacterium-mediated technique was used for transformation of immature embryos of spring barley cv. Golden Promise. Transgenic barley plants of the T0 and T1 generations were evaluated by PCR, real-time PCR, gel electrophoresis, and Western blot. Amino acid content was analyzed by HPLC after HCl hydrolysis. The lysine content in leaves of the T1 generation plant no. 5/5 was 50% higher than in wild-type plants; the lysine content in seeds of T2 generation plant no. 5/16 was 30% higher than in wild-type seeds of spring barley cv. Golden Promise.

An ecofriendly approach to the synthesis of 2-imino- and 2-oxo-3-phenylsulfonyl-2H-chromenes

An efficient synthesis for 2-imino-3-phenylsulfonyl-2H-chromenes was developed from the Knoevenagel condensation of (phenylsulfonyl)acetonitrile and salicylaldehydes in aqueous sodium carbonate solution, at room temperature. Hydrolysis in aqueous HCl, at 80°C, led to the formation of the corresponding 2-oxo-3-phenylsulfonyl-2H-chromenes, also isolated in excellent yields.

Surface Chemistry Determines Electron Storage Capabilities in Alcoholic Sols of Titanium Dioxide Nanoparticles. A Combined FTIR and Room Temperature EPR Investigation

Electron accumulation in TiO2 ethanolic sols prepared by the HCl hydrolysis of a titanium alkoxide has been scrutinized by UV–vis and EPR spectroscopy. Unexpectedly, Ti(III) centers, g = 1.9551, fo...

Prebiotic Organic Microstructures

AbstractMicro- and sub-micrometer spheres, tubules and fiber-filament soft structures have been synthesized in our experiments conducted with 3 MeV proton irradiations of a mixture of simple inorganic constituents, CO, N2 and H2O. We analysed the irradiation products, with scanning electron microscopy (SEM) and atomic force microscopy (AFM). These laboratory organic structures produced wide variety of proteinous and non-proteinous amino acids after HCl hydrolysis. The enantiomer analysis for D-, L- alanine confirmed that the amino acids were abiotically synthesized during the laboratory experiment. Considering hydrothermal activity, the presence of CO2 and H2, of a ferromagnesian silicate mineral environment, of an Earth magnetic field which was much less intense during Archean times than nowadays and consequently of a proton excitation source which was much more abundant, we propose that our laboratory organic microstructures might be synthesized during Archean times. We show similarities in morphology and in formation with some terrestrial Archean microstructures and we suggest that some of the observed Archean carbon spherical and filamentous microstructures might be composed of abiogenic organic molecules. We further propose a search for such prebiotic organic signatures on Mars. This article has been posted on Nature precedings on 21 July 2010 [1]. Extinct radionuclides as source of excitation have been replaced by cosmic radiations which were much more intense 3.5 Ga ago because of a much less intense Earth magnetic field. The new version of the article has been presented at the ORIGINS conference in Montpellier in july 2011 [2] and has since been published in Origins of Life and Evolution of Biospheres 42 (4) 307-316, 2012.DOI: 10.1007/s11084-012-9290-5

Chromatographic fingerprints and quantitative analysis of isoflavones in Tofu-type soybeans

Tofu-type soybeans can differ from conventional soy varieties in seed size, seed composition, flavour and nutrition. Using HPLC coupled with ESI-MS and PDA detection, a total of 19 isoflavones were detected and identified from Tofu-type soybean seeds, more than previously reported forms from many other soybeans. An HPLC/UV fingerprint study was performed for qualitative evaluation, which enabled the isoflavone profile of Tofu-type soybeans to be characterised and differentiated from other vegetative soybeans by similarity comparison. To meet the frequent quantitative application for isoflavone contents, a simple, precise and reliable method using HCl hydrolysis during sample extraction and LC/UV for the detection was developed and validated to quantitate total isoflavones in soybeans, and then applied to determine the total isoflavone contents of different Tofu-type soy varieties grown in different field locations over two growing seasons. A range of total isoflavone contents for Tofu-type soybeans was established to confirm the use of this analytical approach for quality control applications.

Mild acid hydrolysis of fucoidan: characterization by electrophoresis and FT-Raman spectroscopy

Along with proteins, lipids, water and minerals, polysaccharides are the main chemical compounds of which macroalgae are built. Among the chemical compounds now widely examined is fucoidan (fucan, fucosan, sulfate fucan or sulfated fucan), a fucose-containing sulfated polysaccharide. Fucoidans isolated from different species have been extensively studied because of their varied biological properties, including anticoagulant and antitumor effects. Methodology based on mild acid hydrolysis can be used as an efficient tool to study the relationship between molecular weight of the sulfated polysaccharides and their biological activities. Anticancer activity of fucoidans can be significantly enhanced by lowering their molecular weight only when they are depolymerized under mild conditions. In this study, fucoidan was identified during extraction with H2SO4 and HCl; its presence was confirmed by FT-Raman spectroscopy in aqueous solution. In particular, shifts at 840cm−1 were analysed, which are due to the presence of sulfate at the axial C-4 position, as were the shifts at about 811–809cm−1, for which the sulfated fucoidan is responsible. Shifts of electrophoretic bands of fucoidan resulting from mild acid hydrolysis in H2SO4 and HCl were also analysed. The analytical procedure was developed using apparatus for cellulose acetate membrane electrophoresis and this was supplemented by semi-quantitative analysis.

Acid-Catalyzed and Solvolytic Desulfation of H2SO4-Hydrolyzed Cellulose Nanocrystals

Cellulose nanocrystals (CNCs) prepared by H(2)SO(4) hydrolysis have sulfate groups on their surface, which have negative implications for some CNC applications. In this study, two desulfation methods were evaluated, and the properties of desulfated CNCs were compared to those of unsulfated CNCs, prepared by HCl hydrolysis. H(2)SO(4)-hydrolyzed CNCs from softwood sulfite pulp were subjected to either a mild acid hydrolytic desulfation or a solvolytic desulfation in dimethyl sulfoxide via the pyridinium salt. Removal of the sulfate groups was confirmed by conductometric titration and X-ray photoelectron spectroscopy. The effect of the desulfation procedure on the lateral crystallite dimensions was analyzed by X-ray diffraction. The extent of particle aggregation in the samples was assessed by atomic force microscopy and dynamic light scattering. The acid hydrolytic method achieved only partial desulfation and produced gradually decreasing sulfate contents upon successive repetition. The solvolytic method achieved nearly complete desulfation in a single step. The desulfated CNCs showed similar particle aggregation as the HCl-hydrolyzed CNCs, but the extent of aggregation was slightly less.

Fate of aflatoxin B1 in contaminated corn gluten during acid hydrolysis

Aflatoxins are a group of mycotoxins that cause serious chronic disease outbreaks and contaminate several food products such as corn and its by-product, corn gluten. The aim of the current study was to evaluate the effect of hydrochloric acid (HCl) on aflatoxin B(1) (AFB(1) ) degradation in contaminated corn gluten under different HCl concentrations, hydrolysis temperatures and hydrolysis times. During the wet milling process the highest AFB(1) level (45.68 µg kg(-1) ) (37.86%) was found in corn gluten fraction. Treatment with 1 mol L(-1) HCL at 110 °C resulted in degradation of AFB(1) by 27.6% (33.07 µg kg(-1) ) after 4 h and reached 42.5% (26.26 µg kg(-1) ) after 8 h. Increasing HCl concentration from 1 to 3 mol L(-1) HCl resulted in increased degradation of AFB(1) , while complete degradation occurred in the presence of 5 mol L(-1) HCl after 4 h at 110 °C. Meanwhile, half-life time of AFB(1) was recorded after 2 h at 100 °C and was < 2 h at 110 °C in the presence of 3 mol L(-1) HCl. It could be demonstrated that the manufacture of hydrolyzed vegetable protein is a suitable method for decontamination of aflatoxin in highly contaminated grains, especially gluten fractions. The hydrolysis reaction could be considered in terms of first-order reaction kinetics of AFB(1) degradation.

Method development and optimization for isoflavone quantification in dry soy extract containing products

Isoflavones are secondary metabolites belonging to flavonoids found in a variety of plants, especially soy (Glycine max L.). Due to the estrogen-like activity of these compounds several soy products are on the market and applied to relieve menopausal symptoms. The aim of our study was to develop a simple method for the determination of isoflavones in soy extract containing products. The main isoflavones of soybean are genistin, glycitin, daidzin and their respective acetyl, malonyl and aglycone forms. Quantitative measurement of each individual isoflavone is difficult because some of the reference compounds are not widely commercially available [1]. Keeping in mind that the biological effects of soy isoflavones depend upon aglycone form, hydrolysis of glycosides is a practical method for the quantitative determination of total isoflavones and assessment of biological value of extracts [2]. The objective of our investigation was to establish an acid hydrolysis method for the quantitative HPLC determination of total isoflavones including daidzein, genistein and glycitein. To optimize the extraction and hydrolysis of isoflavones, the effect of HCl concentration (1.5–6 N), hydrolysis time (25–210min) and temperature (30–100°C) on total isoflavone aglycone content was studied using an RP-HPLC-DAD method. By mathematical fitting and optimization methods optimum hydrolysis conditions for maximizing the quantification of isoflavones were determined (t=94min, cHCl=5.09 N, T=80°C). The experimentally verified model has a good coefficient of R2 (0,9928) and the recovery of isoflavones was 97.81–102.76%. The developed method allows a reliable and maximum determination of isoflavones in dry soy extract containing products.

Characterization of HKI-272 Covalent Binding to Human Serum Albumin

The study was initiated as an observation of incomplete extraction recovery of N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide (HKI-272) from human plasma. The objective of this study was to 1) identify the binding site(s) of HKI-272 to human plasma protein(s); 2) characterize the nature of the binding; and 3) evaluate the potential reversibility of the covalent binding. After incubation of [(14)C]HKI-272 with human plasma, the mixture was directly injected on liquid chromatography/mass spectrometry (LC/MS), and an intact molecular mass of HKI-272 human serum albumin (HSA) adduct was determined to be 66,999 Da, which is 556 Da (molecular mass of HKI-272) larger than the measured molecular mass of HSA (66,443 Da). For peptide mapping, the incubation mixture was separated with SDS-polyacrylamide gel electrophoresis followed by tryptic digestion combined with LC/tandem MS. A radioactive peptide fragment, LDELRDEGKASSAK [amino acid (AA) residue 182-195 of albumin], was confirmed to covalently bind to HKI-272. In addition, after HCl hydrolysis, a radioactive HKI-272-lysine adduct was identified by LC/MS. After combining the results of tryptic digestion and HCl hydrolysis, the AA residue of Lys190 of HSA was confirmed to covalently bind to HKI-272. A standard HKI-272-lysine was synthesized and characterized by NMR. The data showed that the adduct was formed via Michael addition with the epsilon-amine of lysine attacking to the beta-carbon of the amide moiety of HKI-272. Furthermore, reversibility of the covalent binding of HKI-272 to HSA was shown when a gradual release of HKI-272 was observed from protein pellet of HKI-272-treated human plasma after resuspension in phosphate buffer, pH 7.4, at 37 degrees C for 18 h.