Combination Of EDTA Research Articles

Preventing in vitro lipoperoxidation in the malondialdehyde-thiobarbituric assay.

The malondialdehyde-thiobarbituric acid assay is widely used to study lipid peroxidation. Among the various methods used to perform the assay, the most widely accepted is the quantification of malondialdehyde using the thiobarbituric acid reaction, followed by reversed-phase chromatography. However, unacceptable results may be obtained as malondialdehyde can be produced in vitro. To study the conditions that inhibit in vitro lipid peroxidation, malondialdehyde levels were measured in cultured cells using different concentrations of butylated hydroxytoluene, EDTA or a combination of both. Butylated hydroxytoluene alone inhibits in vitro lipid peroxidation effectively. EDTA reduces artificially produced malondialdehyde, but not totally. Finally, the combination of EDTA and butylated hydroxytoluene does not improve the results obtained using butylated hydroxytoluene alone. The conclusion is that in the malondialdehyde-thiobarbituric acid assay it is necessary to add an inhibitor of the in vitro lipid peroxidation and assay the necessary concentration depending on the specimen used.

Effect of additives on the flow-analysis determination of weak-acid-dissociable and total cyanide.

The effect of reductants, complexants, and nitrite eliminators on the flow-analysis determination of weak-acid-dissociable and total cyanide has been studied for: 1. cyanide recovery from copper, nickel, and iron complexes; 2. cyanide generation from the reagents in the presence of common interferents; and 3. cyanide consumption by the reagents in the presence of those interferents. In the absence of additives the UV-assisted recovery of (total) cyanide from the iron complexes (using a succinate buffer) was insufficient. Arsenite and hypophosphite had no measurable effect on the recovery, ascorbic acid resulted in total recovery but under these conditions nitrite and sulfite seemed to destroy cyanide. Phenanthroline promoted the recovery of cyanide from iron complexes but led to formation of cyanide from thiocyanate. Citrate resulted in good recovery but in the presence of nitrite cyanide was formed; the recovery with EDTA was also good. It proved necessary to destroy nitrite by use of sulfamic acid. If a combination of EDTA, citrate, and sulfamic acid is used rather high concentrations of thiocyanate, nitrite, thiosulfate, and sulfite can be tolerated in the samples. It is strongly advisable to test modifications of the cyanide determination comprehensively, because some surprising results have been obtained.

The Role of Zinc Binding in the Biological Activity of Botulinum Toxin

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.

A transition metal enhanced luminol chemiluminescence in the presence of a chelator

We have investigated the chemiluminescence signal of luminol and hydrogen peroxide in the presence of a transition metal (Co(II), Cu(I), Fe(II), Fe(III)) and of a chelator (EDTA, citric acid) in pH 8.5, 9 and 10 borate buffer solutions. We observed that the chemiluminescence intensities of these systems reached a plateau, where they remained stable for a period of 2–30 s. We also observed linearity between the intensity of chemiluminescence and the hydrogen peroxide concentration. The combination of Co(II) and EDTA at pH 9 was found to give the optimum signal with reference to time stability, intensity and reproducibility. Thus, compared to previous chemiluminescence applications, the present results permit us to propose a simple, enzyme-free and time-independent technique for the detection and quantification of hydrogen peroxide.

Lipid peroxidation in the presence of iron oxides

Exposure to iron oxides dusts may lead to lung cancers among exposed population. To evaluate the involvement of iron‐containing particles in lipid peroxidation by production of reactive oxygen species, hematite, magnetite (iron oxides), and crocidolite (asbestos compound) were tested. The peroxidation was followed by the evaluation of some degradation products of lino‐lenic acid. In a buffered medium, magnetite is higher active than hematite. The addition of an iron chelator (EDTA) or of a reducing agent (ascorbate) in this medium enhances the activity of hematite and magnetite, and the combination of both EDTA and ascorbate increases their activity. Crocidolite is the most active whatever the medium. The appearance of the EDTA‐iron‐oxygen complexes related to the reactivity of these oxides is postulated. These results suggest that the oxidizing activity triggered by hematite and magnetite, in a medium containing an iron chelator and a reducing agent, may lead to damages in biological medium.

Effect of trehalose and edta on cryoprotective action of ram semen diluents

To obtain better ram semen extenders for artificial insemination (AI), we developed effective trehalose-containing hypertonic diluents. The cryoprotective action of trehalose has been explained by its dehydrating activity and interaction with cell membranes. Accordingly, we tested the cryopreserving capacity of different combinations of a Salamon's modified plus trehalose extender with EDTA. Evaluations were based on the percentage of motile spermatozoa and acrosome integrity, measured after thawing and after a 4-h post-thaw resistance test at 37 °C. We conclude that the combination of trehalose plus EDTA confers the highest cryopreserving activity tested, not only for freeze-thawing but also for post-thawing resistance, possibly by removing calcium from the medium thereby preventing cation competition with trehalose for membrane-binding sites.

Amendment Optimization to Enhance Lead Extractability from Contaminated Soils for Phytoremediation

ABSTRACT Eight lead-contaminated soils and one background soil artificially contaminated with several lead compounds were examined to determine the factors that limit lead extractability and thus plant availability during phytoremediation, as lead must be in soluble form for plant uptake to occur. The effect of the chemical form of the lead as well as the association of the lead among the different soil chemical fractions on lead extractability was specifically addressed. Results indicate that all the added lead forms tested except PbCrO4 were readily extracted and believed to be available for plant uptake, operationally defined as EDTA-extractable lead, as EDTA is the primary soil amendment for phytoremediation of lead-contaminated soils. Sequential extraction of the eight lead-contaminated soils that previously had been extracted with EDTA shows that the EDTA-extractable or plant available lead corresponds to mainly the exchangeable and carbonate fractions of each soil. Lead associated with oxide, organic, and residual fractions were less effectively targeted and solubilized by EDTA and therefore are not as readily available for plant uptake. Attempts to increase the available pool of soluble lead included the combination of EDTA with organic acids, reducing agents, and surfactants. Results from these studies indicate that high concentrations or extremely low pH conditions are required to enhance the plant available pool of lead by the organic acids and reducing agents. Surfactants, particularly caprylic acid in combination with 0.25 mM EDTA, were shown to be as effective as 0.50 mM EDTA alone. An amendment formulation combining less EDTA with surfactants is attractive for phytoremediation because of the biodegradability and cost concerns commonly associated with using larger amounts of EDTA.

Shaping and cleaning the root canal system: A scanning electron microscopic evaluation of a new instrumentation and irrigation technique

The purpose of the present scanning electron microscopic study was to investigate the efficacy of a combination of EDTA, NaOCl, and surface-active irrigating solutions during and after root canal preparation with ProFile nickel-titanium rotary instruments. Thirty canals were divided into three groups, instrumented and irrigated as follows: 5% NaOCl and a final flush with 17% EDTA were used for group A; group B specimens were irrigated using 17% EDTA, followed 15 s later by 1% TRITON X-100 (tensioactive agent) and then by 5% NaOCl; and group C specimens were irrigated with the same combination, but once shaping procedures were completed the irrigating sequence was repeated three times. After scanning electron microscopic evaluation, group C specimens exhibited the most effective debridement of the root canals. Results showed that tensioactive agent contributed to enhanced debridement. Cleaning was significantly improved once shaping procedures were completed.

Possible Mechanism for in Vitro Complement Activation in Blood and Plasma Samples: Futhan/EDTA Controls in Vitro Complement Activation

Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. Biotrak(TM) RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 degrees C. In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22-23 degrees C) for 1 h and in plasma held for 24 h at 4 degrees C or -70 degrees C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

Differential Sensitivity of Chromium-Mediated DNA Interstrand Crosslinks and DNA-Protein Crosslinks to Disruption by Alkali and EDTA

Some compounds of hexavalent chromium are well-established carcinogens. Chromium enters mammalian cells in the hexavalent form and is reduced to chromium(III). Treatment of purified DNA with chromium(III) produces DNA-DNA inter-strand crosslinks (DDC) which obstruct the progression of DNA polymerases in vitro. DDC were also detected in chromate-treated cultured normal human lung cells using the renaturing agarose gel electrophoresis (RAGE) assay and correlated with base-specific inhibition of DNA replication. Curiously, DDC have gone undetected in studies of cultured cells using the alkaline elution (AE) technique, whereas chromium-mediated DNA-protein crosslinks (DPC) were readily detected by AE. We tested the hypothesis that AE conditions [60 mM tetraethyl ammonium hydroxide (TEA), 20 mM EDTA, pH 12.6, for 16 h at room temperature] dissociate DDC but not DPC using chromium(III)-treated plasmid DNA and the RAGE assay. Dose-dependent chromium-induced DDC were unaffected by TEA (pH 11.8) alone or by more rigorous alkaline denaturation conditions (200 mM NaOH, pH 13.5, for 16 h). DDC were, however, completely disrupted by EDTA (pH 12.6) alone or the combination of TEA and EDTA (pH 12.6). In contrast, DPC remained largely intact under these conditions. Therefore, past AE-based studies which have failed to detect chromium-induced DDC do not prove the absence of this lesion. AE may not be suitable for detecting DDC induced by EDTA-chelatable agents such as metals.

Stabilization of methionine enkephalin in various rabbit mucosal extracts by enzyme inhibitors

The inhibition of the enzymatic degradation of methionine enkephalin (Met-Enk) was investigated kinetically in nasal, rectal, and vaginal extracts of rabbits with and without inhibitors, such as puromycin (PM), amastatin (AM), thiorphan (TP), Na 2EDTA, and thimerosal (TM), alone or in combination, by analyzing the parent peptide and its hydrolytic fragments by HPLC. The effects of variation of pH in the nasal extracts, and the addition of 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CyD) on the stabilization of Met-Enk were also studied. The degradation of Met-Enk was found to be fastest at around pH 7, indicating that the activity of enkephalin-degrading enzymes is optimal at this pH. Addition of 2-HP-β-CyD (10%) to the nasal, rectal, and vaginal extracts was noted to reduce the first-order degradation rate constants for Met-Enk by 2.5-2.8-fold, compared to the control. AM alone inhibited the enzymatic degradation of Met-Enk with IC 50 values of 3.5 and 0.22 μM for the rectal and vaginal extracts, respectively, whereas PM was found to be approx. 14.2- and 26.8-fold less potent than AM, respectively. The effects of both aminopeptidase inhibitors in the nasal extracts were smaller. Even at 50 μM, TP (a potent enkephalinase A inhibitor) alone revealed only a small increase of Met-Enk stability in the various mucosal extracts, however, EDTA (5 μM) was observed to inhibit enzymatic hydrolysis considerably by blocking both enkephalinase A and B and, to some extent, aminopeptidase. On the other hand, TM (0.05%) was found to be a new and potent inhibitor for enkephalinase B and aminopeptidases, which was more potent than AM (50 μM) in inhibiting the degradation of Met-Enk in various mucosal extracts. Furthermore, the addition of TM (0.01%) to a combination of AM (50 μM) and EDTA (5 μM) was observed to protect Met-Enk from enzymatic degradation in nasal, rectal, and vaginal extracts by more than 90%, after 24 h of incubation, by inhibiting almost completely all the enkephalin-degrading enzymes present in the incubation mixtures.

Enzymatic Browning Control in Minimally Processed Mushrooms

ABSTRACTTreatments to control discoloration of minimally processed mushrooms were investigated. Whole or sliced mushrooms were immersed in browning inhibitor solutions and evaluated for color change during storage. Browning was more intense in first break mushrooms than in second, and in unwashed mushrooms compared to washed. However, washing sometimes induced purple discolorations, associated with bacterial lesions. Other discolorations were induced by hypochlorite, 4‐hexylresor‐cinol, and acidic dips. The most effective treatment was a combination of sodium erythorbate, cysteine, and EDTA at pH 5.5. Addition of preservatives to browning inhibitor dips did not improve storage life. However, dipping in 5% hydrogen peroxide prior to application of browning inhibitors significantly increased shelf‐life.

Transmucosal Delivery of Leucine Enkephalin: Stabilization in Rabbit Enzyme Extracts and Enhancement of Permeation through Mucosae

Leucine enkephalin (Tyr-Gly-Gly-Phe-Leu; Leu-Enk) is a naturally occurring peptide that has been shown to have pain modulating properties. To evaluate the feasibility of using various absorptive mucosae as a route of systemic delivery, the stability of Leu-Enk and the effect of enzyme inhibitors (e.g., amastatin, EDTA, and thimerosal) on stabilization and permeation of Leu-Enk through rabbit mucosae in the presence of dihydrofusidates were investigated. Enzymes in the nasal, rectal, and vaginal mucosae were extracted and Leu-Enk (50 µg/mL) was added to each of the enzyme extracts and incubated to determine the kinetics and mechanism of degradation. The rate of degradation in the extracts in the absence of inhibitors followed the order: rectal > vaginal > nasal.Whereas EDTA had the best stabilizing effect on Leu-Enk, thimerosal was the best stabilizer for the degradation intermediates. A combination of amastatin (50 µM), EDTA (5 mM), and thimerosal (50 µM) had the greatest stabilizing effect on Leu-Enk and its degradation intermediates. For permeation studies, each mucosa was mounted onto a Valia-Chien permeation cell with Leu-Enk (200 µg/mL) in isotonic phosphate buffer (as donor solution). The enhancers used for the study were sodium tauro-dihydrofusidate (STDHF), sodium glycodihydrofusidate (SGDHF), and phosphato-dihydrofusidate (PHDHF). The greatest effect was achieved by PHDHF for all the mucosae. STDHF had a significant effect only on the rectal permeation, whereas SGDHF had significant effects on rectal and vaginal mucosae. Mechanisms by which the dihydrofusidates enhance permeation may involve micelle formation. Thus, the use of enzyme inhibitors and dihydrofusidates in combination has made transmucosal delivery of Leu-Enk a viable option.

Strawberry juice colour: The effect of sulphur dioxide and edta on the stability of anthocyanins

AbstractThe loss of anthocyanins and the formation of colour due to polymeric pigments in juices and purées prepared from strawberries (variety Senga sengana) was studied during storage at — 20°C and + 20°C. Four different treatments were given to the samples immediately after milling: no additions, EDTA addition, sulphur dioxide addition, and a combination of sulphur dioxide and EDTA. The concentration of anthocyanins and the proportion of colour due to polymeric pigments were measured by high‐performance liquid chromatography and spectrophotometry. Losses in anthocyanins were very small during 10 weeks storage at –20°C and there was no effect of the treatment. The addition of sulphur dioxide slowed down the loss of anthocyanins at + 20°C, especially in the purées and correspondingly decreased the rate of polymeric pigment formation. Addition of EDTA had only a slight effect on colour stability.

Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis

We have isolated and characterized two minor extracellular proteases from culture supernatants of a strain of Bacillus subtilis containing deletion mutations of the genes for the extracellular proteases subtilisin (apr) and neutral protease (npr) and a minor extracellular protease (epr) as well as intracellular serine protease-I (isp-1). Characterization studies have revealed that one of these enzymes is the previously described protease bacillopeptidase F. The second enzyme, the subject of this report, is a novel metalloprotease, which we designate Mpr. Mpr is a unique metalloprotease that has been purified to apparent homogeneity by using both conventional and high-performance liquid chromatography procedures. Mpr has a molecular mass of approximately 28 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a basic isoelectric point of 8.7. The enzyme showed maximal activity against azocoll at pH 7.5 and 50 degrees C. Mpr was inhibited by dithiothreitol and a combination of beta-mercaptoethanol and EDTA. Activity was moderately inhibited by beta-mercaptoethanol and EDTA alone as well as by cysteine and citrate and only marginally by phosphoramidon 1,10-phenanthroline and N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine. Mpr was not inhibited by phenylmethylsulfonyl fluoride. In addition, Mpr showed esterolytic but not collagenolytic activities. Our studies suggest that Mpr is a secreted metalloprotease containing cysteine residues that are required for maximal activity.

A scanning electron microscopic evaluation of four root canal irrigation regimens

A scanning electron microscope was used to evaluatethe debridement capabilities of four irrigation regimens on both instrumented and uninstrumented root canal surfaces. A typical smear layer was seen on the instrumented surfaces of specimens irrigated with saline and NaOCl. EDTA demineralized much of the smear layer from the instrumented surfaces and exposed the orifices of some of the underlying dentinal tubules. NaOCl removed all pulpal remnants and predentin from the uninstrumented surfaces of the root canal while EDTA and saline left pulpal remnants and predentin on the uninstrumented surfaces. The combination of NaOCl and EDTA used alternately completely removed the smear layer from the instrumented root canal surfaces as well as the pulpal remnants and predentin from the uninstrumented surfaces. In addition, the combination of NaOCl and EDTA caused the exposed calcospherites on the uninstrumented surfaces to have an eroded appearance.

Release of immune complexes bound to CR1 on erythrocytes; suramin inhibits factor I in the presence of EDTA.

An experimental model was established in order to study the release of immune complexes (IC) bound by complement C3b receptors (CR1) on human erythrocytes (RBC). Soluble tetanus toxoid anti-tetanus toxoid complexes were incubated with RBC in the presence of autologous serum at optimal conditions for binding. The RBC carrying complement-opsonized complexes were incubated with appropriate serum reagents, and it was shown that factor I was required for release of the complexes, which occurred without loss of CR1. Suramin was, irrespective of factor I, found to induce release of CR1-bound IC in the absence of EDTA, whereas factor I-mediated release was inhibited by suramin in the presence of EDTA. EDTA probably interfered through a charge-dependent interaction. These observations are decisive for the interpretation of in vitro experiments involving these reagents. The combination of EDTA and suramin was found inappropriate for use in quantitative determination of in vivo CR1-bound IC.

Quality Improvement of Canned Mung Bean (Vigna radiafa) Sprouts

ABSTRACTThe effect of calcium, ascorbic acid, citric acid and EDTA on texture, color and flavor of mung bean sprouts canned as a low‐acid food (pH > 4.6) and the effect of acidification by acetic, citric, gluconic, lactic and malic acids on quality of mung bean sprouts canned as an acid‐food (pH < 4.6) were examined. Either the addition of calcium or acidification enhanced texture. Texture of products canned as low‐acid food increased with decreasing pH. Color was markedly improved by either EDTA combined with ascorbic acid or acidification. Processing in plain (uncoated) cans or treatment with acetic acid was detrimental to flavor. Overall, the best low‐acid food product was obtained by the combination of calcium, ascorbic acid, citric acid and EDTA while the best acid‐food product was obtained by acidification with gluconic acid.

Radioimmunoassay of angiotensin II in unextracted plasma

A reliable, direct radioimmunoassay of plasma angiotensin II (PAII) is described. No cross-reactivity of human renin, sheep renin substrate, saralasin acetate and angiotensin I with the anti-angiotensin II antiserum was found. However, des- 1Asp-angiotensin II has a stronger binding affinity for the antiserum than angiotensin II. A significant correlation was found between PAII determined in plasma samples without and with extraction on a Dowex 50W-X 2 resin. The combination of EDTA and o-phenanthroline has been found to be a very efficient angiotensinase inhibitor in plasma and is of equal potency as EDTA and diisopropylfluorophosphate in inhibiting the angiotensinase and converting enzyme activity. A significant correlation was found between PAII measurements performed in plasma samples in the presence of both inhibitor solutions. The intra-assay variations were checked by multiple analysis of a plasma pool and the inter-assay variations by measuring the angiotensin II concentration in different plasma samples on two occasions. The mean PAII in 10 healthy, male subjects on an ad libitum diet was 25.1 ± 13.5 (S.D.) pg/ml (range: 10.1 to 51.4). PAII correlated also better with plasma renin activity (active renin) than with plasma renin concentrations, measured after acidification of plasma (total renin).

Ultrastructural study of Salmonella typhimurium treated with membrane-active agents: specific reaction dansylchloride with cell envelope components.

Amino groups of cell envelope proteins, lipids, and lipopolysaccharides cannot be labeled in intact cells of Salmonella typhimurium G 30 by using 5-dimethylaminonaphthalene-1-sulfonylchloride incorporated in lecithin-cholesterol vesicles. However, application of membrane-interacting agents like tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, ethylenediaminetetraacetate (Na salt) (EDTA), divalent cations, and sublethal doses of the cationic antibacterial agents polymyxin B and chlorhexidine induced specific fluorescent labeling of envelope proteins and lipids but not of cytoplasmic compounds, with the exception of a soluble protein with a molecular weight of 46,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with Tris-hydrochloride buffer produced labeling of the heat-modifiable protein B/B(+) and of proteins with molecular weights of 26,000, 22,000, and below 17,000. A combination of Tris-hydrochloride and EDTA induced additional dansylation of the major protein A and of proteins of molecular weights 80,000, 60,000, and 44,000. Polymyxin B and chlorhexidine caused similar labeling patterns. In every case, except with divalent cation treatment, protein B/B(+) was the most prominently labeled species. Phosphatidylethanolamine was dansylated up to 30%. Lipopolysaccharide was not reactive under any condition or treatment. In addition, the peptidoglycan-bound lipoprotein did not react with dansylchloride in either intact or Tris-hydrochloride-treated cells. The results are discussed with regard to a possible localization of labeled and unlabeled compounds of the cell envelope on the basis of a model placing cell envelope amino groups into ion-ion interactions with anionic components of other envelope compounds like phosphate and carboxyl groups.