Trichloroacetic Acid Supernatant Research Articles

Protein–Incorporated Serum Total Antioxidant Capacity Measurement by a Modified CUPRAC (CUPRIC Reducing Antioxidant Capacity) Method

Due to inadequacies in analytical methodology of total antioxidant capacity (TAC) measurement, proteins are initially separated from the human serum matrix by precipitation and are left unmeasured, thereby causing an important “antioxidant gap.” The aim of this work is to measure the TAC of serum with the modified CUPric Reducing Antioxidant Capacity (CUPRAC) method, and to identify the contribution of serum proteins, especially thiol-containing proteins, to TAC. CUPRAC results were statistically compared to those found by reference methods, namely ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power), and Ellman thiols assay. The curves of absorbance vs thiol concentration, as well as of absorbance vs diluted serum (whole serum, trichloroacetic acid (TCA)−precipitated and redissolved serum protein solution, and TCA supernatant fractions) volume, of three distinct serum samples showed excellent linearity and low intercept values only with the modified CUPRAC method. The proposed method will help characterize the “antioxidant gap” of serum TAC originating from protein components which should not be neglected in future antioxidant measurements.

Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.15 to 3mg of N/mL of inoculum) and incubated with 10mL of ruminal inoculum and 5mL of buffer; fermentations were stopped after 2h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer-soluble N and buffer extracts were treated with TCA to determine N degraded at t=0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA; OPA-F), and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was computed from extent of release of 1) ammonia plus free TAA or 2) ammonia plus free TAA and peptides. Rate of degradation (kd) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters Km (Michaelis constant) and kd (Vmax/Km), where Vmax=maximum velocity, were estimated directly; kd values were adjusted (Akd) for the fraction FD0 using the equation Akd=kd − FD0/2. The OPA-C assay yielded faster degradation rates due to the contribution of peptides to the fraction degraded (overall mean=0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were more rapid than rates reported by the National Research Council (NRC). Both assays indicated that the 2 SSBM differed in rumen-undegradable protein (RUP) content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by the NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value. Preliminary studies with 4 additional protein concentrates confirmed that accounting for peptide formation increased degradation rate; however, a trend for an interaction between assay and protein source suggested that peptide release made a smaller contribution to rate for more slowly degraded proteins. The OPA-C assay is a simple and reliable method to quantify formation of small peptides.

Characterisation and quantification of the reaction(s) catalysed by transglutaminase using the o-phthaldialdehyde reagent.

The potential application of the o-phthaldialdehyde (OPA) reagent for quantification of the type and extent of the reaction(s) catalysed by transglutaminase (TGase) during incubation with sodium caseinate (NaCN) was investigated. Initial studies were performed to ensure that NH3, a by-product of TGase activity, could be determined with the OPA reagent in trichloroacetic acid (TCA) supernatants of NaCN solutions. The detectable concentration of exogenously added NH3 (at NH3 concentrations > 10 mM) was found to decrease during extended incubation at 23, 37 and 50 degrees C and at either pH 7.0 or 8.0 in 4% w/v NaCN solutions, even when taking into account the evaporation of water from the sample. The recovery of NH3 from 12% w/v TCA supernatants of NaCN solutions spiked with 5 mm NH3 at 23 degrees C and pH 7.0 was found to be 88%. The release of NH3 and the decrease in epsilon-amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23 degrees C resulted in progressive increases and decreases, respectively, in NH3 and -amino group concentration with increased incubation time. These changes were dependent on TGase:NaCN. It was estimated that approximately 20% of the available Lys residues in NaCN were involved in TGase-catalysed cross-links. However, the observed decrease in epsilon-amino group concentration was higher than expected. This may be due to concealment of noncross-linked amino groups in polymerised NaCN, making them unavailable for reaction with the OPA reagent.

Evidence that both receptor- and heparan sulfate proteoglycan-bound basic fibroblast growth factor are internalized by cultured immature Leydig cells

The present studies examined how 125I-labeled basic fibroblast growth factor (bFGF) bound to high affinity receptors and with lower affinity to heparan sulfate proteoglycans (HSPG) of cultured immature rat Leydig cells was processed. Following incubation for 2 h at 4°C with 125I-bFGF, cells were washed to remove unbound radioactivity. Fresh medium was added, and cells were incubated at 4° and/or 37°C. At time zero and at specific intervals over the next 6 h, the incubation medium was saved and cells washed to quantitate 125I-bFGF released into the medium, associated with HSPG of the cell surface or extracellular matrix (radioactivity released by washing cells with 2 M NaCl, pH 7.4), associated with cell surface receptors (radioactivity released by washing cells with 2 M NaCl, pH 4.0) or internalized (radioactivity resistant to high salt and acid washes, and solubilized with 0.5 M NaOH). Radioactivity released into the initial medium and the pooled washes was further divided into a trichloroacetic acid (TCA)-precipitated form (radioactivity precipitated by 10% TCA) and a TCA-soluble form (radioactivity remaining in the TCA supernatant). 125I-bFGF associated with both HSPG and surface receptors declined progressively during the first 4 h of incubation before stabilizing when cells were transferred to 37°C. These declines were associated with a corresponding increase in intracellular 125I-bFGF. These changes were blocked by maintaining cells at 4°C. The majority of internalized 125I-bFGF appeared to originate from the HSPG-bound fraction as there was a greater decline in HSPG-associated radioactivity and most of the increase in internalized radioactivity could be blocked by the inclusion of 10 μg/ml heparin (which mainly blocks 125I-bFGF binding to HSPG but not to high affinity receptors) during the initial incubation with 125I-bFGF for 2 h at 4°C. Furthermore, HSPG-mediated internalization appeared to have two components: the major fraction was blocked by the inclusion of 10 μg/ml heparin, while a heparin-resistant fraction, appeared to be closely linked both quantitatively and temporarily to receptor-mediated internalization. A minor fraction of internalized 125I-bFGF was metabolized in lysosomes, as the inclusion of 50 μM chloroquine during the 6 h incubation at 37°C inhibited most of the increase in TCA-soluble radioactivity appearing in the incubation medium. Exposure of cultured cells to increasing concentrations of bFGF (0.1–10 ng/ml) for ~ 16 h decreased bFGF receptor levels in a dose-dependent manner, suggesting that internalization, in part, is associated with down-regulation of bFGF receptors.

Effects of cyclic adenosine 3′,5′-monophosphate on amino acid transport and incorporation into protein in the rat aorta

To assess the effects of cyclic AMP on amino acid transport and incorporation into aortic tissue protein, rat aortic rings were incubated with the cyclic AMP analog, N6-monobutyryl cyclic AMP (MBcAMP), the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX), and radiolabeled amino acids. Subsequently, the aortic rings were homogenized in 5% trichloroacetic acid (TCA) and processed for liquid scintillation counting. Radioactivity present in the TCA supernatant following centrifugation was used to estimate amino acid transport. TCA-precipitable radioactivity was used as a measure of amino acid incorporation into protein. MBcAMP induced an increase in the uptake of [3H]alpha-aminoisobutyric acid into aortic rings and an increase in the incorporation of radiolabeled proline and leucine into TCA-precipitable protein. Similar effects were observed with low concentrations of MIX (0.025-0.25 mM); however, at higher concentrations of MIX, there was an attenuation of the effect or frank inhibition. Maximum stimulation of transport was observed within 90-120 min of the addition of MIX or MBcAMP to the incubation medium, whereas the effect on amino acid incorporation was not detectable until after 12 h of exposure to MIX or MBcAMP. The effects of cyclic AMP on transport were observed in both the tunica media and the tunica adventitia, whereas the effects on amino acid incorporation into protein were observed only in the tunica media. These data are consistent with a possible role for cyclic AMP in promoting changes in the tunica media that could lead to the development of vascular hypertrophy.

Bioreactivity of intratracheally administered slate dust in rats: Incorporation of 14C‐acetate into lung lipids

The effect of intratracheally instilled slate dust on the phospholipid profile, and 14C-acetate incorporation into the lipids of lung lavage, whole lung tissue and its subcellular fractions, has been studied in rats. The acellular fraction of lung lavage showed a decrease in the phospholipid content at 4 days and then an increase at 40 days of dust exposure, whereas the cellular fraction showed the reverse. The order of 14C-acetate incorporation into total lipids and individual phospholipids showed a parallel trend. The rate of incorporation with total lipids of lung tissue was higher at the two stages of dust exposure and a similar pattern prevailed in the subcellular fractions, i.e. mitochondrial, microsomal and cytosolic fraction. Acetate incorporation was highest in mitochondria, followed by the microsomes. An increase in the microsomal and mitochondrial cholesterol levels was also observed. There was no significant change in the solvent-extracted 14C-counts of whole plasma, trichloroacetic acid (TCA) precipitate and TCA supernatant of plasma. The results indicate that slate dust causes an enhanced synthesis of pulmonary surfactant and other lung lipids and, therefore, has an effect on the metabolism of type II alveolar epithelial cells.

Inhibition of polyamine biosynthesis by dicyclohexylamine in cultured cotyledons of Pinus radiata

Biondi, S., Torrigiani, P., Sansovini, A. and Bagni, N. 1988. Inhibition of polyamine biosynthesis by dicyclohexylamine in cultured cotyledons of Pinus radiata. ‐ Physiol. Plant. 72: 471–476.The effect of 1 mAf dicyclohexylamine (DCHA) on the synthesis of spermidine and spermine was examined in excised cotyledons of radiata pine (Pinus radiata D. Don) cultured under shoot‐forming (with cytokinin) and non‐shoot‐forming (minus cytokinin) conditions by incubation with [14C]‐putrescine. In control cotyledons incorporation into spermidine showed a peak at day 2 in the presence and at day 5 in the absence of N6‐benzyldenine (BA). DCHA‐treated cotyledons gave the same labeling pattern, both in the presence and absence of benzyladenine, with a much smaller peak at day 2. The incorporation into spermidine and spermine was insignificant at day 5 and later. The total radioactivity in the trichloroacetic acid supernatant indicated that precursor uptake was strongly reduced by the drug. In addition, the percentage label found in the benzene phase and combined in the 3 polyamines was lower in DCHA‐treated cotyledons. Thus, treatment with DCHA not only inhibited the conversion from putrescine to spermidine and spermine, but also reduced its conversion to other benzene‐extractable compounds. S‐Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity, which furnishes the propylamine group to spermidine and spermine synthases (EC 2.5.1.16 and EC 2.5.1.‐), was not significantly affected by DCHA and appeared to be independent of the spermidine and spermine synthase reactions, suggesting that spermine synthesis decreased as a result of substrate depletion. The correlation between morphological development and polyamine biosynthesis is discussed.

Toasted soybean flour components with trypsin inhibitor activity

AbstractCompounds in toasted soybean flour having trypsin inhibitor (TI) activity were isolated and characterized. Sodium hydroxide (0.01N) extracts of toasted soybean flour had an average of 2.59 mg TI/g sample. These extracts, after trichloroacetic acid (TCA) precipitation and dialysis, yielded supernatant and precipitate fractions. Addition of polyvinylpyrrolidone to eliminate free tannins and phenolics in the extracts, which may lead to overestimation of TI activity, was unnecessary. Material balance studies revealed 91% protein recovery and 92% recovery of TI activity in the TCA supernatant (1.1% protein, 2.0% TI) and precipitate (89.8% protein, 90.0% TI) fractions. Column chromatography and electrophoresis showed the TCA supernatant and precipitate fractions to contain proteins, including those having TI activity. Kunitz type TI and Bowman‐Birk type protease inhibitors accounted for most residual TI activity of toasted soybean flour, as verified by column chromatography, isoelectric focusing, sodium dodecyl sulfate polyacrylamide electrophoresis, and size‐exclusion high performance liquid chromatography, using the two similarly treated protease inhibitors as standards. Immunoblotting was also used to detect and identify Kunitz type TI’s in toasted soybean meal extracts. This study established the proteinaceous nature of residual trypsin inhibitor activity in toasted soybean flour and the presence of both Kunitz and Bowman‐Birk inhibitors.

Plasma clearance and tissue uptake of native and desialylated equine gonadotropins

Equine luteinizing hormone (eLH), equine follicle stimulating hormone (eFSH) and equne chorionic gonadotropin (eCG) were desialylated and their plasma disappearance, tissue uptake and degradation determined. Both native hormones and their desialylated derivatives were radioiodinated and injected intravenously into male rats. Plasma samples were taken at different time intervals and examined for both total and trichloroacetic acid (TCA) precipitable radioactivity. Disappearance curves for each hormone consisted of two exponential components: a fast clearance followed by a slow phase. The apparent t 1 2 of the fast component for native eCG, eLH and eFSH were 18.3 min, 17.0 min and 17.1 min, respectively; this is in contrast to their desialylated derivatives which had a t 1 2 of 2.8 min, 4.9 min and 7.1 min, respectively. The slower component for native and desialylated eCG had at t 1 2 of 355.5 min and 171.2 respectively. After TCA precipitation more than 90 percent of the radioactivity in plasma was in the pellet, whereas in urine almost 95 percent of the radioactivity was in the TCA supernatant. Coincident with the disappearance of the hormone from the plasma its uptake and release was observed in liver, kidney, testis, and muscle tissue. Gel filtration data on plasma and urine samples indicated that the former had mostly intact hormone in contrast with the latter. Together these results indicate that eLH, eFSH and eCG disappear from the plasma of male rats in a biphasic manner with a rate slower than their desialylated derivatives.

Vitamin K-dependent carboxylase.

Vitamin K-dependent carboxylase.

Protein dependence of digoxin determination on the Abbott-TDX

The protein dependence of digoxin quantitation on the Abbott TDX was evaluated. An inverse relationship was found to exist between protein concentration and digoxin levels in samples that were deproteinized with 50 g/L trichloroacetic acid prior to assay. This was especially evident in the protein range from 30-60 g/L where a 30% decrease in digoxin concentration was observed. Recovery studies using 125I-digoxin showed that this decrease could be totally accounted for by a decreased recovery of digoxin in the trichloroacetic acid supernatant. The serum-based calibrators used to establish the standard curve on the instrument showed similar recoveries to serum samples. The reduced recovery does not pose accuracy problems as long as the protein concentrations of the patient samples and calibrators are similar. The clinical and operational implications of this observation are discussed.

Faecal chymotrypsin--a new photometric method using N-acetyl-L-tyrosine ethyl ester as substrate.

A photometric method for chymotrypsin is proposed, using the substrate N-acetyl-L-tyrosine ethyl ester, which is already used in the titrimetric procedure. Hydrolysis of the ester bond produces equal amounts of ethanol and acetyltyrosine, the latter being measured in the titrimetric method. The ethanol can easily be measured by the alcohol dehydrogenase method in the trichloroacetic acid supernatant. Suitable test conditions are reported.

The simultaneous quantitative determination of cephalothin and cefazolin in serum by high pressure liquid chromatography

Conventional microbiological assay procedures for cephalosporins in serum do not allow the determination of serum concentrations if more than one cephalosporin is present in a single sample. An HPLC procedure has been developed which permits the simultaneous quantitative determination of cefazolin sodium and cephalothin sodium in serum. Reverse phase chromatography using methanol in 0.2 M ammonium acetate as the mobile phase was employed to separate and quantitate the two cephalosporins in a trichloroacetic acid supernatant solution prepared from serum.

Modulation of the root effect in goldfish by ATP and GTP

Both ATP and GTP are present in considerable amount in red cells of the common goldfish Carassius auratus. They both influence the Root effect of the single major fish hemoglobin, but GTP is, depending on pH, 2–6 times more effective than ATP. The two triphosphates account for 3 4 of the effect of trichloroacetic acid supernatant obtained from hemolysate which contains still some compound(s) which can influence the shift of the Root effect toward higher pH.

Free Amino Acid Patterns and the Distribution of 14C from [U-14C]-L-Leucine, [U-14C]-L-Lysine, and [U-14C]-L-Alanine in the Tissues of Young Adult Rats

In studies designed to evaluate the role of the liver and muscle in modifying the plasma amino acid response, three levels of dietary amino acids, 3.6%, 4.8% and 6.0% were fed to three groups of young adult male rats for 2 weeks. After fasting, either [U-14C]-L-leucine, [U-14C]-L-lysine or [U-14C]-L-alanine was administered intragastrically with a portion of diet. After a 4.5 hour fast, rats were killed. Distribution of radioactivity was evaluated in trichloroacetic acid (TCA) supernatants and lipid-extracted precipitates of plasma, liver and gastrocnemius muscle, and in expired CO2. Free amino acids were measured and specific activities of amino acids were determined. Rats fed 6.0% or 4.8% amino acids lost less weight than those fed 3.6%. A high percentage of radioactivity from 14C-leucine and 14C-lysine was recovered in the lipid-extracted precipitate. TCA supernatants from rats fed 14C-leucine contained low levels of radioactivity. A large percentage (60%) of the radioactivity from 14C-alanine was expired in 14CO2. The percentages of alanine found in TCA precipitates were very low. Significant increases in concentration associated with increments in dietary amino acids occurred in plasma free isoleucine, leucine, valine and tyrosine; liver free histidine; and muscle free leucine and threonine. Concentrations of many amino acids were depressed in the muscle of rats fed 4.8% amino acids whereas they increased in response to 6.0%. Protein and free amino acid specific activities indicated no change in rates of protein synthesis. Data from this type of experiment may assist in interpreting the role of the liver and muscle in modifying the plasma amino acid response to dietary amino acids.

A comparative study of two procedures used in the determination of hepatic microsomal aniline hydroxylation

The hepatic microsomal parahydroxylation of aniline is measured by the determination of its product p-aminophenol (pAP). The pAP formed in incubation mixtures is assayed either after ether extraction or after trichloroacetic acid (TCA) precipitation. The TCA-precipitation method is simpler and less time-consuming than the ether-extraction method, but it has been found that the apparent recovery of pAP was lower in TCA supernatants than with the ether-extraction method. In the present work, it was found that the recovery of pAP by the TCA-precipitation method depended on the nature of NADPH-generating system used in the incubation mixture. When “soluble fraction” was used as a source of glucose-6-phosphate dehydrogenase for the reduction of NADP to NADPH, the recovery of pAP using the TCA-precipitation method was less than that with the ether-extraction method. But when “soluble fraction” was replaced by yeast glucose-6-phosphate dehydrogenase or by chemically prepared NADPH, the recovery of pAP was approximately equal with both the methods. Lowered recovery of pAP when added to the soluble fraction or its dialyzate might be due to the presence of sulfhydryl reacting groups. The addition of mercuric chloride to the soluble fraction dialyzate resulted in almost “full” recovery of pAP by the TCA-precipitation method, i.e., the apparent loss of pAP was prevented.

Reevaluation of the method of Kunitz for the assay of proteolytic activities in liver and brain homogenates

The assay of proteolytic activities in liver and brain homogenates by determining the change in absorbancy at 280 mμ in the trichloroacetic acid supernatants of incubation mixtures has been reevaluated. It has been shown that this assay is satisfactory for the study of proteolytic activities with a pH optimum in the acid range. On the other hand, it has been shown that this method does not measure the hydrolysis of protein at neutral pH values, but the hydrolysis of tissue RNA by tissue neutral ribonucleases when the blank is taken from the same incubation mixture at the beginning of the incubation period. In this case, the addition of ATP, coenzyme A, reduced gluthatione, and phosphocreatine to the system induced the release of ninhydrin positive material during the incubation of total homogenates with either hemoglobin or albumin, indicating the activation or the release of inhibition of the neutral proteolytic activity.

Isolation of threonyl adenylate-enzyme complex

The aminoacyl-RNA synthetases have been shown to carry out the following two-step reaction: 1. 1) amino acid + ATP + enzyme ⇌ aminoacyl-AMP-enzyme + PP i 2. 2) aminoacyl-AMP-enzyme + sRNA ⇌ aminoacyl-sRNA + AMP + enzyme Evidence for the formation of the intermediate aminoacyl-AMP-enzyme complex has been reported by several investigators ( Hoagland, 1955, De Moss and Novelli, 1955, Berg, 1956). Tryptophanyl adenylate (Karasek, et , al. , 1958) and seryl adenylate (Webster and Davie, 1961) were isolated in trichloroacetic acid supernatant fractions after incubation of amino acid and ATP with substrate amounts of the respective enzymes. The present communication describes the isolation of enzyme-bound threonyl adenylate and the capacity of this complex to transfer the threonine directly to sRNA.

Release of inorganic iodine from thyroid gland in initial phase of TSH action.

SummaryMeasurement of inorganic I131 and PBI131 in thyroid venous blood obtained by catheterization was performed in 17 dogs to study the early effect of TSH. Following a latent period of about 15 minutes, output of both inorganic I131 and PBI131 started to increase sharply. The increased output continued for about 10 hours, and thereafter the thyroid gland started to accumulate inorganic iodine. The inorganic I131 fraction, represented in the trichloroacetic acid supernatant fraction, was confirmed by radiopaperchromatography. TSH apparently releases inorganic iodine, in addition to PBI, from the thyroid in the early phase of its action.

Selective capacity of components of the aqueous extract of the electric organ to bind curarizing quaternary ammnium derivatives

1. 1. Trichloroacetic acid supernatants of aqueous extracts of the electric organ, when purified by acid alcohol precipitation or by column chromatography, show increased capacity for binding quarternary ammonium compounds. 2. 2. This increase in binding capacity is proportional to the concentration of acid mucopolysaccharides in the purified material. 3. 3. The results presented here suggest that the binding of quarternary ammonnium compounds in tissue extracts is mainly due to the presence of acid mucopolysaccharides. 4. 4. In vitro experiments with several types of acid mucopolysaccharides do not lead to the establishment of a specific type of mucopolysaccharide responsible for neuromuscular transmission. Several hypotheses are discussed in an attempt to elucidate the importance of those substances in neuromuscular transmission.