Chymotrypsin Inhibitory Activity Research Articles

Molecular cloning and functional analysis of a novel type of Bowman-Birk inhibitor gene family in rice.

Bowman-Birk inhibitor (BBI) genes encode serine protease inhibitors that have repetitive cysteine-rich domains with reactive sites for the trypsin or chymotrypsin family. We have identified seven BBI genes from japonica rice (Oryza sativa subsp. japonica var Teqing). All of the genes identified were found in a single cluster on the southern end of the long arm of rice chromosome 1. Four of the seven BBI genes have two repetitive cysteine-rich domains, whereas one has a truncated domain with only one reactive site. We have also identified three novel BBI genes, each of which possesses three repetitive domains instead of two. In situ hybridization analyses indicated that the accumulation of rice BBI transcripts was differentially regulated in germinating embryos and also in the leaves, roots, and flower organs at later developmental stages. Different members of the rice BBI gene family displayed different expression patterns during rice seed germination, and wounding induced the expression of rice BBI transcripts. The three-domain BBIs had higher expression levels than the two-domain BBIs. It was also found that the mRNA of rice BBI genes was present in abundant amounts in scutellar epithelium and aleurone layer cells. RBBI3-1, one of the three-domain RBBI, exhibited in vitro trypsin-inhibiting activity but no chymotrypsin-inhibiting activity. Overexpression of RBBI2-3 in transgenic rice plants resulted in resistance to the fungal pathogen Pyricularia oryzae, indicating that proteinase inhibitors confer resistance against the fungal pathogen in vivo and that they might play a role in the defense system of the rice plant.

Selection of low-molecular-mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry methods

Using a combinatorial chemistry approach, a decapaptide library containing the N-terminal fragment of trypsin inhibitor CMTI-III was synthesized by the solid-phase method. The peptide library was screened for trypsin and chymotrypsin inhibitory activity applying the iterative method in solution. Two decapeptides were selected and resynthesized for each enzyme. The association equilibrium constants ((1.1 ± 0.2) × 10 8 and (7.3 ± 1.6) × 10 7) determined for peptides with trypsin inhibitory activity indicate that they are 3–4-fold less active than the CMTI inhibitors. On the other hand, they are significantly more effective as compared with the starting sequence. Two peptides selected as chymotrypsin inhibitors displayed about 10 times higher activity (1.7 ± 0.4) × 10 7 and (1.1 ± 0.2) × 10 7, respectively) than those monosubstituted in position P 1 of the CMTI-III analogue. Considering low molecular weight of peptides selected and the lack of conformational constraints in their structures, the results are promising. They are good templates as starting sequences for further selection of small, peptidomimetic proteinase inhibitors.

Molecular cloning, characterization and gene expression of a water deficiency and chilling induced proteinase inhibitor I gene family from sweet potato ( Ipomoea batatas Lam.) leaves

A 7-kDa-proteinase inhibitor, designated SPLTI (S̱weet P̱otato Ḻeaf Ṯrypsin I̱nhibitor) was partially purified from sweet potato ( Ipomoea batatas Lam.) leaves under water deficiency. The N-terminal amino acid sequence was determined and used to design two overlap degenerate primers for isolation of the SPLTI gene. Two full-length cDNA clones encoding proteinase inhibitor I (PI-I), designated SPLTI-a and SPLTI-b, were isolated. Both SPLTI-a and SPLTI-b are 98% identical to each other in both levels of nucleotide and amino acid sequence. Different from most of PI-Is that exhibit chymotrypsin-inhibitory activity, SPLTI from sweet potato and recombinant SPLTI-a overexpressed in Escherichia coli showed mainly trypsin-inhibitory activity. Furthermore, site-directed mutagenesis analysis of an Arg 46–G1u 47 motif of SPLTI-a, based on amino acid sequence alignment with other PI-Is, indicated that Arg 46–G1u 47 of SPLTI is a novel reactive site for PI-I family conferring the trypsin-specific inhibitory activity. Using SPLTI-a as a probe, we found that SPLTI gene exhibited a leaf-specific expression pattern. Additionally, this was the first report that the SPLTI genes were up-regulated by water deficiency and chilling as well as osmoticant treatments in the PI-I family in plants. As other PIs, the SPLTI transcripts were induced by wounding and also by exogenous applications of abscisic acid and methyl jasmonate; however, accumulation of the wound-induced transcripts were restricted locally in the injured leaves, but not systemically. These distinct expression patterns provided a new insight to the regulation of PI-I gene family in response to environmental stresses. Our results suggested that SPLTI could participate in defense systems against invasions of insects or bacteria as other PI-Is. Moreover, it may play a role against environmental stresses through regulation of endogenous proteolytic activities during leaf development.

A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin–agarose or a trypsin–agarose column. In both cases, SDS–PAGE showed two bands of M r 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K is for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6×10 −7 and 1.3×10 −5 M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.

Protease Inhibitors in Porcine Colostrum: Potency Assessment and Initial Characterization

Porcine colostrum and milk were separated into the acid-soluble and casein fractions by acidification followed by centrifuge. The acid-soluble fraction of porcine colostrum was further separated by liquid chromatography and anisotropic membrane filtration. Trypsin and chymotrypsin inhibitory capacity in porcine colostrum, milk and their components was determined by incubating bovine trypsin or chymotrypsin in a medium containing their corresponding substrates with or without addition of various amounts of porcine colostrum, porcine milk or their components. The inhibition of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) degradation in pig small intestinal contents by porcine colostrum was measured by incubating iodinated IGF-I or EGF with the intestinal contents with or without addition of porcine colostrum. Degradation of labeled IGF-I or EGF was determined by monitoring the generation of radioactivity soluble in 30% trichloroacetic acid (TCA). The results showed that porcine colostrum had high levels of trypsin and chymotrypsin inhibitory activity and increased the stability of IGF-I and EGF in pig intestinal contents. The inhibitory activity declined rapidly during lactation. It was also found that trypsin and chymotrypsin inhibitory activity and the inhibition on IGF-I and EGF degradation in the acid-soluble fraction were higher than that in the casein fraction. Heat-resistance study indicated that trypsin inhibitors in porcine colostrum survived heat treatments of 100°C water bath for up to 10 min, but exposure to boiling water bath for 30 min significantly decreased the inhibitory activity. Compared with the trypsin inhibitors, the chymotrypsin inhibitors were more heat- sensitive. Separation of the acid-soluble fraction of porcine colostrum by liquid chromatography and anisotropic membrane filtration revealed that the trypsin and chymotrypsin inhibitory capacity was mainly due to a group of small proteins with molecular weight of 10,000-50,000. In conclusion, the present study confirmed the existence of high levels of protease inhibitors in porcine colostrum, and the inhibition of porcine colostrum on degradation of milk-borne growth factors in the pig small intestinal tract was demonstrated for the first time. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 12 : 1822-1829)

Effect of sprouting on nutrients, antinutrients and in vitro digestibility of the MACS-13 soybean variety

MACS-13, a recently developed soybean variety, was analyzed for nutrient and antinutrient compositions and the changes brought about by sprouting for 24 h, 48 h, 72 h along with in vitro digestibility. Of the proximate components, sprouting resulted in a significant gradual increase of 4 to 8 fold in free amino acids and amino N content as well as reducing sugars (13.33%). A gradual reduction was observed in crude fiber (69%), crude fat (9.74%) and ash (6.62%). The trypsin inhibitor activity (TIA), chymotrypsin inhibitor activity (CIA), amylase inhibitor activity (AIA) and phytate P/ total P gradually decreased by 51.68%, 16.53%, 27.51%, 25.04%, respectively, with sprouting. Cyanogens (135.30%) and phenolics (23.75%) gradually increased. The resultant improvement in the nutritional quality due to sprouting was confirmed by increased in vitro digestibility carried out with trypsin, pepsin and pepsin followed by trypsin. Similar results have been obtained for digestibility of protein isolates.

Proteinase and alpha-amylase inhibitors of sweet potato: Changes during growth phase, sprouting, and wound induced alterations

Changes in proteinase (trypsin and chymotrypsin) and α-amylase inhibitors were monitored during the growth phase and sprouting of sweet potato, with a view to establishing the biological role of these secondary plant principles. Although the trypsin inhibitor (TI) activity of tuberising roots of sweet potato was very low in four genotypes at 15 days after planting (DAP), a steep increase was observed during the tuber-hulking phase, indicating a possible role for the inhibitor in regulating plant proteinases. In contrast, the chymotrypsin inhibitor (CI) activity decreased from 40-90 DAP, indicating an inconsequential role in protease regulation. With initiation of tuber bulking, the α-amylase inhibitor (AI) increased sharply in three sweet potato genotypes, and after completion of the bulking phase, the activity came down sharply at 105 DAP. A pronounced role for regulation of endogenous a-amylases by the AI of sweet potato could be postulated from the study. The TI activity increased in the tubers until the sixth day of planting in vermiculite, and it decreased with the emergence of sprouts, indicating the possibility of proteolytic cleavage at the time of sprouting. Amylase inhibitor activity continuously decreased until sprouting, indicating the role of these proteins as N-reserve proteins in sweet potato. A significant increase in proteinase inhibitor levels in all the sweet potato leaves was observed within four hours of artificial wounding. The rise in inhibitor levels was more evident in leaves far off from the wounded leaf. The fact that proteinase and amylase inhibitors of sweet potato are wound-inducible points to a major defensive role for them. This information on the proteinase inhibitor levels in sweet potato leaves and vines and their change with plant growth is of interest, especially due to their use as animal feed.

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.

Inhibition of bovine α-chymotrypsin by cyclic trypsin inhibitor SFTI-1 isolated from sunflower seeds and its two acyclic analogues

Trypsin inhibitor SFTI-1 isolated from sunflower seeds (comprising 14 amino acid residues and two cycles: head-to-tailcyclisation and disulfide bridge) is the smallest naturally occurring plant serine proteinase inhibitor. In our recent paperwe have shown that the elimination head-to-tail cyclisation did not change trypsin inhibitory activity as judged by measuredby association equilibrium constants Ka. The removal of disulfide bridge produced 2.4-fold lower activity. In the present paper we described chymotrypsin inhibitory activity. SFTI-1 inhibits significantly lower bovine α-chymortypsin(Ka = (5.20±1.56) × 106 M-1). The activity of the analogue with disulfide bridge only was practically the same, whereas the Ka value determined forhomodetic peptide was almost 3-fold lower. Considering the results obtained and the recent literature data we postulate thelower inhibitory activity against both enzyme of the analogue with head-to-tail cyclisation only reflect its lower proteolytic stability.

Agrobotanical traits and chemical composition of Cassia obtusifolia L.: a lesser-known legume of the Western Ghats region of South India.

Four accessions of the lesser-known legume, Cassia obtusifolia L. (Sickle pod), collected from four different agroclimatic regions of Western Ghats, were evaluated for agrobotanical traits and chemical composition. Among the four accessions, the Keriparai accession had the highest values for plant height (cm), number of flowers per cluster, number of pods per cluster, pod length (cm), seeds per pod, seed weight (g) per pod and seed recovery percentage. Crude protein ranged from 18.56-22.93%, crude lipid was between 5.35-7.40%, crude fiber ranged from 6.83-9.45%, ash content ranged from 5.14-5.83% and carbohydrate varied from 57.00-60.69%. Globulins constituted the bulk of the seed protein as in most legumes. Mineral profiles, viz., sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc and manganese ranged from 42.92-84.83, 758.05-1555.79, 559.92-791.72, 456.36-709.47, 629.13-947.79, 8.42-12.35, 0.93-2.06, 10.60-30.04 and 2.12-4.12 mg/100 g seeds flour, respectively. Seed proteins of all accessions exhibited relatively high levels of non-essential and essential amino acids, with the exception of threonine. The in vitro protein digestibility of the legume ranged from 74.66 to 81.44%. Antinutritional substances such as total free phenolics ranged from 0.34-0.66%; tannins were between 0.47-0.60%; L-DOPA content ranged from 0.98-1.34%; trypsin inhibitor activity varied from 11.4-13.5 TIU/mg protein and chymotrypsin inhibitor activity ranged from 10.8-12.3 CIU/mg protein. Phytohemagglutinating activity also was assayed. In conclusion, the accessions of C. obtusifolia, collected from Western Ghats, South India, could serve as a low-cost source of some important nutrients for humans. The antinutritional factors might have little nutritional significance, if the seeds are processed properly.

Chemical composition, protein fractionation, essential amino acid potential and antimetabolic constituents of an unconventional legume, Gila bean (Entada phaseoloides Merrill) seed kernel

AbstractPhysical characteristics of pods and seeds, proximate composition, different protein fractionation, SDS‐PAGE analysis of proteins, amino acid composition, starch content, fatty acid profiles and various antimetabolic substances of Gila bean (Entada phaseoloides Merrill) were studied. The pod length and the number of seeds per pod ranged from 55 to 90 cm and from 5 to 11 respectively. The kernel comprised 66.1% of the seed weight (18.41 ± 1.14 g). The seed kernels contained 256.7 g kg−1 crude protein, 108.1 g kg−1 lipid, 27.3 g kg−1 ash and a high content of carbohydrate (585.7 g kg−1). The levels of potassium, phosphorus, zinc and iron were similar to those in conventional pulses. Among the different protein fractions of seed kernels, albumins constituted the major storage proteins (69.7%). The kernel proteins were rich in essential amino acids, particularly sulphur‐containing amino acids, and their values appeared to be higher than the FAO/WHO (1990) reference protein for a 2–5‐year‐old growing child and soybean, and comparable to hen egg. Seed kernel lipids contained high levels of unsaturated fatty acids, oleic and linoleic acids, which accounted for 83% of the total fatty acid recovered. The kernel exhibited high trypsin and chymotrypsin inhibitor activities (96.65 mg TI g−1 and 30.02 CIU mg−1 sample respectively) in addition to containing phenolics, phytic acid, lectins and oligosaccharides. Another major toxic constituent was identified as a group of triterpenoid saponins (3.21%), which had high haemolytic activity (HeU) against cattle erythrocytes and caused high mortality in fish. The in vitro digestibility of the kernel protein was low (67%).© 2001 Society of Chemical Industry

Serine protease inhibitors from Amazon leguminosae seeds: purification and preliminary characterization of two chymotrypsin inhibitors from Inga umbratica

Nine species of Leguminosae seeds from Amazon forest have been screened for trypsin and chymotrypsin inhibitory activity. The species involved were Cassia basselari, Cassia grandis, Cassia occidentalis, Dialium guianense, Inga fagifolia, Inga rubiginosa, Inga umbratica, Inga velutina and Mimosa guillandinae. Extracts from all the species, except I. umbratica, contained significant activity against trypsin, chymotrypsin and blood human clotting factors Xa. Two new chymotrypsin inhibitors (IuCI-1 and IuCI-2) have been purified to homogeneity from I. umbratica with molecular masses of 20,088.6 and 20,271.2, respectively.

Association of the Brazil Nut Protein Gene and Kunitz Trypsin Inhibitor Alleles with Soybean Protease Inhibitor Activity and Agronomic Traits

The use of soybean [Glycine max (L.) Merr.] in animal feed without heat treatment may be possible by reducing protease inhibitors. The objectives of this study were to determine to what extent soybean protease inhibitors would be reduced genetically by combining the Brazil nut protein (BNP) gene from the transgenic line BX4P9341B6 with the ti allele for the Kunitz trypsin inhibitor from the cultivar Kunitz and their effect on agronomic traits. Soybean seed from 42 F2‐derived lines from the cross BX4P9341B6 × Kunitz were evaluated in replicated trials in 1995 for trypsin inhibitor (TI), chymotrypsin inhibitor (CI) activity, and agronomic traits. There were six lines homogeneous for presence of the BNP gene and the Ti allele (BNP+, Ti), 18 lines homogeneous for presence of the BNP gene and the ti allele (BNP+, ti), six lines homogeneous for absence of the BNP gene and presence of the Ti allele (BNP−, Ti), and 12 lines homogeneous for absence of the BNP gene and presence of the ti allele (BNP−, ti). The mean TI activity of the BNP+, ti lines was 85.1% less than for the BNP−, Ti lines representative of conventional soybean cultivars. The mean CI activity of the BNP+, ti lines was 61.4% less than the BNP−, Ti lines. The means of the four genotypic classes were not significantly different (P > 0.05) for seed yield, maturity, lodging, and protein content. It should be possible to develop high‐yielding cultivars with the BNP+, ti genotype that have major reductions in TI and CI activity.

Effect of various domestic processing methods on antinutrients and in vitro protein and starch digestibility of two indigenous varieties of Indian tribal pulse, Mucuna pruriens Var. utilis.

The effect of various domestic processing methods on antinutrients and starch fractions and in vitro protein and starch digestibilities of white and black varieties of Mucuna pruriens var. utilis was studied. Cooking or autoclaving of both raw seeds and presoaked seeds in different solutions (water, tamarind extract, sodium bicarbonate, and citric acid) significantly (p < 0.05) reduced the content of total phenolics, phytic acid, trypsin inhibitor and chymotrypsin inhibitor activities, and L-dopa compared to soaking or dry heating techniques. The germination processes (24 and 48 h) were also effective in the reduction of various antinutrients, although this reduction appeared to be more pronounced in a prolonged period of germination (72 h). Water soaking followed by dehusking was found to be ineffective in the reduction of trypsin and chymotrypsin inhibitor activities in both varieties. All of the treatments were effective in significantly (p < 0.05) reducing the resistant starch content in the presently investigated samples. Cooking as well as autoclaving brought about a more significant (p < 0.05) improvement in the digestibility of protein and starch compared to germination and dry heat treatment. Moreover, among the different processing techniques, soaking in sodium bicarbonate solution followed by cooking (29.6-34.8%) or autoclaving (33.0-37.2%) seemed to be the best method for improving starch digestibility.

Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana.

Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.

Bowman–Birk inhibitors in soybean seed coats

Soybean Bowman–Birk inhibitor (BBI), possessing a molecular weight (MW) of 8 kD, is a known cancer chemopreventative and anticarcinogenic agent Aqueous extracts of soybean seed coats, rigorously screened to remove cotyledons, contained peroxidase along with three unknown proteins each of which possessed an estimated MW of <10 kD when compared with known standards subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because these extracts tested positively for trypsin inhibitor (TI) activity, our objective was to isolate and characterize the low MW inhibitor(s). To eliminate the solubilization of peroxidase from soy seed coats, we applied the classic methodology for BBI isolation which utilizes 60% ethanol. Soybean seed coats, which represent 8% of the whole seed, were defatted, then twice extracted with 60% aqueous ethanol to yield 6.96% sugary solids with 1.56% Kjeldahl nitrogen and a TI activity of 44 mg TI/g solids. Water extracts of the alcohol extracted residues provided an additional 4.55% solids with 2.71% nitrogen and 27 mg TI/g solids. The protein components in the 60% ethanol solubles were further concentrated by acetone precipitation. The acetone precipitates and water extractables were each fractionated on a CM Sephadex C-25 column eluted with a salt gradient. The consequent column fractions from the acetone precipitates when subjected to SDS-PAGE yielded three proteins, one of which possessed the mobility similar to a commercially available soybean BBI. Because BBI can simultaneously inhibit both trypsin and chymotrypsin, we verified the presence of BBI in those column fractions that eluted after 0.21M NaCl concentration based on colorimetric assays for TI and chymotrypsin inhibitor (CTI) activities. Chymotrypsin negative stain technique applied to native PAGE of the TI and CTI-active fraction from acetone precipitates showed evidence for seven isoforms of BBI. Two CM Sephadex column fractions eluting prior to 0.21 M salt concentration showed two low MW proteins (<7 kD) when stained for protein and compared with MW standards subjected to SDS-PAGE. Based on the summation of CTI active components in the CM Sephadex C-25 column fractions from 60% ethanol extract, their CTI activity relative to CTI in a commercial BBI standard was 61.9%, whereas, the CTI activity in fractions of column chromatographed water extracts that eluted after 0.21M salt concentration was 86.1%. Soybean seed coats may provide a new, inexpensive source of BBI concentrate for use as a potential anticancer agent.

Nutritional and anti-nutritional attributes of the under-utilized legume, Cassia floribunda Cav.

Abstract Three germplasm seed samples of the under-utilized legume, Cassia floribunda Cav., collected from three locations of South India were analyzed for proximate composition, mineral profiles, protein fractions, amino acid profiles of total seed protein, in vitro protein digestibility and certain anti-nutritional factors. The accessions contain (%) 19.9–21.7 crude protein, 2.1–3.1 crude lipid, 11.7–13.8 total dietary fiber, 3.4–5.6 ash and 58.0–60.5 carbohydrates (by difference). Globulins and albumins constituted the predominant fractions of the seed protein. The contents of essential amino acids, valine, phenylalanine, tyrosine, isoleucine, histidine and lysine, were high in all three germplasm seed samples compared to FAO/WHO (1991), [FAO/WHO. (1991). Protein quality evaluation . Rome, Italy: Food and Agricultural Organization of the United Nations requirement pattern. In vitro protein digestibility ranged from 81.4-85.9%. Anti-nutritional factors such as total free phenolics, tannins, L-DOPA (3,4-dihydroxyphenylalanine), trypsin inhibitor activity, chymotrypsin inhibitor activity and haemagglutinating activity also were analyzed and resulting levels found were not considered a threat to human health if the seeds were properly processed.

Some nutritional properties of the seeds of three Mucuna species

The seeds of Mucuna nivea, M. pruriens and M. utilis showed ash 4.3-5.1%, oil 4.9-5.5%, protein 25.9-27.5%, L-dopa 3.6-4.2%, trypsin 28.5-39.7 mg/g and chymotrypsin inhibitor activity 19.3-24.6 mg/g. The trypsin and chymotrypsin inhibitor activity increased in pod hull and seeds while the amount of protein increased in seeds and decreased in pod hull with maturity. The essential amino acid profile was comparable to the FAO pattern (lysine 6.0-6.4%). The fatty acid composition had total unsaturated acids 51.9-55.9%, but were poor in oil contents.

Studies on the nutritional composition and antinutritional factors of three different germplasm seed materials of an under-utilized tropical legume, Mucuna pruriens var. utilis.

Two different germplasms of a white variety and one germplasm of a black variety of Mucuna pruriens var. utilis were evaluated for their physicochemical properties as well as their nutritional and antinutritional characteristics. All germplasms had higher grain weight, density, hydration, and swelling capacity than other common legumes. The dehulled samples contained 303.2-335.5 g(-1) protein and 46.1-53.5 g x kg(-1) lipid, and these values were higher than the respective whole seeds. The levels of macro- and microelements in both whole and dehulled seeds were comparable to those in common pulses. All germplasms had a high dietary fiber content (18-19.5%), made up of mainly insoluble dietary fiber (DF). Seed lipids were high in unsaturated fatty acids (64.7-66.9%), specifically linoleic acid (48-49%). Whole and dehulled seeds of the white variety from Salem were particularly rich in sulfur-containing amino acids with significantly higher levels of in vitro protein digestibility than the other two germplasms. All germplasms had high levels of total phenols and phytate, trypsin, and chymotrypsin inhibitor activities, but were low in tannins, saponins, and alpha-amylase inhibitor activity. Only weak hemagglutinating activity against cow erythrocytes and no hemagglutinating activity against human erythrocytes (O) was observed in all the samples. Dehulled seeds were higher in total starch, including resistant starch and oligosaccharides (with verbascose as the major fraction) than the respective whole seeds. Both whole and dehulled samples of the white variety of Salem germplasm showed significantly lower concentrations of L-dopa, nonmethylated, and methylated tetrahydroisoquinolines than the respective whole and dehulled samples of other germplasms. In general, dehulling didn't affect the overall nutritional status in any of the presently investigated samples.

Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome.

The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.