Low Lysine Content Research Articles

The Hepatitis B Virus Precore Protein Is Retrotransported from Endoplasmic Reticulum (ER) to Cytosol through the ER-associated Degradation Pathway

The hepatitis B virus precore protein is closely related to the nucleocapsid core protein but is processed distinctly in the cell and plays a different role in the viral cycle. Precore is addressed to the endoplasmic reticulum (ER) through a signal peptide, and the form present in the ER is the P22 protein. P22 is then cleaved in its C-terminal part to be secreted as HBe antigen. In addition, a cytosolic form of 22 kDa less characterized has been observed. Precore gene was shown to be implicated in viral persistence, but until now, the actual protein species involved has not been determined. Our work focuses on the cytosolic form of precore. Using human cells expressing precore and a convenient fractionation assay, we demonstrated that the cytosolic form is identical to the ER form and retrotransported in the cytoplasm through the ER-associated degradation pathway. This cellular machinery translocates misfolded proteins to the cytoplasm, where they are ubiquitinated on lysine residues and degraded by proteasome. We showed that precore escapes proteasome due to its low lysine content and accumulates in the cytosol. The role of this retrotransport was investigated. In the presence of precore, we found a specific redistribution of the Grp78/BiP chaperone protein to cytosol and demonstrated a specific interaction between precore and Grp78/BiP. Altogether, these data support the idea that the hepatitis B virus develops a strategy to take advantage of the ER-associated degradation pathway, allowing distinct subcellular localization and probably distinct roles for the viral precore protein.

The Effect of Maillard Conjugation of Deamidated Wheat Proteins with Low Molecular Weight Carbohydrates on the Secondary Structure of the Protein

A soluble isolated wheat protein fraction (sIWP) prepared from isolated wheat protein (30–35% deamidation) was incubated alone or in the presence of glucose or maltodextrins of various molecular weights (MW 1, 1.9 and 4.3 kDa) at 60 °C and 75% relative humidity to promote the formation of Maillard conjugates. The formation of Maillard conjugates was confirmed by the loss of available -NH2 groups on incubation. Approximately 3–4 carbohydrate moieties (glucose or low molecular weight carbohydrates in the commercial maltodextrin) were attached per mole of sIWP after 24 h incubation. Principal component analysis of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra measured in the dry state showed that there were no major structural changes among non-incubated sIWP, sIWP incubated alone, sIWP–glucose conjugate and sIWP–maltodextrin (MW 1 kDa) conjugate. Structural changes were observed when the protein was incubated with larger molecular weight maltodextrin (MW 1.9 kDa or 4.3 kDa). However, there were no detectable differences in their circular dichroism (CD) spectra suggesting the absence of conformational changes in proteins with or without attached carbohydrates in solution state. The differences between the FTIR and CD results are possibly due to differences in water content of the samples although pressure-induced changes to protein structure induced in the ATR cell and the influence of unreacted maltodextrins cannot be discounted. Attachment of low molecular weight carbohydrate moieties on a relatively large molecular weight protein (i.e. sIWP with average MW of 40.4 kDa) with low lysine content (average three per mole of protein) is not sufficient to have an impact on the secondary structure of the protein.

Bayberry ( Myrica rubra Sieb. et Zucc.) kernel: A new protein source

Bayberry ( Myrica rubra Sieb. et Zucc.) kernel protein isolate (BKPI) was isolated from bayberry kernel defatted flour (BKDF) by isoelectric precipitation. BKPI was evaluated for chemical composition and selected functional properties with defatted kernel flour as reference. BKPI contained over 90% dry weight (DW) of protein versus 60.5% DW of protein in BKDF. It possessed a well-balanced amino acid composition according to the FAO/WHO reference except for a low content of lysine. BKPI had a solubility profile similar to that of BKDF, with minimum solubility observed at pH 4.0 and maximum solubility at pH 12.0. BKPI exhibited minimum foaming capacity (FC) (31.1%) and maximum foaming stability (FS) (72.7%) at pH 4.0. Minimum emulsifying capacity (EC) and emulsifying stability (ES) of BKPI and BKDF were observed at pH 4.0. BKPI had a least gelation concentration of (LGC) of 6% (w/v) at pH 4.0. Results indicated that bayberry kernel has potential to be exploited as a new protein source in China.

Approaches in breeding for high quality protein maize

Maize is the principal crop and major staple food in the most African and South American countries. The main problem in human nutrition in developing countries, and in livestock feed in developed countries, is insufficient production and poor quality of cereal proteins. In the case of maize, due to the very low content of essential amino acids lysine and tryptophan in grain endosperm, biological value is very low, which is main limiting factor of common maize in human nutrition and feeding of monogastric animals. Quality protein maize (QPM) can help in solving of this problem. Maize production also faces serious constraints caused by agro-ecological conditions and poor socio-economic situation. To alleviate the effect of the constraints, selected genotypes with more desirable traits and appropriate field-plot techniques to create multiple-stress conditions, were used. It was found that, in downy mildew nursery distance up to 35 m from spreader plot is providing sufficient down load of spores for plant infection, provided that the testing breeding materials are planted towards to down-stream direction of the dominant wind. Using these breeding approaches large number of early, white and flint synthetics, composites and inbred lines were created with resistance or tolerance to downy mildew (DMR), maize streak virus (SR) and drought (DT). Created genotypes exhibited very good kernel modification and yield potential under low and normal inputs. In the case of synthetics and composites, besides tolerance to multiple stress factors, they were competing in yield with local QPM and normal maize checks. In the case of created inbred lines high combining ability was exhibited both in non-conventional and conventional maize hybrids. Trial data revealed that in the most cases the best entries were over-yielding the best checks.

Modifying lysine biosynthesis and catabolism in corn with a single bifunctional expression/silencing transgene cassette

Although it is one of the major crops in the world, corn has poor nutritional quality for human and animal consumption due to its low lysine content. Here, we report a method of simultaneous expression of a deregulated lysine biosynthetic enzyme, CordapA, and reduction of a bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharophine dehydrogenase (LKR/SDH), in transgenic corn plants by a single transgene cassette. This is accomplished by inserting an inverted-repeat sequence targeting the maize LKR/SDH gene into an intron of a transgene cassette that expresses CordapA. This combination of LKR/SDH silencing and CordapA expression led to the accumulation of free lysine to over 4000 p.p.m. in transgenic corn grain, compared to less than 100 p.p.m. in wild-type controls. This intron-embedded silencing cassette design reduces the number of transgene cassettes needed in transgenic approaches for manipulating metabolic pathways that sometimes require expression of one gene and silencing of another.

Nutritional value of diets containing graded levels of wheat distillers grains with solubles fed to broiler chicks

AbstractThis feeding trial was conducted to determine the effects of graded levels of wheat distillers dried grains with solubles (DDGS) on nutrient digestibility and performance when fed to broiler chicks. A total of 125, day‐old, male broiler chicks (52.8 ± 0.6 g) were fed wheat/soybean meal‐based diets containing 0, 5, 10, 15 or 20% wheat DDGS. There were five birds per pen and five replicate pens per treatment. Digestibility coefficients for dry matter (P = 0.001), energy (P = 0.001) and phosphorus (P = 0.001) declined in a linear manner with increasing levels of wheat DDGS in the diet. Despite the lower digestibility of nutrients in diets containing wheat DDGS, there were no significant differences in weight gain (P = 0.721), feed intake (P = 0.748) or feed conversion (P = 0.766) due to the incorporation of wheat DDGS in the diet, although weight gain and feed conversion showed a tendency to decline at the highest inclusion level (20%) in the diet. The overall results of this study indicate that wheat DDGS can be successfully incorporated into diets fed to broiler chicks. Potential detrimental factors contained in wheat DDGS, including a low energy and low lysine content, can be compensated for in diet formulation. Therefore, when the price of alternative feed ingredients dictates, wheat DDGS can be incorporated into diets at levels as high as 15% without detrimental effects on broiler performance. Copyright © 2007 Society of Chemical Industry

Efecto del nitrógeno y la densidad de población en el contenido de lisina en la semilla de maíz en México

Efecto del nitrógeno y la densidad de población en el contenido de lisina en la semilla de maíz en México. El objetivo del presente trabajo fue identifi car la disponibilidad de lisina en cultivares de maíz y su relación con la fertilización nitrogenada y densidad de población. Para ello, se evaluaron variedades de grano blanco, amarillo, QPM's y el Opaco-2 en la Universidad Autónoma Agraria Antonio Narro (UAAAN), Coahuila, México, en el año 2005; el factor, densidad de población estuvo a tres niveles y la fertilización nitrogenada en cuatro dosis. Para la evaluación se empleó un método microbiológico (Schizophyllum commune) y a través de una escala se realizó la selección de bajo y alto contenido de lisina. En campo se consideró un diseño experimental de bloques completos al azar con arreglo de parcelas subdivididas. Para el laboratorio se aplicó un diseño completamente al azar con la prueba múltiple Duncan. Se encontró una composición de proteína en el germen y el endospermo que varió de 18,73% a 21,29% y 9,04% a 10,36%, respectivamente. Para el cultivar Opaco-2, se encontró 35,1% de proteína en el germen y 60,7% en el endospermo. Los valores para lisina variaron del 17% en la variedad de grano blanco hasta el 58% para el Opaco-2. El porcentaje de lisina fue fuertemente infl uenciado por el genotipo, la densidad de población y fertilización nitrogenada.

Significance of quality of truly digestible protein on performance of ewes at late pregnancy and early lactation

Two trials were conducted in ewes at late pregnancy and early lactation in order to evaluate the effects of substituting the moderate degradable protein of soy bean (SBM), with a low degradable protein of corn gluten meal (CGM), accordingly that both diets were calculated to provide equal truly digestible protein (PDI). At the first trial thirty multiparous ewes were examined in an 8-week study that started at parturition. Animals were fed in groups (15 ewes), with two isocaloric-isonitrogenous-isofibrous rations. Treatments were: (A) SBM, (B) CGM. Diets were different at PDIA/PDI ratio (0.52 versus 0.63). The second trial lasted 7 weeks, 5 weeks pre partum till 2 weeks post partum. Three rations were tested by the use of 48 pregnant ewes. Ewes were allotted to three equivalent groups: (A) SBM and wheat bran, (B) CGM and corn gluten feed, (C) CGM and corn gluten feed plus additional lysine (CGML). Data from both trials were analyzed as repeated measures. CGM compared to SBM decreased milk yield and fat corrected milk (FCM) yield (2.25 and 1.98 kg of milk; 2.47 and 2.17 kg of FCM), while tended to decrease milk's protein content (5.45 and 5.36%, for the SBM and CGM group). The daily production of fat, protein and lactose were higher in SBM than in CGM. CGM ration decreased body weight (BW) gain of ewes during late pregnancy and mean birth BW of litter, and also caused a 30% increase of abortions-dead births. CGM cannot totally replace SBM, in ewes’ diets at early lactation, because of the decrease in milk yield as well as the milk's protein content, even when equal PDI is provided, due to low lysine content. This trial has extended the knowledge from cattle, that supplementation of lysine, usually the first limiting amino acid, is also crucial in ewes during pregnancy and lactation.

Use of Cottonseed Meal in Aquatic Animal Diets: A Review

AbstractCottonseed meal is a plant protein source that is abundant in most parts of the world, relatively high in protein, and generally less expensive per unit of protein than soybean meal. It is highly palatable and readily digested by most aquatic animals, though generally not as digestible as soybean meal. Its use in aquaculture feeds may be limited because of the potential toxic effects of free gossypol and its relatively low available lysine and methionine content. However, depending on economics, the protein quality of cottonseed meal can be improved by supplementing it with lysine or both lysine and methionine. Also, the concentrations of free gossypol in diets containing significant levels of cottonseed meal are typically below the levels that are detrimental to many aquatic animals. However, even as a partial replacement of soybean meal, cottonseed meal has generally been used at relatively low levels in aquatic animal diets, partially because the safe levels of cottonseed meal appear to differ for various fish and crustaceans. Although some cultured aquatic species can tolerate relatively high dietary levels of cottonseed meal, its use will probably continue to be limited to about 10–15% of their diets. Although this is largely related to the higher nutritive value of soybean meal, it is, to a lesser degree, because of the variation of free gossypol levels in various sources of the meal and the potential toxic effects of gossypol on certain aquatic animals. However, when market conditions are favorable, the use of cottonseed meal will increase because some aquatic animals are largely unaffected by free gossypol, and for them cottonseed meal can lower rearing costs.

High‐lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation

Corn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis.

Proteins of Thermus thermophilus Are Resistant to Glycation‐Induced Protein Precipitation: An Evolutionary Adaptation to Life at Extreme Temperatures?

In thermophilic bacteria, formation of Maillard products may occur at increased rates because this reaction is favored at higher temperatures. Therefore, specific protective mechanisms against glycation-induced protein precipitation are likely to exist in thermophilic bacteria. Indeed, Thermus thermophilus proteins remained soluble when a cell-free extract of T. thermophilus was incubated at 37 degrees C in the presence of glucose, fructose, or methylglyoxal; whereas E. coli proteins precipitated. In E. coli cell-free extracts, sugar-induced precipitation was accelerated by the addition of 5 microM Fe2+ and inhibited by metal chelators, suggesting that glycoxidation processes are involved in the formation of the precipitate. A low lysine content, endogenous small scavenger molecules, or enzymatic "antiglycation" mechanisms for the degradation of AGEs or their precursors could be excluded as possible causes for the resistance to protein precipitation in T. thermophilus. Therefore, the resistance to glycation-mediated protein precipitation is an endogenous property of thermophilic proteins that was acquired during evolution in environments with high glycation activity.

Quantitative dietary lysine requirement of juvenile grass carp Ctenopharyngodon idella

A 90-day feeding trail was conducted to determine the dietary lysine requirement of juvenile grass carp using six isonitrogenous and isoenergetic diets (38% protein, 14 MJ digestible energy kg − 1 ) with wheat gluten–casein–gelatin as protein resource supplemented with six graded levels of crystalline lysine (from 0.69% to 3.08% of dry diet). Crystalline amino acid mixtures were supplemented to simulate the amino acid pattern found in the whole-body protein of grass carp except for lysine. Each diet was randomly assigned to triplicate groups of 20 fish each (3.15 ± 0.01 g, mean ± S.E.M.) and fish were fed five times daily at 2.5% body weight per day. No mortality or nutritional deficiency signs were observed except for growth depression in fish fed the diet with low content of lysine. Weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio, nutrient retention, proximate body composition, morphometry and hematology were significantly ( P < 0.05) affected by the dietary lysine concentrations. WG, SGR and FE were significantly higher in fish fed diet containing lysine 2.18% of the diet. Second-degree polynomial regression analysis of the WG and FE data indicated that the minimum recommended dietary lysine requirement for optimal growth of juvenile grass carp was 2.24% of the dry diet, corresponding to 5.89% of dietary protein. While the lysine availability (92.37%) of the protein sources was considered, the optimal lysine requirement of juvenile grass carp was calculated to be 2.07% of the diet, corresponding to 5.44% of the dietary protein. Additionally, the estimated requirements for the other essential amino acids were calculated from A/ E ratios of whole-body amino acid profile based on the lysine requirement determined from the present experiment.

Mammary nutrient uptake in response to dietary supplementation of rumen protected lysine and methionine in late and early lactating dairy goats

The objective of the present study was to evaluate how supplementation of lysine and methionine would affect mammary supply and uptake of these two amino acids in early and late lactation, and the impact this would have on milk synthesis. Four dairy goats were used in a 4 × 4 Latin square design in early and late lactation. The goats were fed a diet low in lysine and methionine and supplemented with rumen protected lysine and methionine in order to get four treatments with low lysine and low methionine content (C), low lysine and high methionine content (M), high lysine and low methionine content (L), and high lysine and high methionine content (ML). Feed supplements of rumen protected lysine and methionine increased arterial lysine and methionine concentrations, but mammary AV-differences did not change. However, only in early and not late lactation lysine and methionine were found to be co-limiting for milk protein production on the basal diet. Mammary uptake of lysine and methionine seem to be affected not only by supply, but also mammary synthetic capacity, since mammary tissue to some extent seems capable of extracting these and possibly other amino acids according to its needs independently of arterial supply. Mammary synthetic capacity seems to play a similar role for uptake of glucose and TAG, whereas uptake of acetate and BHB mainly depend on arterial supply in both early and late lactation. The present results also indicate that mammary uptake of lysine and possibly methionine is more sensitive to changes in arterial concentration in late compared to early lactation.

Brassica carinata protein isolates: chemical composition, protein characterization and improvement of functional properties by protein hydrolysis

Brassica carinata defatted flour has been used to prepare protein isolates by alkaline extraction and precipitation at low pH. Different extraction parameters have been tested, and the chemical composition and functional properties of the resulting isolates have been analyzed. All the isolates had a protein content above 90% and a well balanced amino acid composition according to FAO standards except for a low content of lysine. The extraction process reduced antinutritional components by more than 90%. Solubility, water and fat adsorption, emulsifying and foaming properties of the original flour and the isolates have been compared. In addition, hydrolysis of B. carinata protein during extraction using the endoprotease preparation Alcalase was performed, and functional properties of hydrolysates with low and high degrees of hydrolysis were studied and compared with those of the original isolates.

Retrograde transport of toxins across the endoplasmic reticulum membrane.

Several protein toxins, including the A chain of the plant protein ricin (RTA), enter mammalian cells by endocytosis and catalytically modify cellular components to disrupt essential cellular processes. In the case of ricin, the process inhibited is protein synthesis. In order to reach their cytosolic substrates, several toxins undergo retrograde transport to the ER (endoplasmic reticulum) before translocating across the ER membrane. To achieve this export, these toxins exploit the ERAD (ER-associated protein degradation) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates in order to intoxicate the cell. Toxins that translocate from the ER have an unusually low lysine content that reduces the likelihood of ubiquitination and ubiquitin-mediated proteasomal degradation. We have changed the two lysyl residues normally present in RTA to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect on the protein, suggesting that the endogenous lysyl residues are not sites for ubiquitin attachment. However, when four additional lysyl residues were introduced into RTA in a way that did not compromise the activity, structure or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasomal inhibitor lactacystin increased the cytotoxicity of the lysine-enriched RTA to a level approaching that of wild-type RTA.

The Low Lysine Content of Ricin A Chain Reduces the Risk of Proteolytic Degradation after Translocation from the Endoplasmic Reticulum to the Cytosol

Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin beta-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.

Purification and biochemical characterization of a cadmium metallothionein from the digestive gland of the Antarctic scallop Adamussium colbecki (Smith, 1902)

A cadmium-binding protein was purified from the digestive gland of the Antarctic scallop, Adamussium colbecki, and biochemically characterized. Purification procedures included gel permeation and anion exchange chromatography, followed by preparative polyacrylamide gel electrophoresis. Our results demonstrate that the A. colbecki cadmium-binding protein has the general properties of metallothioneins: low molecular weight of about 10 kDa, spectroscopic features typical of cadmium thiolate clusters and high metal (cadmium) content. Analysis of amino acid composition reveals the absence of aromatic amino acids, histidine, methionine and arginine. Asparagine and glutamine are also absent. The A. colbecki metallothionein shows high levels of glycine (14%), aspartic acid (14%), glutamic acid (11%) and a low lysine content (4%); the A. colbecki metallothionein shows a lower cysteine content (12%) compared to other metallothioneins (17–30%) purified from both vertebrate and invertebrate organisms. The presence of a metallothionein in the digestive gland of A. colbecki suggests that in cold-ocean-adapted molluscs the heavy metal homeostasis mechanisms may have evolved similarly to those of organisms living in temperate marine environments, although the A. colbecki cadmium-binding protein shows a typical amino acidic composition that might reflect a peculiar physiological role.

Use of Sunflower Seed Meal (SSM) in broiler ration

Soyabean meal (SBM) is used extensively in poultry rations. As the cultivation of soya bean is limited in Iran, annually a large amounts of SBM is imported. In recent years cultivation of some oil seed such as sunflower seed (Heliantus annus) is undertaken in some provinces of Iran. This plant will grow in cooler and dried climates than the soya bean or cotton plants. It contains 45 percent oil and a good source of protein and B-group vitamins. Due to high fibre content and low lysine content, use of sunflower meal (SSM) is limited in poultry feeds, but dehulled SSM is suitable for broiler feeds (Church, 1988 and Scott et al., 1982). The objective of the present study was to investigate the effect of SSM on performance of broiler chicken.

Influence of processing conditions on the nutritive value of Ogi-baba, a Nigerian fermented sorghum gruel.

The effect of germination, cowpea fortification and fermentation on the chemical and amino acid composition of ogi-baba, a Nigerian fermented sorghum gruel was investigated. The lowest protein value of 1.4% was obtained in the traditionally prepared sample, while samples prepared from germinated, fortified sorghum had 13%. The ash ranged from 0.9% to above 2%, while the fat was in the range of 1.2% to 2.8%. Generally, fortification, in addition to germination, improved the chemical composition of ogi-baba relative to the control sample. There was improvement in the amino acid profile of all the ogi-baba samples compared to the unprocessed sorghum grains. Lysine, a limiting amino acid in sorghum, increased more than 50% in ogi-baba. However, samples prepared using germinated sorghum without the addition of cowpeas had a lower lysine content.

The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).