Search

Many people, particularly in Africa, suffer various conditions from eating bitter cassava which contains poisonous cyanogens. As well as poisoning, which sometimes causes death, these conditions include konzo, an irreversible paralysis of the legs, which affects mainly children and young women, impaired neurocognition in children, tropical ataxic neuropathy in older people, and aggravation of iodine deficiency disorders (such as goitre and cretinism) in iodine deficient areas. The wetting method removes residual cyanogens, and is an additional method of processing cassava flour after its preparation by one of the traditional methods. The wetting method is simple and easy to use, the traditional thick porridge (fufu or ugali) made from the treated flour tastes very good, and the method is readily accepted by rural women in East and Central Africa. Regular use of the wetting method by rural women in 13 villages in the Democratic Republic of Congo has prevented konzo amongst more than 9,000 people. We recommend that the wetting method be used as an additional method to traditional methods to remove cyanogens from cassava flour in tropical Africa.

The lactic acid content of gari, was determined by pH titration to be about 10 g lactic acid/kg gari. As the size of the gari particles increased from 400 to >1000 μm their total cyanide content increased from 5 to 21 ppm. The acetone cyanohydrin content of gari samples exposed to ambient laboratory conditions of temperature and relative humidity gradually decreased by 58% in 38 weeks whereas linamarin in cassava flour is 100% stable for 6 months. Cyanogens could not be removed from wet gari samples at 30 or 50 °C and were only slowly removed by repeated heating at 100 °C and rewetting. By mixing equal weights of gari (pH 4.1) and low cyanide cassava flour (pH 6.5), wetting and heating at 50 °C for 5 h, the cyanide content was reduced by about one half. Wetting a gari/flour (1:1) sample with 1.5 times its weight of water and standing in a 1 cm thick layer in the sun for 4 h reduced the cyanide content by about one half. This treated mixture may be cooked to prepare stiff porridge with lowered cyanide content, and may help reduce tropical ataxic neuropathy in West Africa.

In 2005 a simple wetting method was developed that reduced total cyanide content of cassava flour 3–6-fold. The method involved spreading wet flour in a thin layer and standing in the shade for five hours to allow evolution of HCN gas. We found that breakdown of linamarin catalysed by linamarase to acetone cyanohydrin, followed by its spontaneous decomposition to HCN and acetone was greatly increased by standing the wet flour in the sun. Treatment for two hours in the sun gave the same amount of total cyanide remaining as five hours in the shade. This rapid treatment in the sun may be more acceptable to rural women in Democratic Republic of Congo, than five hours in the shade. The two methods are offered as alternatives for use in rural Africa. With adequate linamarase present the residual cyanogen remaining after the wetting treatment was acetone cyanohydrin.

The sensitivity of the normal picrate method for determination of total cyanide in cassava was increased tenfold using a small 1 cm 2 picrate paper, eluted using 0.5 mL instead of 5 mL of water as in the normal method. The absorbance was measured in a 2 mm cuvette in the spectrophotometer. The sensitive method was calibrated against the normal picrate method. The total cyanide content in mg HCN equivalents/kg sample = ppm, is calculated from the absorbance (A) by the equation ppm = A × 45.7 which is applicable from 0.1 to 50 ppm. A new method to determine acetone cyanohydrin was developed based on irreversible denaturation of linamarase in 0.1 M HCl at 30 °C for 1 h. Five gari samples from Mozambique gave a mean total cyanide content of 12 ppm (range 6–15 ppm) and mean acetone cyanohydrin content of 11 ppm (range 5–14 ppm). Acetone cyanohydrin liberates cyanide quantitatively in the human intestine.

Cassava is the staple food of tropical Africa and its production, averaged over 24 countries, has increased more than threefold from 1980 to 2005, and the population has more than doubled over that time compared with a 1.5 times increase worldwide. Agriculturally, cassava performs very well but the roots and leaves contain cyanogenic glucosides that are dangerous to human health. These cyanogens sometimes produce acute intoxication leading to death, they exacerbate goitre and cretinism in iodine-deficient regions, cause konzo and are implicated in the occurrence of tropical ataxic neuropathy and stunting of children. Konzo is an irreversible paralysis of the legs with many thousands of cases, mainly amongst children, in Mozambique, Tanzania, Democratic Republic of Congo, Cameroon, Central African Republic and probably other tropical African countries. Attempts to alleviate cassava cyanide toxicity have included the development of an information network and distribution in developing countries of picrate kits, which measure total cyanide in cassava and urinary thiocyanate. A simple wetting method that reduces total cyanide in cassava flour three- to sixfold has been successfully field tested and is being introduced in Mozambique. Transgenic technology shows promise in increasing the rate of loss of cyanide from roots during processing. World health and agricultural bodies should pay more attention to emerging health problems associated with toxicity of cyanogens in cassava. Copyright © 2008 Society of Chemical Industry

A simple equation is developed between the total cyanide contents of cassava root parenchyma and the processed product with the % retention of cyanide on processing. This equation is applied to different methods of processing used worldwide. Thus to produce cassava flour of 10 mg HCN equivalents/kg flour (ppm), the WHO safe level, by sun drying or heap fermentation requires starting with sweet cassava containing 12–32 ppm total cyanide. In an average year only 14% of flour samples in our study areas in Nampula Province of Mozambique had total cyanide contents of Processing methods used to produce farinha in Brazil and gari in West Africa reduce the total cyanide content to less than one eighth of that using heap fermentation and less than one sixteenth of that using sun drying. Heap fermentation and sun drying, commonly used in eastern and southern Africa, do not adequately remove cyanide in a normal year and are hopelessly inadequate when used on cassava grown during drought. New and greatly improved processing methods are urgently needed. The high levels of cyanide intake in central, eastern and southern Africa from high cyanide flour are the most likely cause of konzo in young people and the very long term consumption of gari of lower cyanide content in West Africa is the most likely cause of TAN in older people.

A general method has been developed for determination of the total cyanide content of all cyanogenic plants and foods. Ten cyanogenic substrates (cassava, flax seed, sorghum and giant taro leaves, stones of peach, plum, nectarine and apricot, apple seeds and bamboo shoot) were chosen, as well as various model compounds, and the total cyanide contents determined by the acid hydrolysis and picrate kit methods. The hydrolysis of cyanoglucosides in 2 M sulfuric acid at 100 o C in a glass stoppered test tube causes some loss of HCN which is corrected for by extrapolation to zero time. However, using model compounds including replicate analyses on amygdalin, the picrate method is found to be more accurate and reproducible than the acid hydrolysis method. The picrate kit method is available free of charge to workers in developing countries for determination of cyanide in cassava roots and cassava products, flax seed, bamboo shoots and cyanide containing leaves. For eleven different samples of flax seed and flax seed meal the total cyanide content was 140–370 ppm. Bamboo shoots contained up to 1600 ppm total cyanide in the tip reducing to 110 ppm in the base. The total cyanide content of sorghum leaves was 740 ppm 1 week after germination but reduced to 60 ppm 3 weeks later. The acid hydrolysis method is generally applicable to all plants, but is much more difficult to use and is less accurate and reproducible than the picrate method, which is the method of choice for plants of importance for human food.

Background Konzo is an irreversible paralysis of the legs that occurs mainly among children and young women in remote villages in tropical Africa and is associated with a monotonous diet of bitter cassava. Konzo was discovered in 1938 by Dr. G. Trolli in the Democratic Republic of Congo (DRC). It also occurs in Mozambique, Tanzania, Cameroon, Central African Republic, and Angola. It was first controlled in Kay Kalenge village, DRC, in 2011 with the use of a wetting method to remove cyanogens from cassava flour. Fourteen months later, another visit was made to Kay Kalenge. Objective To determine whether Kay Kalenge women were still using the wetting method, whether there were new cases of konzo, and whether the wetting method had spread to other villages. Methods Meetings were held with chiefs, leaders, and heads of mothers' groups, women from 30 households were interviewed, and three nearby villages were visited. Total cyanide and thiocyanate were analyzed in cassava flour and urine samples, respectively. Results The women in Kay Kalenge village still used the wetting method. There were no new cases of konzo. The mean cyanide content of the flour samples was 9 ppm, and no child had a mean urinary thiocyanate content greater than 350 μmol/L. The use of the wetting method had spread naturally to three adjacent villages. Conclusions The wetting method has been readily accepted by rural women as a simple and useful method to control konzo by removing cyanide from cassava flour, and its use has spread to nearby villages. The wetting method should be promoted by health authorities to control konzo and reduce cyanide poisoning from high-cyanide cassava flour.

The total cyanogen content of 90 cassava flour samples from Mujocojo, Terrene A and Acordos de Lusaka in Nampula Province of Mozambique was determined in 1997 using the simple picrate kit method. The results at the first two locations were compared with those obtained at the same locations at the same time of the year in 1996. There was a significant decrease of about 50% in the cyanogen levels at Mujocojo and Terrene A in 1997. Results from Acordos de Lusaka and Mujocojo showed that flour processing by heap fermentation gave a substantially lower cyanogen content compared with sun drying. This confirms the conclusion found in our previous study. The cyanogen levels of flour from Acordos de Lusaka were very high (mean value 67 (39) ppm). The amount of available cyanogen (acetone cyanohydrin +HCN/CN − ) and partially available cyanogen (linamarin) was determined for 37 samples of cassava flour. There was no correlation between the amounts of available cyanogen and partially available cyanogen. Twenty-nine samples of urine from school children at Acordos de Lusaka gave a mean value of thiocyanate content of 512(277) μmolL −1 .

Background: It would be useful to develop a simple kit method for determination of thiocyanate in urine, which could be used to monitor cyanide overload in cassava-consuming populations. Methods: The method was based on the quantitative oxidation of thiocyanate in acid permanganate at room temperature in a closed vial with liberation of HCN, which reacted with a picrate paper. For semiquantitative analysis in the field, the colored picrate paper was matched with a color chart prepared using known amounts of KSCN. In the laboratory, a more accurate result was obtained by elution of the colored complex in water and measurement of the absorbance at 510 nm. Over the range 0–100 mg/L, there was a linear relationship given by the equation: thiocyanate content (mg/L) = 78 × absorbance. Results: The picrate thiocyanate method gave no interference with urine samples containing protein at less than 7 g/L, 21 amino acids, histamine, glucose, NaCl, urea, blood, and linamarin. For 53 urine samples analyzed by an accurate column method and the thiocyanate picrate method, a regression line gave very good agreement (r2 = 1.000). Quantitative recoveries of thiocyanate added to urine samples were obtained with the picrate method. Conclusions: A simple picrate kit for determination of thiocyanate in urine was developed and is available free of charge for workers in developing countries.

The corms and leaves of most cultivars of the edible aroids, the major staple food of about 200 million people in the tropics, are acrid. This means that if eaten raw they cause swelling of the lips, mouth and throat. This effect is related to the presence of needle-like raphides of calcium oxalate. A new method was developed for the separation of raphides using petroleum ether (PE) and then purification by centrifugation. The acrid raphides were shown by X-ray powder diffraction to consist of calcium oxalate monohydrate (whewellite) and their morphology was studied by scanning electron microscopy. Two distinct types of raphides (thick and thin) were observed and druses (rosette like structures of calcium oxalate) were found only in taro leaves. A comparative survey of raphide morphology was made over four species of aroids. The removal of the acridity of raphides by immersion in hydroxylic solvents, but not in PE, indicates the presence of an irritant on the raphides. Acridity is due to the dual action of the sharp raphides in puncturing soft skin and the irritant (a protease) in causing swelling and soreness. © 1998 SCI.

A simple picrate paper kit method was developed for the semi- quantitative determination of the cyanogenic potential for cassava Nour. The method involved the addition of linamarase and pH 8 phosphate bu†er (absorbed in a -lter paper disc) to 100 mg of Nour )0E5 ml water placed in a small vial. A rectangle of yellow picrate paper attached to a plastic strip was added and the vial capped and left overnight. The yellow paper changed colour towards brown and its colour was compared with that of a standard colour card with 10 shades of colour which corresponded with cyanogenic potentials of 0E 800 mg HCN equivalents kg~1 Nour (ppm). To obtain a more accurate measure of cyanogenic potential (^20%) the paper was eluted with water and the absorb- ance measured at 510 nm. The cyanogenic potential in ppm was determined from a calibration graph. The methodology is available in kit form. The simple method may be used in the -eld by a relatively unskilled person. The more accurate method requires a spectrophotometer and is suitable for use in simple laboratories in developing countries. 1998 SCI. (

A simple method was developed for isolating cassava leaf linamarase. It involved homogenising the leaves in a bu†er containing polyvinylpolypyrroli- done, followed by ammonium sulphate precipitation, -ltration and partial puri-- cation of the enzyme by hydrophobic interaction chromatography. All stages of the enzyme preparation were carried out at room temperature and it was com- pleted within 90 min. A linamarin-indicator strip for checking the activity of the enzyme preparation was also developed. This isolation technique which has been developed into a simple kit should be suitable for laboratories in developing countries where the enzyme is needed to determine the cyanogenic potential of cassava and its products.

A mild method was developed to remove cyanogens from cassava leaves that involved three consecutive steps (1) pounding, (2) standing for 2 h in the sun or 5 h in the shade in the tropics and (3) washing three times in water. Four cassava cultivars were used and the mean residual total cyanide content after steps 1, 2 and 3 was 28%, 12% and 1%, respectively. The pounded cassava leaves retained their bright green colour and texture. The traditional method for removing cyanogens from pounded cassava leaves is by boiling in water which removed all cyanogens in 10 min. However this method caused the pounded leaves to become dull green in colour and would cause considerable losses of vitamins, protein and methionine, which are already in short supply in the diet of poor village people in tropical Africa.

A simple method is described for the preparation, from very young cassava leaves, of a dilute hydrochloric acid solution of linamarin. Linamarin extraction from the leaves is virtually quantitative. The linamarin solution is used in the preparation of standard linamarin filter paper discs that are needed to monitor the performance of picrate kits for determination of the total cyanide content of cassava roots and cassava products. These standard discs are stable indefinitely if stored in the refrigerator, but very slowly lose linamarin activity if stored for more than one month at room temperature.

Three villages in Boko Health Zone, Bandundu Province, Democratic Republic of Congo (DRC), had 61 konzo cases and konzo prevalences of 2.5%, 4.1% and 7.5% respectively. Konzo cases occurred every year for 10 years and every month, peaking in July. The high mean cyanide content of cassava flour of 50 ppm was due to short soaking of cassava roots for 1–2 days instead of 3–4 days. Konzo cases were examined and village women taught the wetting method that removes cyanogens from flour. The villages were visited every month for 1 year following previous methodology. No new konzo cases occurred during the intervention, mean flour cyanide levels reduced from 50 to 14 ppm and mean urinary thiocyanate levels of school children reduced from 930 to 150 μmole/L. The percentage of children with urinary thiocyanate levels of >350 μmole/L was reduced from a maximum of 80 in Ikialala before the intervention to 0 in Ikusama, Ikialala and 3 in Imboso Mwanga 1 year later. This is the second time that konzo has been controlled and success depends on regular use of the wetting method by village women. The methodology is now being used in other villages in DRC with financial support of AusAID.

Compounds extracted by methanol from cassava parenchyma and cortex have been separated on a preparative HPLC column and identified by 1 H and 13 C NMR spectroscopy. A new compound isopropyl-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (IAG, structure I) has been found as well as small amounts of phenylalanine and tryptophan. The composition of another HPLC fraction has not been elucidated. The amounts of the identified compounds and of linamarin, lotaustralin, citrate, malate and the various sugars present in cassava have been determined by HPLC methods. The threshold levels of bitterness of aqueous solutions of linamarin, lotaustralin and IAG, have been determined and together with published data on L-phenylalanine and L-tryptophan have allowed our evaluation of their contributions to the bitterness of cassava. Linamarin is the sole contributor in the parenchyma but (with two cultivars out of six studied) IAG contributes more to the bitterness of the cortex than does linamarin. The perception of bitterness of linamarin solutions is confounded in the presence of neutral citrate and malate which have a sour taste. These modify the taste of cassava tubers. There are many compounds that contribute to the taste of cassava tubers, hence it is not surprising that the bitterness of cassava is not always correlated positively with the cyanide potential.

Fifty konzo cases were identified in four villages in Popokabaka Health Zone, DRC. One third of people had only one meal per day, mainly of cassava flour consumed as a thick porridge (fufu) and pounded, boiled cassava leaves. Retention of cyanogens in flour resulted from short soaking of cassava roots. A 1.5 years intervention was made in the largest village Kay Kalenge, where the wetting method was taught to all women of the village, who accepted it willingly. The total cyanide content of cassava flour was reduced to below 10 ppm. Fufu from treated flour tasted and stored better than fufu from untreated flour. The mean urinary thiocyanate content of 100 school children reduced from 332 to 130 lmole/L and the number of samples exceeding 350 lmole/L decreased from 26 to 0 during the intervention. No new konzo cases occurred, which included two dry seasons when konzo peaks. Konzo was first identified by Dr. Trolli in 1938 in Popokabaka Health Zone and it has now been prevented for the first time in the same area. The methodology is now in use in Boko Health Zone and we believe it is the most effective way to control konzo in tropical Africa.

Cassava leaves and tubers from six cultivars were analysed for carotenoids by both open-column chromatography and high-performance liquid chromatography (HPLC). The major carotenoids of leaves were the non-vitamin A carotenoid lutein (86-290 mg kg−1 fresh weight (FW)) and the pro-vitamin A carotenoid β-carotene (13-78 mg kg−1 FW). Immature leaves contained less than mature leaves. Tubers contained 0.1-3 mg kg−1 FW of β-carotene and 0.05-0.6 mg kg−1 FW of lutein. Carotenoids present in small amounts included α-, γ-, and §-carotenes and β-cryptoxanthin and others were separated and partially identified. Yellow tubers contain much more β-carotene than white tubers and cassava is a good source of pro-vitamin A carotenoids compared with other root crops. Both methods gave similar results for β-carotene and lutein, but the open-column method did not allow separation and quantitation of carotenoids present in small amounts. HPLC separated many carotenoids, but most peaks could not be identified for lack of reference compounds. A great advantage of open-column chromatography for developing countries is its use of simple equipment and readily available reagents and solvents compared with HPLC, which requires complex equipment needing careful maintenance and expensive solvents. The open-column method is more labour intensive but this is not a constraint in developing countries.

A rapid, inexpensive screening assay was developed in order to measure the cyanide content of cassava (Manihot esculenta Crantz) tubers. A small disc of parenchyma tissue cut with a cork borer or alternatively grated tissue was placed in a stoppered glass tube and the hydrogen cyanide liberated produced a blue colour on a filter paper previously spotted with a drop of tetra-base [4,4'-methylenebis-(N,N-dimethylaniline)] and cupric acetate. The intensity of the blue colour which developed within one hour was rated visually on a graded scale from 0 to 5. The correlation coefficient between samples accurately analysed for total cyanide and also tested using the rapid assay was 0.77.

The heat stability of trypsin inhibitors from rice (Oryza sativa L), sweet potato (Ipomoea batatas (L) Lam), taro (Colocasia esculenta var esculenta (L) Schott), giant taro (Alocasia macrorrhiza (L) G Don) and giant swamp taro (Cyrtosperma chamissonis (Schott) Merr) was studied. Rice trypsin inhibitor (TI) occurred only in the germ, so it is absent from white rice. Taro, giant taro and giant swamp taro (but not rice or sweet potato) showed an initial large increase (one- to tenfold) in the amount of TI present on heating at about 80°C. The heat release mechanism, which was accompanied by a change to a rubbery texture in the food, amounted to a breakdown of the cellular structure making TI available for extraction. Such heat release of TI would cause partially cooked food to be deleterious for human consumption. Brown rice, sweet potato, taro and giant swamp taro contained moderate amounts of TI, which was fully inactivated when the boiled or baked food was soft enough to eat. Giant taro, however, contained 10–100 times as much TI (which in this case also inhibited chymotrypsin) as the other foods, and a boiling time of at least 30 min was needed for its inactivation.

Cassava contains cyanogenic glucosides which are hydrolysed by an enzyme linamarase to produce cyanohydrins which breakdown to toxic cyanide. Cyanide ingestion from bitter cassava can cause cyanide poisoning sometimes leading to death and also konzo, an irreversible paralysis of the legs which occurs mainly in children and young women. In 2005 we developed a simple wetting method that reduces the total cyanide content of cassava flour 3–6-fold. It involves wetting the flour, spreading it in a thin layer in the shade for 5 h and using it the same day to make traditional thick porridge (ugali). The method was readily accepted by rural women and requires no additional equipment or water. Laminated, illustrated posters describing the method are available for free in ten languages, see http://online.anu.edu.au/BoZo/CCDN/ . An equally effective treatment method is to expose wet flour in a thin layer in the sun for 2 h. Projects for rehabilitation and prevention of konzo occurred in Mozambique in 2007 and in 2008–2009 in Tanzania, funded by AusAID. The Ministry of Health in Mozambique is now using our posters in Macua. In Uvira DRC, the wetting method has been taught in many villages and over 1200 posters distributed.

The maximum daily cassava flour intake of children may be calculated from determination of the total cyanide content of cassava flour and urinary thiocyanate levels of school children in samples collected at the same time and place. Four sites, two with and two without recent konzo cases, were chosen for study. In two sites with recent konzo cases, 84% and 93% of school children consumed cassava the previous day, and the calculated maximum daily consumption of cassava was over 700 g. In two sites without recent konzo cases, about 50% of school children consumed cassava the previous day and the calculated daily consumption of cassava flour was less than 150 g. By measurements of cyanide in flour and urinary thiocyanate we are therefore able to distinguish between communities whose diet is almost totally reliant on cassava, and who are therefore susceptible to konzo, and those who have a broader diet and are free from konzo. In another calculation it is shown that 4-23% of the essential S-containing amino acids in the cassava flour consumed by children is used up to detoxify and flour cyanide to thiocyanate. This depletion of methionine and cystine may leads to protein deficiency and may contribute to onset of konzo.

We aimed to detect new cases of konzo and monitor cyanogen exposure from cassava flour in communities previously affected by konzo epidemics in Nampula Province, northern Mozambique. Other objectives were to detect subclinical upper motor neuron damage in schoolchildren and test a new kit to measure urinary thiocyanate concentration. In 1999 and 2000, we carried out active and passive case detection for konzo in Memba and Mogincual Districts. In July and October, 1999, we collected cassava flour from 30 houses in three communities and measured cyanogen concentrations with a picrate kit. In October 1999, we examined all schoolchildren in three communities for ankle clonus and measured urinary thiocyanate concentration in thirty schoolchildren in each of five communities with a picrate kit. We found 27 new cases of konzo in Mogincual District. Mean total cyanogen concentrations in cassava flour varied between both seasons and years, but were always high, ranging from 26 to 186 ppm. Very high mean levels at three sites in November 1998 and July 1999 were probably due to low rainfall in the 1997–1998 season. The proportion of schoolchildren with ankle clonus varied from 8 to 17%. The new picrate kit for urinary thiocyanate worked well; mean concentrations in schoolchildren ranged from 225 to 384 mol l − 1 . Konzo and sub-clinical upper motor neuron damage persist in poor rural communities in northern Mozambique, associated with high cyanogen concentrations in cassava flour and high urinary thiocyanate concentrations in schoolchildren. © 2002 Elsevier Science B.V. All rights reserved.

A trisaccharide, which is a major carbohydrate component of the milk of the tammar wallaby and the grey kangaroo, has been identified by chemical, enzymic, g.l.c.-m.s., and n.m.r. methods as O-beta-D-galactopyranosyl-(1 yields 3)-O-beta-D-galactopyranosyl-(1 yields 4)-D-glucose (3'-galactosyl-lactose).

The proton chemical shifts of the glycosidic protons have been measured for solutions of oligosaccharides in D 2 O; to some degree, the shifts are diagnostic of both the nature of the sugar and of the type of linkage. To determine the sequence of the sugar residues, the aldose or ketose is first treated with cyanide to produce a terminal carboxylic acid. Then Gd 3+ is added, which is bound on the carboxyl group, causing line broadening of the proton resonances and a decrease in the spin-lattice relaxation-time ( T 1 ) of the glycosidic protons. These effects fall off progressively along the oligosaccharide chain, hence allowing the sequence to be determined. The method, which is illustrated for maltotriose, has been used at the milligram level, and may be applicable up to pentasaccharides. Difficulties occur in the treatment of non-reducing sugars (1→31 and 1→32 glycosides), and with certain sugar residues that bind Gd 3+ on three adjacent hydroxyl-groups of particular stereochemistry. The possible usefulness of 13 C-n.m.r spectroscopy is also considered.

Trypsin inhibitors from three edible aroids of the family Araceae, viz. taro ( Colocasia esculenta ) var. esculenta ) giant taro ( Alocasia macrorrhiza ) and giant swamp taro ( Cyrtosperma chamissonis ) obtained from the Pacific region, were isolated by affinity chromatography and purified by gel filtration. The M r of these inhibitors, as determined by gel filtration were 35 000–38 000, but were ca 20 000 by SDS gradient PAGE. A time course of heating in SDS showed a ready dissociation of the native protein into subunits of equal size. Further experiments showed that there were no disulfide bonds between these subunits. A single N -terminal sequence was found for each inhibitor showing that the two subunits had similar primary structure. Each of the N -terminal sequences showed homology with that of soybean trypsin inhibitor. To our knowledge, this finding follows only one other example of a Kunitz family inhibitor being located in a monocotyledonous, rather than dicotyledonous, plant species, and indicates that the ancestral gene from which Kunitz family inhibitors originate predates the evolutionary divergence of flowering plants into monocotyledons and dicotyledons.

Determination, par CLHP, de la teneur en oxalate, malate, citrate et succinate de differentes varietes de tubercules du Pacifique sud : taro, ignames, patate douce

The amino groups of insulin have been studied by 1H and 13C nuclear magnetic resonance spectroscopy in insulin, zinc-free insulin and methylated insulin. By difference spectroscopy it is possible to follow the shift with pH of the epsilon-CH2 and delta-CH2 proton resonances of lysine-B29 in insulin. In methylated insulin the dimethyl proton resonances of glycine-A1, phenylalanine-B1 and lysine-B29 can be followed as a function of pH. In native insulin pKapp values of 6.7 and 8.0 are obtained for phenylalanine-B1 and glycine-A1 (the assignment is tentative) and 11.2 for lysine-B29. Separate resonances have been observed from the lysine-B29 Nepsilon-(CH3)2 group for the monomeric and dimeric forms of methylated insulin, which indicates a small change in the environment of lysine-B29 on dimerisation. The nuclear magnetic resonance spectral characteristics of these groups are, in general, consistent with the overall structure of the crystal form of the 2-zinc insulin hexamer.

Kinetic equations previously developed for the H–D exchange of the C-2 protons of an isolated imidazole group or for an imidazole group with an adjacent charged group of pK 6–12, have been analysed in detail. By measurement of the pseudo-first-order rate constant k for exchange in D2O as a function of pD, it is possible to obtain the secondorder rate constant k2 and the pK of the histidine residue. A nearby charged group with a pK two or more units greater than that of the histidine residue, causes a double S shaped curve when k is plotted against pD, from which the pK of the nearby charged group may be determined. The size of the second step in the curve is related to the closeness of the charged group to the histidine residue. Kinetic equations have been developed for more complex cases involving (a) two adjacent imidazole rings and one adjacent charged group of pK 6–12 and (b) an imidazole group with two adjacent charged groups of pK 6–12. The equations involve numerous second-order rate constants and equilibrium constants which cannot be evaluated explicity using experimental data obtained for L-histidyl-L-histidine, L-histidyl-L-tyrosine, and L-histidyl-L-lysine. For histidine residues in proteins it is possible to determine their pK and k2 values for H–D exchange. From the magnitude of these values one can infer the type of environment of the histidine residue. Thus the data for certain histidine residues in Iysozyme, ribonuclease, myoglobin, and leghaemoglobin have been rationalised in terms of their known environments obtained from X-ray structural studies.

The methods which have been used for the observation and assignment of resonances in the NMR spectra of proteins are reviewed. One such method, the selective deuteration of the aromatic protons of tryptophyl residues, is studied by NMR spectroscopy in model compounds in this paper, and in proteins in the following paper. On the basis of a reassignment of the PMR spectrum of the aromatic protons of L-tryptophan, the relative rates of H-D exchange in deutero-trifluoracetic acid (d-TFA) are H-2 greater than H-5 greater than H-6 greater than H-4 approximately H-7. The energies of activation for the first order exchange of both the H-2 and H-5 protons is 12 k.cal.mol-1. The rate constant for exchange of the H-2 protons of tryptophyl residues in peptides is much greater than in the amino acid itself and 5-10 times that for exchange of the H-5 protons. This suggests that the method can be used to label tryptophyl residues in proteins rapidly and specifically.

The changes in nutrient content of sweet potato (Ipomoea batatas), taro (Colocasia esculenta), yam (Dioscorea alata) and cassava (Manihot esculenta) as a result of boiling, steaming and baking were determined. These changes were similar for all root crops (except in the case of sweet potato) and may be explained largely in terms of breakdown of nutrients with heat and in solubilisation of water-soluble constituents. Major changes observed were (1) an approximate doubling of the apparent dietary fibre content due to formation on cooking of starch which was resistant to degradation by the enzymes used in the starch assay procedure; (2) a decrease in the content of potassium by up to one-fifth and smaller decreases of other water-soluble salts; (3) a very large increase in the maltose content of sweet potato on cooking, which did not occur with the other root crops. In terms of human nutrition dry baking, generally speaking, appears to be the best method of cooking, followed by steaming, then boiling with retention of water and, finally, boiling with discarding of the water. However, in the particular cases of cassava and the edible aroids, which include taro, antinutritional factors may need to be removed by special cooking methods.

1. 1. The interaction of butacaine with isolated rat liver mitochondria was investigated by monitoring the proton magnetic resonance spectrum of butacaine at 10° in 0.12 M KCl in D 2 O. 2. 2. Addition of mitochondria (1 mg mitochondrial protein to 0.3 μmoles butacaine) reduces by 50 per cent the intensity of each peak of the butacaine spectrum. The residual butacaine spectrum obtained in the presence of mitochondria exhibits no chemical shift. The resolution of the butacaine spectrum is partially lost in the presence of mitochondria, but is regained following separation of the mitochondria from the mixture. The entire butacaine molecule and not specific portions of it, appears to bind to mitochondria in such a manner that the bound butacaine contributes little to the spectrum of butacaine observed in the presence of mitochondria. 3. 3. Under conditions similar to those used to study the effect of mitochondria on the spectrum of butacaine, concentrations of the local anaesthetic within the range of 100–400 μM inhibit ADP translocation by about 40 per cent. 4. 4. The results are briefly discussed in relation to the mechanism by which butacaine inhibits adenine nucleotide translocation in mitochondria.

Revue bibliographique (104 ref.) des diverses methodes d'epoxydation par des peroxydes et des peracides

The deuteration of the tryptophan residues of hen egg white lysozyme, bovine alpha-lactalbumin and bovine beta-lactoglobulin in d-TFA has been studied by PMR spectroscopy. It is found that short times of exposure to d-TFA allow selective deuteration at the C-2 position with only a small amount of deuteration at the C-5 position, as expected from studies on model peptides described in the previous paper. The proteins studied essentially regained their native structures after the treatment, except for broadening and shifting of the histidine resonances in the case of alpha-lactalbumin. Selective deuteration at the tryptophan C-2 position was readily observed by difference spectroscopy of the denatured protein, but PMR difference spectra of the same proteins in benign solvents did not contain resonances from all of the exchanged protons. Some resonances would not be observed because of line broadening, which causes the resonances to fall below the limit of sensitivity of detection at 100 MHz. Deuteration by brief exposure to d-TFA should be useful for the identification of tryptophan resonances in the PMR spectra of native proteins. The deuteration of all the aromatic protons of tryptophan residues in proteins by immersion in d-TFA for 4 hours at room temperature was studied. This technique is unlikely to be of general use for the simplification of the aromatic region of the PMR spectra of native proteins because of the degradation of tryptophan residues which results from the acid treatment.

Natural rubber dissolved in chloroform was reacted with aqueous NaOH using a phase transfer reagent or with solid NaOH to produce dichlorocarbene modified natural rubber. The 13C n.m.r. spectrum of the 100% modified natural rubber showed the presence of diastereoisomers due to two modes of addition of dichlorocarbene units as previously observed in epoxidation. There was no evidence of cis-trans iso-merisation, occurrence of other side reactions or of chain scission (shown by measurements of intrinsic viscosity). Partially dichlorocarbene modified natural rubber gave rise to a complex 13C n.m.r. spectrum that was assigned in terms of triad structures (for methylene carbon atoms)and pentad structures (for olefinic carbon atoms). Using 13C n.m.r. and measurements of glass transition temperature, it was found that reaction with aqueous NaOH and with solid NaOH both gave a random distribution of dichlorocarbene units, as compared with a block copolymer structure observed by previous workers with dichlorocarbene modified polybutadiene. The thermal stability of natural rubber is intermediate between that of the more stable 100% dichlorocarbene modified rubber and the less stable partially modified rubber.

The 13C NMR spectrum of d-biotin is completely assigned, by a study of 1H-coupled and broadband-decoupled spectra of d-biotin and four of its derivatives and also by assignment of the 1H NMR spectrum of norbotin and single-frequency 13C-{1H} decoupling of the 13C spectrum of norbotin.

Etude des structures par spectrometrie RMN de 13 C d'un pentasaccharide et d'un hexasaccharide obtenus par addition d'un ou deux groupes D-galactosyl respectivement sur l'une ou les deux bouts de chaines du lacto-N-novotetraose

The 13C nuclear magnetic resonance (n.m.r.) spectrum of sugars normally present in foods, viz. glucose, fructose, sucrose, maltose and raffinose contains resonances that are diagnostic of each sugar. The quantity of each sugar is determined by measurement of the ratios of the peak heights of the sugar resonances compared with peak height of an internal standard, present at constant concentration in all solutions. A linear calibration relationship between the peak height ratio and concentration is established for single resonances of glucose and fructose and for two resonances of each of sucrose, maltose and raffinose. Samples of sweet potato and taro flour dried at 40°C, were blended with 80% ethanol, the sugars were extracted and analysed by 13C n.m.r. and h.p.l.c. Both methods gave similar results and have similar degrees of reproducibility. The n.m.r. method is useful for quantitation of sugars and identification of other molecules extracted from the food, but requires more material than does h.p.l.c. Drying at 40°C causes a decrease in sucrose content and increases in glucose, fructose and maltose which partly compensate for the loss in sucrose. There is considerable variability of the sugar content between different sweet potato tubers and between different taro corms of the same cultivar as well as across different cultivars. For sweet potato the total sugar content is 0.6-3.6% fresh weight, with considerably more sucrose present than glucose, fructose and maltose. With taro total sugar content is 0.2-0.8% fresh weight. Raffinose is present in one sample only.

Amino groups of a series of model compounds and proteins are methylated by reductive condensation with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance experiments. The extent of methylation varies from 92% for ribonuclease A to 66% for bovine serum albumin. As observed by PMR difference spectroscopy, the conformation of most of these proteins is unaltered by the modification. Retention of native conformation by methylated lysozyme is also shown by enzymatic activity measurements and PMR observation of thermal denaturation. Resonances corresponding to each of the six Nɛ-dimethyllysine residues in methylated lysozyme are resolved at 270 MHz and titrated to obtain individual pK values. A correction factor of 0.6 pH units obtained from model compound studies is applied to obtain apparent pK values ranging from 10.1 to 10.7 for the ɛ-amino groups of lysozyme. Assignment of resonances to specific residues is accomplished using the selective broadening effect of Gd3-. These results indicate the utility of the N-dimethyl group for probing the environment of lysine residues in proteins.