Dye-binding Capacity Research Articles

Relationship Between Nutritional Quality Characters and Grain Yield in Pearl Millet1

Before selecting genotypes for such quality characteristics as protein or amino acid contents, it is important to know how such selection is likely to affect yield. Using a diverse range of germplasm grown over seasons, locations and years, the relationships that exist between protein content, dye binding capacity (DBC) per unit protein (an estimate of the basic amino acids in protein), grain yield and grain size in pearl millet [P. americanum (L.) Leeke] were investigated. Protein content was estimated as total N ✕ 6.25, total N being measured with an automatic analyzer. Basic amino acid content was estimated using the Udy DBC technique. Grain yield and protein content were not significantly correlated and the relationship between grain yield and DBC/protein was nonsignificant and negative. The relationship between protein and DBC/protein was strongly negative. These results suggest that in pearl millet it should be possible to select for increased protein content or basic amino acid content (of which lysine is the most important) by monitoring levels of lysine and protein, respectively, without detrimental effect on grain yield or grain weight.

Effects of maternal nutritional stress on synaptic plasma membrane proteins and glycoproteins in offspring

An earlier study in this laboratory demonstrated a 33 to 50% deficiency of synaptic plasma membrane (SPM) proteins in the brains of developing offspring of rats that were fed a protein-deficient diet during lactation. We have demonstrated that an SPM deficit persisted if the nutritional stress (in this case postnatal protein-calorie deficiency) was continued on a long-term basis. If nutritional rehabilitation was instituted on the 21st postnatal day, however, the SPM deficit was reversible. We also demonstrated abnormalities in the synthesis of fucosylated SPM glycoproteins in the developing offspring rats subjected to protein or protein-calorie deficient diets and in rehabilitated offspring. These abnormalities were detected using sensitive double-label isotopic procedures. Few abnormalities were found in the relative distribution of the dye-binding capacity of SPM proteins from developing control, undernourished, or rehabilitated rats on polyacrylamide gels that had been stained with Coomassie blue. The potential cause and importance of abnormalities in synaptic glycoproteins is discussed.

Changes in the stainability and sulfhydryl level in the sperm nucleus during sperm‐oocyte interaction in mice

AbstractDuring maturation in the epididymis, mouse sperm nuclei become difficult to stain with Giemsa and its component basic dyes. Mature sperm from the cauda epididymis can be stained only after DTT treatment. Stainable sperm such as those from the testis accumulate 3H NEM when examined by autoradiography, while unstainable sperm do not, indicating a close correlation between the basic dye binding capacity and SH levels in the sperm nuclei. During insemination of zonafree ovarian oocytes with a germinal vesicle (GV), mature sperm nuclei become stainable and capable of binding with 3H NEM. At the same time, sperm have established pronase‐resistant contact with the oocyte. Similarly, sperm nuclei become stainable during fertilization when the sperm attachment to the egg becomes pronase resistant. However, these changes occur before sperm chromatin decondensation begins. Therefore, it is suggested that S‐S bonds in sperm nucleoproteins are reduced when the sperm establish a stable contact with the egg plasma membrane, thus reversing sperm maturational changes. The reduction of S‐S bonds may be a prerequisite for sperm chromatin decondensation.

Indomethacin and bilirubin-albumin binding.

We studied the effects of indomethacin on the bilirubin binding of albumin using the hydroxybenzene azobenzoic acid (HBABA) dye binding method, the estimation of unbound bilirubin concentration (UBC) by the horseradish peroxidase assay and the whole blood fractionation (WBF) technique. The addition of up to 2 micrograms/ml of indomethacin, which is the level obtained with an 0.2 mg/kg dose, did not decrease the mean HBABA dye binding capacity of 29 icteric sera or increase the mean UBC of 11 icteric sera. There was also no additional displacement of bilirubin in 5 icteric sera measured by the WBF technique. However, the addition of 10-40 micrograms/ml of drug, which is the level that would be achieved if a 2 mg/kg dose of indomethacin were administered, resulted in a significant reduction of HBABA in 15 icteric sera (81.74-57.3%, p less than 0.005) and a significant increase of UBC in 11 icteric sera (6.17-37.2 nM/l, p less than 0.001). Therefore, no displacement of bilirubin from albumin occurred in the presence of usual therapeutic level of indomethacin; higher levels displaced bilirubin.

Methods for the estimation of protein in pigeonpea [Cajanus cajan (L.) Millsp.] and the relationship between whole grain and dhal protein contents

AbstractProtein determinations of 172 grain and dhal (decorticated dry split seed) pigeonpea samples were carried out using three methods: (a) micro‐Kjeldahl (MKJ), (b) colorimetric estimation of NH4+ with phenol hypochlorite reagents using the Technicon Auto‐Analyser (TAA), and (c) dye‐binding capacity (DBC) method using Acid Orange 12 dye. Protein percentages determined by the TAA and MKJ methods were highly correlated for whole‐grain (0.948**) and dhal (0.967**) samples. The DBC method gave reliable results for dhal samples only. In the DBC procedure, higher protein percentages were recorded with smaller flour particles and longer mixing time, but different temperatures and durations of heating had no effect. Positive and highly significant correlations were obtained between the protein values of whole‐grain and dhal samples in all the methods. Small grains gave a lower correlation between whole‐grain and dhal protein content due to the observed negative correlation between grain size and percentage of seed‐coat.

Relative changes in the RNA contents of oocytes and preimplantation embryos of the mouse.

Changes in Azur B basophilia of isolated oocytes and preimplantation embryos of the mouse have been quantitated cytophotometrically. Components of the nuclei and cytoplasm bound Azur B but most of the dye-binding capacity was lost after RNase treatment indicating that RNA was the main basophilic constituent. The amount of dye bound by oocytes of the resting pool was relatively low but increased during growth reaching a peak in oocytes of about 100 microns in diameter. Thereafter, there was a significant decline during progression through to ovulation. No change in dye-binding was observed between ovulation and the 1 cell embryo stage. However, there were significant declines between the 1 and 2 cell stages and between the 2 and 4 cell stages. Azur B basophilia increased again in the 8 cell embryo and was very high in the blastocyst 5 days after ovulation. These data are discussed in relation to the storage of developmental information in the oocyte and its utilization during early embryonic development.

PROTEIN QUALITY EVALUATIONS OF COMMERCIAL RAPESEED MEALS BY CHEMICAL AND BIOLOGICAL ASSAYS

Fourteen commercial rapeseed meals (RSM), selected from a total of 63 samples received from six rapeseed processing plants, and one sample of soybean meal (SBM) were tested for protein quality in a chick growth trial. The RSM studied consisted of six pairs, of which each pair of meals represented the meals with the highest and lowest protein solubilities in 0.2% KOH solution among samples received from each plant and one pair of RSM selected in a similar manner from among a second set of RSM samples supplied by one of the processing plants. The protein solubilities of the RSM used in the growth trial ranged from 33.9 to 72.6%. However, the protein quality of the same meals determined biologically by the total protein efficiency (TPE) method were not significantly different (P < 0.05). The TPE values of the RSM varied from 2.57 to 2.71 and were not significantly different from that of soybeam meal (2.65). These results indicate that determination of protein solubility of RSM in 0.2% KOH solution is not a suitable laboratory method of predicting the protein quality of commercial RSM. Other analyses performed on the RSM samples included determination of basic amino acid composition, available lysine content and dye-binding capacity of protein (DBCP) with Acid Orange 12. Of these analyses, only the DBCP values of the meals were found to be significant (P < 0.05) correlated with the TPE values.

Effect of 5-iodo-2′-deoxyuridine on physical properties and nonhistone chromosomal proteins of chromatin from Burkitt somatic cell hybrids

A Burkitt somatic cell hybrid line, D98/HR-1, contains latent Epstein-Barr virus genome which can be induced to express by 5-iodo-2′-deoxyuridine (IdUrd). Chromatins were isolated from confluent monolayers of D98/HR-1 grown in the presence and absence of IdUrd and its effect on the chromatin was studied by circular dichroism, ethidium bromide binding capacity, and template activity. IdUrd-Treated chromatin consistently exhibits a 20 to 25% decrease in positive ellipticity of circular dichroism at 275 nm. Scatchard plots of spectrophotometric titration of chromatin with ethidium bromide indicate a 25% reduction of the number of primary binding sites in IdUrd-treated chromatin which corresponds with a 25% reduction in template activity of the same chromatin. These effects are reversible when IdUrd-treated cells were released into IdUrd-free medium for 3 h. However, the differences in circular dichroism spectra and dye binding capacity are not observed between DNA isolated from IdUrd-treated and nontreated cells. These results suggest that chromosomal proteins or modification of protein-DNA interactions are responsible for the alterations. Polyacrylamide gel electrophoretic analysis of the chromosomal proteins reveals that a class of nonhistone chromosomal protein in the high molecular weight region (above 130K) is lacking in IdUrd-treated cells.

Evaluation of rapid methods for the estimation of protein in chickpea (Cicer arietinum L.)

AbstractProtein content in chickpea (Cicer arietinum L.) was determined by the following rapid procedures: (a) colorimetric estimation of ammonia with phenol‐hypochlorite reagents using the Technicon auto‐analyzer (TAA) method, (b) dye‐binding capacity (DBC) method using acid orange‐12 dye, and (c) modified biuret methods of Pinckney (B1) and Johnson and Craney (B2). Results obtained with the above procedures were compared statistically with the standard microKjeldahl (MKJ) procedure. Correlation of MKJ protein values with TAA, DBC, B1 and B2 methods were 0.99, 0.98, 0.96 and 0.95, respectively. Standard errors of estimation obtained by methods B1 (0.99) and B2 (0.95) were high when compared to the TAA (0.55) and DBC (0.69) methods. Possible interference of seed‐coat pigments, effect of flour particle size, and time of shaking on protein estimation by the DBC and B1 methods were also studied. Implications of these results are discussed with reference to adapting any of these rapid methods as a routine screening procedure for the estimation of protein in large numbers of chickpea samples in a breeding programme.

Immediate Effect of Male and Female Gametes and N on Five Pearl Millet Grain Characteristics1

Pearl millet, Pennisetum americanum (L.) Leeke, inbreds ‘Tift 12,’ ‘Tift 13’, ‘Tift 18DB’, ‘Tift 23DB’, ‘Tift 239DB’, and ‘Bil 3B’ were grown in the field in plots fertilized with 15, 80, or 150 kg/ha of N with P and K adequate to study the immediate effect of male and female gametes and N on five pearl millet grain characteristics. Plants were replicated four times. Within each N‐treatment plot selfed, sibbed, open‐pollinated, and intermated seed was produced so that each genotype supplied both female and male gametes. Grain characteristics determined were thousand‐kernel weight (TKW), total amino N and proline (Pro) N of grain acid hydrolysates, N/Pro ratio, and dye‐binding capacity (DBC). Nitrogen treatments affected all grain characteristics except the N/Pro ratio, female gametes affected all grain characteristics, and male gametes affected TKW, proline and the N/ Pro ratio significantly. The female effect was greater than the male effect. Open‐pollinated seed was significantly higher than self‐pollinated seed in all characteristics measured. The data show that pollen source has an immediate effect on three of the pearl millet grain characteristics measured. They also indicate that selfpollinated seed of pearl millet and perhaps other cross‐pollinated crops should be used for the most accurate assessment of grain characteristics of any genotype. A minimum seed population and a standard pollen source could serve as an alternate but less desirable substitute for selfed seed for crops with a high degree of self‐incompatibility.

REACTIVE LYSINE IN FOODSTUFFS AS MEASURED BY A RAPID DYE‐BINDING PROCEDURE

ABSTRACTThe dye‐binding capacity (DBC) of basic groups in the proteins of test materials for Acid Orange 12 is measured before and after reaction with propionic anhydride to block the lysine groups. The difference in moles dye bound as a result of the reaction agrees well with the lysine moles present, as determined with fluoridinitrobenzene and by ion‐exchange chromatography, for over 50 samples‐fish, meat, milk, barley, peanut, grain legumes etc. The procedure takes approximately 40 min using commercial dye‐binding equipment and longer using normal laboratory apparatus. It appears suitable for plant breeding studies and for quality control of damage to lysine in processed foods.

DYE BINDING PROCEDURE FOR THE ESTIMATION OF PROTEIN CONTENT OF MEAT COMPONENTS AND SAUSAGE EMULSIONS

ABSTRACTThe dye binding technique was investigated for its ability to determine the protein content in a processed meat product. Frankfurter emulsions were prepared with varying protein content by adjusting the fat content of the emulsions. The unprocessed (raw) emulsion and the processed (cooked, smoked) emulsion were tested with the dye binding technique using Acid Orange 12 (C.I. 15870). In both the processed and in processed emulsion products it was found that the bound dye parameter alone could be used to monitor the protein content. A regression equation containing two variables, the reciprocal of the dye binding capacity and the bound dye, would account for over 90% of the variation in the protein content of a processed emulsion product.

APPLICATION OF THE DYE-BINDING TECHNIQUE FOR QUANTITATIVE AND QUALITATIVE ESTIMATION OF RAPESEED MEAL PROTEIN

The linear equation Y = 2.2 + 0.27X relating dye-binding capacity with Acid Orange 12 (X) and Kjeldahl crude protein content (Y) of rapeseed meal, derived previously, was applied to 126 commercial samples for estimating the protein contents of the meals. Results indicated that the means of crude protein contents obtained by both dye-binding and Kjeldahl nitrogen analyses were comparable. However, comparisons based on individual samples showed that the dye-binding method underestimated or overestimated the protein content of about 20% of the samples by 1% or more. The deviation was caused mainly by the atypical content of basic amino acids, particularly of lysine, in these meals. Application of the equation for quantitative prediction should, therefore, be limited to rapeseed meals which have been properly processed. On the other hand, the correlations noted between the dye-binding capacity of protein (DBCP, mg Acid Orange 12/g protein) of 21 selected rapeseed meals and the lysine and available lysine contents of the meals (r = 0.84 and 0.79) showed that the ability of the protein to bind Acid Orange 12 may be used as a protein quality index of the samples. This potential was further investigated by studying the effects of autoclaving for varying periods of time at 121 °C on the DBCP of rapeseed meal protein. In this regard, a significant reduction in DBCP of the meals was noted after 45 min of heating. Available lysine values were reduced by autoclaving at a more rapid rate than DBCP values.

THE APPLICATION OF THE DYE-BINDING METHOD FOR MEASURING PROTEIN DENATURATION OF RAPESEED MEALS CAUSED BY AUTOCLAVE OR OVEN HEAT TREATMENTS FOR VARYING PERIODS OF TIME

Laboratory-prepared rapeseed meals (RSMs) with moisture contents ranging from 2 to 40% of the meal were heated in an autoclave or an oven at 121 °C from 15 min to 4 h. The degree of protein denaturation of the RSMs was estimated by nitrogen solubility (NS) in water and by dye-binding capacity of the protein (DBCP) with Acid Orange 12. The NS study indicated that heating RSM for 15 min to 1 h in an autoclave produced more severe denaturation than heating RSM at a similar temperature for the same periods of time in an oven. When heating in the autoclave was prolonged, the nitrogen solubility increased. The ability of the RSM protein to bind Acid Orange 12 was more severely affected by the autoclave treatment than by oven heating. In this regard, the DBCP of the RSMs was at the lowest after 2 h of heating in the autoclave and after 4 h of heating in the oven. Since the NS values for the RSMs include both protein and non-protein nitrogen while DBCP values measure mainly basic amino acids, the latter values are likely to be more valid for estimating the nutritive value of RSM. The higher correlation coefficient between DBCP and available lysine than between NS and available lysine (0.90 vs. 0.63) would seem to support this conclusion.

Application of a semi-automatic micro-DBC analyzer system for screening basic amino acids in single grains

The micro-DBC method (dye binding capacity) developed for single seeds is based on the FP-9 Analyzer System, which was originally developed for medical research. With a few extra devices it was adapted for seed protein micro-DBC determination. With the new analyzing system it is possible to calculate the amount of acid orange 12 dye bound by a single grain, determined per unit weight of the grain. In the FP-9 Analyzer System, nine samples are handled simultaneously at all working stages. The system includes 9-tipped pipettes, a 9-tipped automatic diluter, scales, a grinder for 9 grains, a mixer for 8 x 9 samples and a centrifuge for 4 x 9 samples, and a photometer for 9 samples as well as a programmable desk calculator connected to it. The largest volume to be pipetted is 1 ml. Twentyone barley lines, donated by Dr. L. Munck, Carlsberg Laboratory, Denmark, were studied with the FP-9 Analyzer System. From each barley line, 4—12 grains were studied. The correlation coefficient (r) between their means and the results obtained by the macro-DBC system was 0.9600***. The coefficient of variation (C.V. %) within the barley lines was 4.74 —23.28 %. With homogeneous standard flour the coefficient of variation was 1.81 —3.79 % for eight cuvette blocks with 9 samples each. The micro-DBC system has been specially developed for plant breeding purposes. Screening of material can be carried out on single or half grains in segregating early generations (F2—F5). Thus the cultivation of large line materials can partly be avoided.

Human cytomegalovirus-induced chromatin factors responsible for changes in template activity and structure of infected cell chromatin

Human cytomegalovirus (HCMV) infection in human embryo lung cells caused rapid changes in conformation of chromatin as judged from circular dichroism (the ellipticity at 272 run) of the infected cell nuclei or chromatin determined at intervals after infection. The infection also caused a rapid increase in chromatin template activity determined by (i) the incorporation of [ 3H]UTP into RNA in the presence of saturating levels of E. coli RNA polymerase and (ii) the number of binding sites for ethidium bromide in chromatin. These conformational and functional changes did not occur when cycloheximide was present in the cultures or when cells were infected with UV-irradiated virus, suggesting the dependency of the chromatin changes on HCMV-coded functions expressed after infection. Chromatin reconstitution experiments in which NaCl extracts from HCMV-infected cell chromatin were added to uninfected cell chromatin and the reconstituted chromatin was subjected to the determination of ellipticity at 272 nm and dye-binding capacity showed that HCMV induced at least two chromatin factors; one responsible for an increase in ellipticity at 272 nm of chromatin was selectively extractable with 0.35 – 0.6 M NaCl from HCMV-infected cell chromatin and the other responsible for an increase in dye-binding capacity (corresponding to chromatin template activity) was extractable with 0.35 M NaCl. These chromatin factors had properties of DNA-binding proteins; the former was dissociated from DNA at 0.6 M NaCl and the latter at 0.35 M NaCl.

The effect of chlorhexidine and cetylpyridine on the binding of amaranth to saliva-coated hydroxyapatite.

The effects of chlorhexidine (CHX) and cetylpyridine (CPC) on the binding of an acidic food‐dye (amaranth) to hydroxyapatite treated with salivary proteins were studies. Apatite treated with proteins and CHX or CPC bound considerable amounts of amaranth, whereas protein‐coated apatite did not. The dye binding capacity of untreated apatite was dependent on the amount of CHX or CPC absorbed. CHX displayed much greater efficiency than CPC in promoting binding of dye. A mechanism of bisbiguanide‐ or quaternary ammonium‐mediated tooth stain formation is discussed.

Use of Aspergillus flavus to evaluate relative nutritive value of barley cultivars intended for incorporation in animal diets

Thrity-six barley cultivars were analyzed for relative nutritive value, using the fungusAspergillus flavus. The protein content ranged from 7.8 to 14.7% and dye-binding capacity (DBC) ranged between 180 to 260. When additional glucose was not supplied in the medium containing barley samples at equal nitrogen level the biomass was low (76.3 mg average) and the correlation between the biomass and protein % and DBC were r=−0.97** and −0.65** respectively. When additional glucose was provided, theAspergillus biomass increased significantly (114.5 mg average) but the correlation between the biomass and protein % or DBC was reduced. When extra glucose was supplied at the start of the experiment, some native starch was left unutilized and this was avoided by incorporating glucose 43 h after the start of the experiment. In such experiments the biomass improved significantly in some samples and a better correlation between the biomass and protein % and DBC was noticed (r=−0.70** and −0.53**) respectively.

THE ESTIMATION OF CRUDE PROTEIN IN RAPESEED MEAL BY A DYE-BINDING METHOD

The relationships between Kjeldahl protein content and the dye-binding capacity (DBC) with Orange G and Acid Orange 12 of 15 rapeseed meals were studied. The unbound dyes were measured with a Beckman spectrophotometer and also by the Udy colorimeter in the case of Acid Orange 12. The correlation between DBC and Kjeldahl protein content or total basic amino acids was highly significant. The results favored the Udy method with Acid Orange 12.

Peptidases and Trypsin Inhibitor in the Developing Endosperm of Opaque‐2 and Normal Corn1

AbstractTo determine the nature of the association of trypsin inhibitor content to lysine content in high‐lysine corn (Zea mays L.), the relationship of proteolysis, trypsin inhibitor content, and lysine content was studied during endosperm development in opaque‐2 and genetically similar normal corn. Peptidases with pH optima of 4.0, 5.5, 7.5, 8.4, and 10.0 capable of hydrolyzing α‐N‐benzoyl‐dl‐arginine‐p‐nitroanilide (BAPA) were extracted from the endosperm of developing field grown corn. These peptidases were inhibited by the endogenous trypsin inhibitor and were also unable to hydrolyze bovine albumin, wheat (Triticum aestivum L.) gliadin, zein, and Azocoll (Cal biochem general proteolytic substrate). Significant correlations were found between trypsin inhibitor content and dye binding capacity, a measure of lysine content, between trypsin inhibitor and pH 9.0 optimum peptidase, between the pH 9.0 optimum peptidase and lysine content, and between the pH 5.0 optimum peptidase and water‐soluble protein content. Higher trypsin inhibitor levels found in opaque‐2 corn were associated with higher albumin levels. Normal corn was lower in peptidase, lysine, trypsin inhibitor, and moisture than the genetically similar opaque‐2 line.