Etsushiro Doi

Modified colorimetric ninhydrin methods for peptidase assay.

Abstract Four colorimetric procedures suitable for the determination of peptidase activity on peptides having a free α- or e-amino group are described. Two of the methods (A and B) are modifications of the conventional ninhydrin method described by S. Moore and W. H. Stein ((1948) J. Biol. Chem.176, 367–388; (1954) Ibid.211, 907–913); the heating time is shortened to 5 min at 100°C and the pH of the buffer in the reagent is lowered to 4.0. Method A differs from method B in buffer concentration. The other two methods (C and D) are modifications of the Cd-ninhydrin method described by A. P. Tsarichenko ((1966) Nauch. Tr. Krasnodar. Gos. Pedagog. Inst.70, 86–88, as cited in Chem. Abs.67, 79479c); the water content in the reagent is reduced to 1 20 of the original reagent and the sample is heated for 5 min at 84°C. Method C differs from method D in the ratio of sample to reagent. In contrast to the free amino acids which are sufficiently colored, various peptides and amino acid derivatives except for the glycylpeptides give only a faint color with these methods. These four methods are not only useful for the determination of peptidase activity on peptides (e.g., Leu-Gly and tert-butyloxycarbonyl-glycyl-lysyl-leucine), but are also useful for the determinations of amidase activity on amino acid amides (e.g., Leu-NH3) and esterase activity on amino acid esters (e.g., tyrosine ethyl ester).

Gels and gelling of globular proteins

Food gels have many useful properties. Globular protein gels are more nutritious than polysaccharide gels, but most are not as transparent as agarose or gelatin gels. However, by controlling the pH, ionic strength and heating procedure, it is possible to obtain desirable transparent gels from solutions of globular proteins, currently an unused source. The transparent protein gels are interesting substances in which beat-denatured proteins appear to form highly ordered linear polymers.

Irreversible thermal denaturation and formation of linear aggregates of ovalbumin

Abstract Changes in several physicochemical properties related to the thermal denaturation of ovalbumin have been investigated at neutral pH and low ionic strength. The far-UV circular dichroism (CD) spectrum at 80°C indicated small secondary structural changes compared with those induced by addition of guanidine hydrochloride (GuHCl). The near-UV CD spectrum and difference absorption spectrum (250–320 nm) showed completely irreversible micro-environmental changes around the aromatic amino acid residues upon heat treatment (at ≥67°C). In the sedimentation measurements of heated ovalbumin solutions a sharp, single peak, corresponding to soluble aggregates of low polydispersity, appeared, and these aggregates were observed as linear polymers by transmission electron microscopy. After 2 h of heating at 75°C at pH 7.0, the intrinsic viscosity was ~20 times higher than the native one. We conclude that, under these conditions, although the globule form of ovalbumin molecule did not alter drastically upon thermal denaturation, partially denatured molecules which would expose the hydrophobic area(s) aggregate immediately and linear polymers (high-molecular-weight soluble aggregates) were formed.

Structure of glycinin and ovalbumin gels

Abstract Research on the structures of heat-induced gels of glycinin and ovalbumin is reviewed. Both glycinin and ovalbumin gels are thought to have a so-called ‘string of beads’ structure. In glycinin gels one building unit of the string (a bead) has been thought to be a whole molecule. This means that heat-denatured glycinin molecules do not dissociate into their constituent subunits. An alternative explanation which includes dissociation and reassociation of glycinin has been presented. Heated ovalbumin solutions make transparent gels, turbid gels, clear solutions or turbid suspensions containing aggregates, depending on the pH, ionic strength and protein concentration of the medium. The transparent solutions contain linear polymers of high molecular weight. Ovalbumin gels are thought to be formed by a network of linear polymers. The ‘string of beads’ model has been accepted as the structure of some globular protein gels including glycinin, ovalbumin, serum albumin, ribonuclease and lysozyme. However, how the heat-denatured asymmetric protein molecules combine to make an ordered string has not been explained.

Heat-induced Transparent Gel Formation of Bovine Serum Albumin

The formation of transparent gels by 6% bovine serum albumin (BSA), pH 7.5, was examined by one- and two-step heating methods. Heating of the BSA solutions at various NaCl concentrations produced transparent gels at 25-50mM NaCl and transparent sols at 0-20 mM NaCl (one-step heating method). The transparent sol obtained by heating without NaCl was reheated after mixing with various amounts of NaCl (two-step heating method I). The result was almost identical to that obtained by the one-step heating method. However, when the first heating was done with 10 mM NaCl, transparent gels were obtained over a wide range of NaCl concentrations with a second heating (two-step heating method II). Analyses of sols obtained at various NaCl concentrations by gel permeation chromatography and transmission electron microscopy showed the presence of linear polymers in the heated BSA sol (10 mM NaCl) and gel networks formed by the linear polymers (20 mM NaCl). The mechanism of transparent gel formation in BSA may be similar to that in ovalbumin.

Melting of heat-induced ovalbumin gel by pressure

The effects of pressure on heat-induced gels of ovalbumin were examined. A heat-induced gel containing 7% ovalbumin and 10 mmol/dm3 Na-phosphate buffer, pH 7.0, melted completely with pressure at 600 MPa for 20 min at 20°C. The melted sample gelled again after the pressure ceased. Most of the heat-induced gels of ovalbumin prepared under various conditions (different protein concentrations, ionic strengths, pHs and heating methods) melted partly with pressure at 600 MPa for 20 min at 20°C, and gelled again at atmospheric pressure. However, a heat-induced gel of glycinin and cold-set gels of gelatin or agarose did not melt with pressure at 400–700 MPa for 20 min at 20°C. The pressure denaturation of ovalbumin and the renaturation of heat-denatured ovalbumin caused by pressure were also examined. The pressure denaturation of ovalbumin was partly reversible and heat-denatured ovalbumin renatured somewhat with treatment at 600 MPa for 20 min at pH 7.0 and low ionic strength.

Effects of limited proteolysis on functional properties of ovalbumin

We examined the limited proteolysis of ovalbumin by pepsin and its effect on the functional properties of the ovalbumin. Pepsin hydrolyzed only the single peptide bond of ovalbumin between His-22 and Ala-23. This provided a large intermediate (MW 42,500), P-ovalbumin. A P-ovalbumin solution gave a transparent gel when heated. Under the same conditions, an ovalbumin solution gave a turbid gel. We studied the physicochemical properties of P-ovalbumin and the formation of the transparent gel.

Improvement of protein gel by physical and enzymatic treatment

Abstract To improve the functional properties of food protein, physical and enzymatic treatments are effective and attractive. As an example of physical treatment, heat treatment of egg protein under various conditions is shown. Egg albumin and egg white usually give turbid (white) gel on heating. However, we have learned that a transparent gel can be prepared by regulating conditions of the medium. The molecular mechanism for formation of such gels and the physical properties of transparent and turbid gels are shown. Proteolytic enzymes are often used to improve the properties of food protein, but sometimes bitter peptides are formed. Therefore, a limited pro‐teolysis rather than nonspecific hydrolysis is preferable and effective in order to change the functional properties. We have shown that pepsin promotes limited proteolysis under pH control. Not only proteolytic enzyme, but other enzymes are also useful for improving food protein. Possibilities of the use of enzymes for food processing are shown here.

Formation of a new toxic compound, citrinin H1, from citrinin on mild heating in water

Citrinin, a relatively weak mycotoxin, can be detoxified by heating; however, a compound having high toxicity was formed on heating at 140 °C in the presence of water. The toxic compound was isolated from heated citrinin and its structure was determined. Its toxicity, evaluated by cytotoxicity assay, was 10-fold higher on a weight basis than that of citrinin. This new compound was named citrinin H1, which was also formed by heating citrinin at 100 °C for 30 min.

Toxicity evaluation of the mycotoxins, citrinin and ochratoxin A, using several animal cell lines

1. The cytotoxicities of the nephrotoxic mycotoxins, citrinin and ochratoxin A were assayed on HeLa, C3H/10T1/2, NIH/3T3, MDCK (canine kidney), and HeLa P3 cell lines, using the MTT colorimetric assay. 2. Citrinin was less toxic than ochratoxin A in all of the cell lines examined. 3. The MDCK cells were more susceptible to both citrinin and ochratoxin A, in comparison with other cell lines. 4. Dose-responses, as measured by activities of leucine aminopeptidase and alkaline phosphatase of MDCK cells, were less sensitive than MTT colorimetric assay, indicating that these enzymes were not specifically inhibited in MDCK cells. 5. The LD50 of both toxins, calculated at 72 hr incubation, was in the same order as those reported from animal experiments using rats and mice.