James C. Zahnley

Estimation of the disulfide content of trypsin inhibitors as S-β-(2-pyridylethyl)-l-cysteine

Abstract Disulfide bonds in soybean trypsin inhibitor were reduced with tri- n -butylphosphine in 50% ethanol at pH 7.6. The generated SH groups were simultaneously alkylated with 2-vinylpyridine. Cystine residues were thus transformed to residues of S -β-(2-pyridylethyl)- l -cysteine. The extent of reductive alkylation was determined by both amino acid analysis and ultraviolet spectroscopy. The modified inhibitor was inactive against trypsin. It also lost resistance to proteolysis. Analogous results were obtained with partially reduced alkylated lima bean and ovomucoid trypsin inhibitors. These results show that S -2-pyridylethylation is a convenient method for measuring the half-cystine content of a protein.

A mutant strain of Leuconostoc mesenteroides B-1355 producing a glucosyltransferase synthesizing α(1→2) glucosidic linkages

A mutant strain (R1510) of Leuconostoc mesenteroides B-1355 was isolated which synthesized primarily an insoluble polysaccharide and little soluble polysaccharide when grown in sucrose-containing medium. Glucose or sucrose cultures of this strain produced a single intense band of GTF-1 activity of 240 kDa on SDS gels, and a number of faint, smaller bands. Oligosaccharides synthesized by strain R1510 from methyl-α-D-glucoside and sucrose included a trisaccharide whose structure contained an α(1→2) glucosidic linkage. This type of linkage has not been seen before in any products from strain B-1355 or its mutant derivatives. The structure of the purified trisaccharide was confirmed by 13C-nuclear magnetic resonance. The insoluble polysaccharide also contained α(1→2) branch linkages, as determined by methylation analysis, showing that synthesis of the linkages was not peculiar to methyl-α-D-glucoside. GTF-1, which had been excised with a razor blade from an SDS gel of a culture of the parent strain B-1355, produced the same trisaccharides as strain R1510, showing that GTF-1 from the wild-type strain was the same as GTF-1 from strain R1510. Mutant strains resembling strain R1510, but producing a single intense band of alternansucrase (200 kDa) instead of GTF-1 were also isolated, suggesting that mutations may be generated which diminished the activities for any two of the three GTFs of strain B1355 relative to the third. Strain R1554 produced a soluble form of alternansucrase, while strain R1588 produced a cell-associated form. The mechanism(s) by which specific GTFs become associated with the cells of L. mesenteroides was not explored.

Effects of manganese and calcium on conformational stability of concanavalin A: A differential scanning calorimetric study☆

The effect of degree of saturation of concanavalin A with Mn2+ or Ca2+, or both, on its thermal denaturation was investigated by differential scanning calorimetry. Acid-demetallized concanavalin A was partly or fully remetallized in acetate buffer (pH 5.0) containing 0.4 to 0.5 M NaCl. Under these conditions, native dimeric concanavalin A is highly stable, undergoing heat denaturation at 101 degrees C, with an enthalpy of denaturation of 7.4 cal/g. Removal of metal ions lowered stability considerably; concanavalin A with 0.06 Mn2+/monomer and 0.23 Ca2+/monomer (mol/mol) was denatured at 74 degrees C with an enthalpy of denaturation of 3.2 cal/g. Added Mn2+ stabilized demetallized concanavalin A, but added Ca2+ alone (up to 2 mol/mol monomer) did not. The Ca2+/ concanavalin A ratio influenced stabilization by Mn2+. In the presence of 1 to 2 Mn2+/ monomer and 0.5 or less Ca2+/monomer (mol/mol), stabilized concanavalin A was denatured at 85-88 degrees C and at 94-97 degress C, indicating presence of two stabilized metallo-concanavalin A species. At 1.0 or more mole each of Mn2+ and Ca2+ per monomer, one endotherm was observed at or above 98 degrees C and the enthalpy of denaturation was increased to 5.3 cal/g from less than 3.6 cal/g at lower metal ion/protein ratios. Stabilization was greater with Mn2+ plus Ca2+ than with Mn2+ alone, consistent with intrasubunit cooperativity in metal ion-induced stabilization of concanavalin A.

Association of Tenebrio molitor L. α-amylase with two protein inhibitors - one monomeric, one dimeric - from wheat flour: Differential scanning calorimetric comparison of heat stabilities

Thermal stabilization resulting from protein . protein association between two protein inhibitors (coded as 0.19, a dimer, and 0.28, a monomer) from wheat flour and the alpha-amylase from Tenebrio molitor L. (yellow mealworm) larvae was investigated by differential scanning calorimetry (heating rate 10 degrees C/min). Thermograms (plots of heat flow vs. temperature) for the two inhibitors showed broad endothermic peaks with the same extrema (denaturation temperatures) at 93 degrees C, and equal, small enthalpies of denaturation (2 cal/g). The amylase produced a sharp endotherm at 70.5 degrees C, but a larger enthalpy change on denaturation (6 cal/g). The amylase . inhibitor complexes differed in thermal stability, but both showed significant stabilization relative to free enzyme. The complex formed with monomeric inhibitor 0.28 showed a higher denaturation temperature (85.0 degrees C) than that formed with dimeric inhibitor 0.19 (80.5 degrees C). This order of stabilization agrees with the relative affinities of the inhibitors for the amylase. These thermograms are consistent with previous results which indicated that 1 mol of amylase binds 1 mol of inhibitor 0.19.

A simple preparation of β-trypsin based on a calorimetric study of the thermal stabilities of α- and β-trypsin

Abstract Bovine trypsin preparations contain, in addition to the single chain form of the enzyme, an active two-chain autolysis product (Schroeder, D. D., and Shaw, E., J. Biol. Chem. (1968), 243 , 2943–2949). Differential scanning calorimetric (DSC) studies showed that the single chain form, β-trypsin, is more stable to thermal denaturation than the two-chain form, α-trypsin. Rate constants and activation energies for the thermal denaturation of β-trypsin are 5 × 10 −5 sec −1 and 69 kcal/mole and of α-trypsin are 5 × 10 −3 sec −1 and 38 kcal/mole at pH 4.4 and 48 °C. Preparation of pure β-trypsin can be greatly simplified by prior thermal denaturation of the α form. At least 75% of the α form is denatured by heating a 10–15% solution of commercial crystalline trypsin for 30–45 min at 48 °C, pH 4.4, 0.02 m Ca 2+ . The native β-trypsin is then easily isolated from the denatured α-trypsin by batchwise adsorption onto ovoinhibitor-agarose at pH 8. After elution at pH 2, dialysis, and lyophilization an average preparation contained approximately 85% β-trypsin, 10% α-trypsin, and 5% inactive material. Benzamidine was used during the isolation to decrease the rate of conversion of β- to α-trypsin. Because the separation of active β-trypsin from heat-denatured α-trypsin is relatively easy, the total preparation time has been reduced to 1 day.

Production of glucosyltransferases by wild-type Leuconostoc mesenteroides in media containing sugars other than sucrose

Leuconostoc mesenteroides produces glucosyltransferases (GTFs) and fructosyltransferases (FTFs) which are inducible enzymes which respectively synthesize dextrans and levans from sucrose. Except for a few mutant strains which produce high activities in glucose medium, L. mesenteroides is thought not to produce GTFs and FTFs unless sucrose is present. We show here that cultures of eight strains produced low, but detectable GTF activity when glucose, maltose or melibiose replaced sucrose as the growth substrate. Four strains also produced FTFs of approximately 130 kDa in medium with or without sucrose. The GTFs and FTFs produced on sugars other than sucrose could be detected as bands on SDS gels even when not detected by other methods. Except for strain B-523, the number, sizes and relative intensities of the bands were independent of the sugar used for growing the cultures. Alternansucrase from strains B-1355 and B-1501 in glucose or maltose medium was almost entirely associated with the cell fraction, ruling out binding to glucans as the cause of the association.

Stability of Enzyme Inhibitors and Lectins in Foods and the Influence of Specific Binding Interactions

Proteins with actual or potential antinutrient or toxicant activity found in foodstuffs include (1) enzyme inhibitors, especially those specific for serine proteinases and alpha-amylases, and (2) lectins (hemagglutinins). These inhibitors and lectins must be inactivated during processing or food preparation, usually by heat, to avoid possible undesirable effects. Knowledge of their heat stabilities thus helps determine conditions required for their inactivation or denaturation. Many are heat-stable proteins, and their conformations can be stabilized or destabilized by interactions with other constituents present in the food or the digestive tract. Differential scanning calorimetric (DSC) results show that specific binding interactions can lead to substantial increases in kinetic thermal stability of proteins. Examples of such stabilization include serine proteinase-proteinase inhibitor, alpha-amylase-amylase inhibitor, and metal ion-lectin complexes. The extent of thermal stabilization of proteinases in complexes with inhibitors is correlated with the equilibrium association constant. Presence of more than one denaturing unit revealed by DSC in complexes involving multiheaded inhibitors can be interpreted in relation to domain structures of the inhibitors. Basic information on stability of the enzyme inhibitors and lectins is relevant to food processing, quality, and safety.

Cellular association of glucosyltransferases in Leuconostoc mesenteroides and effects of detergent on cell association

Most glucosyltransferase (GTF) activity in sucrose-grown cultures of some strains of Leuconostoc mesenteroides is found with the cell pellet after centrifugation. GTFs are known to bind to dextrans, and it was traditionally assumed that cell-associated GTFs were bound to those dextrans that cosedimented with the cells. We used a mutant strain (LC-17), derived from strain NRRL B-1355, which produced dextransucrase in the absence of dextrans, to investigate the extent to which GTFs were bound to cells or dextrans. Much of the GTF activity in glucose-grown cultures of strain LC-17, which do not produce dextran, was located in the cell pellets. Soluble enzyme activity increased when cell suspensions from glucose- or sucrose-grown cultures were incubated with mild nonionic detergents or zwitterionic reagents. Alternansucrase produced by the parent strain B-1355 was almost entirely associated with cells under conditions in which dextrans were or were not produced. Alternansucrase, but not dextransucrase, tended to be enriched in the particulate fraction of B-1355 cells that had been broken in a French press. The distribution of alternansucrase and the effects of detergents on the distribution of GTFs suggest that soluble GTFs sequestered in the cytoplasm, and GTFs bound or adsorbed to the cell membrane are probably the major contributors to the cell-associated GTF activity.

Inactivation of Metalloenzymes by Lysinoalanine, Phenylethylaminoalanine, Alkali-Treated Food Proteins, and Sulfur Amino Acids

Synthetic lysinoalanine (LAL) may be a more effective inhibitor of the zinc-containing enzyme carboxypeptidase A than is ethylenediamine tetraacetic acid (EDTA). The enzyme is also inactivated by alkali-treated, lysinoalanine-containing food proteins such as casein, high-lysine corn protein, lactalbumin, soy protein isolate, and wheat gluten, and by alkali-treated zein, which contains no lysinoalanine. Zinc sulfate regenerates only part of the enzymatic activity after exposure to the treated proteins. The extent of inhibition increases with protein concentration and time of treatment. Any inhibition due to phytate is distinct from that due to the treatment. Phenylethylaminoalanine (PEAA), derived from biogenic phenylethylamine, inhibited enzymatic activity of the metalloenzyme carboxypeptidase A (CPA). The inhibition was maximal at pH 7.0 in the pH range 7 to 8.5. The extent of inhibition increased with time of treatment and PEAA concentration. N-acetyl-PEAA did not inhibit the enzyme, suggesting that the free alpha-NH2 group is required for inhibition. PEAA, LAL, sodium phytate, and cysteine also inactivated the copper enzyme, polyphenol, oxidase (tyrosinase) which plays a major role in enzymatic (oxidative) browning of foods. Analogous comparative studies with LAL, EDTA, and sodium phytate suggest that the potency of PEAA as an inhibitor of CPA is similar to that of sodium phytate, and that of the four compounds tested, PEAA is least effective against tyrosinase. Related studies of the iron and copper containing enzyme cytochrome C oxidase showed that EDTA was not inhibitory, PEAA was slightly inhibitory, and LAL and sodium phytate were stronger inhibitors. Mechanistic explanations are offered to account for some of these observations. The possible relevance of these findings to in vivo protein digestion, enzymatic (oxidative) browning of foods, and the mechanism of the lysinoalanine effect on kidney cells are also discussed.

Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor

Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S-β-2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H2SO4 was directly proportional to concentration in the range 0.4–8 μm 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.