Racemization kinetics of free and protein-bound amino acids under moderate alkaline treatment

Abstract

557reagents, loss in activity on reduction by mercaptoethanol, and electrophoretic studies confirm that the new inhibitor is proteinaceous in nature.We could not unequivocally demonstrate the formation of an amylaseinhibitor complex during our studies. When inhibitor and human pancreatic amylase were chromatographed on Red-Sepharose, all the protein was eluted from the column whereas the native inhibitor was bound tightly to the immobilized ligand. While it should be expected that the inhibitor-enzyme complex would be eluted from Sephadex G-100 column earlier than the free enzyme and inhibitor, in our studies such a protein peak could not be identified. Instead, the inhibitor enzyme mixture was eluted in two protein peaks. The first peak with a V, value corresponding to the native inhibitor had measurable amylase activity. The second peak corresponding to native human pancreatic amylase had neither amylase activity nor inhibitory activity. However, treatment of this fraction with mercaptoethanol resulted in the appearance of amylase activity. These data suggest that the two protein peaks obtained during gel chromatography of amylaseinhibitor mixture represent two types of complexes. The fraction with amylase activity eluted first during gel chromatography could represent a complex formed due to loose interaction of inhibitor and enzyme dissociating under assay conditions exhibiting amylase activity. The second protein fraction may represent a complex formed by strong noncovalent interaction between the inhibitor and amylase, which exhibited amylase activity on inactivation of the inhibitor by treatment with mercaptoethanol. Further studies are needed to substantiate this view. Registry No. a-Amylase, 9000-90-2. Mundy, J.; Hejgaard, J.; Svendsen, I. FEBS Lett. 1984,167, 210. Petrucci, T.; Rab, A.; Tomasi, M.; Silano, V. Biochem. Biophys. Shainkin, R.; Birk, Y.Amino acid racemization was measured in a-lactalbumin, 0-lactoglobulin, a-casein, lysozyme, and BSA and in a free amino acid mixture exposed to pH 9 at 83 "C for times ranging from 0.5 to 24 or 96 h.Inversion rate constants were determined for 14 to 15 amino acids in the six models. Conclusions derived from these data were as follows: (1) Free amino acids racemize about 10 times more slowly than bound residues.(2) The main driving force in free amino acid racemization is the electron-withdrawing ability of the side chain (a* constant), except for Asp, the inversion of which seems to involve an intramolecular assistance effect.(3) The racemization of bound residues is governed by both amino side chain effects and protein-related factors. (4) Bound amino acid racemization is also affected by alkali-induced denaturation of the proteins (e.g., partial hydrolysis).

A novel pathway for the production of hydrogen sulfide from D-cysteine in mammalian cells

“…Discussion L-Amino acids are nonenzymatically racemized by heat and alkaline conditions applied during food processing, and 21-44% of L-cysteine is changed to D-cysteine by alkaline treatment 28,29 . D-cysteine may thus be mostly provided from food.…”

Theoretical Inversion of Amino Acids (Alanine and Aspartic Acid) by Semi-Empirical Methods

“…The greater the electron withdrawing capacity of the R group of an amino acid the more stable the carbanion will be and the faster the racemization rate will be (Bada and Shou (10) ; Bada (11) ; Liardon and Lederman (12) ). Smith and Evans (7) added that the overall effect of an R group depends on the combination of its electron withdrawing or donating characteristics (electronic factors) and its bulk and shape (steric factors).…”

Effects of processing on soybean nutrients and potential impact on consumer health: An overview

“…Since D isomers have no nutritional value, racemization of an essential amino acid thus severely reduces its nutritional value [23]. Residues such as Asp, Ser, Cys, Glu, Phe, Asn, and Thr are racemized at a faster rate than other amino acid residues due to the higher electron withdrawing power of the peptide chain and the higher hydroxyl concentration; however, racemization rate is reported to be independent of protein concentration [36]. Racemization reduces the digestibility of the protein and therefore its bioavailability (due to the lower potential hydrolysis by gastric and pancreatic proteases) of the D forms in vivo compared to those containing only the L residues [16].…”