Heat-Induced Redistribution of Disulfide Bonds in Milk Proteins. 1. Bovine β-Lactoglobulin

Abstract

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.

Determination of Exposed Sulfhydryl Groups in Heated β-Lactoglobulin A Using IAEDANS and Mass Spectrometry

“…Cys106 seems to be always involved in different disulfide bonds, 384 while the other four cysteine residues reacted with IAEDANS indicating that they 385 were sometimes generated in free sulfhydryl form following sulfhydryl/disulfide 386 exchange reactions. In addition to the role of Cys160 as a major player of 387 sulfhydryl/disulfide exchange reaction [10], results obtained highlight the prevalent 388 occurrence of Cys66 as an exposed cysteine under our conditions of heat-treatment. 389 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 …”

“…Exposed sulfhydryl groups are further able to react with other proteins 69 leading to the formation of dimers, oligomers and larger aggregates [3,4,7]. When 70 the dimers and trimers were digested with trypsin and analyzed by mass spectrometry 71 in MALDI and electrospray modes, it was found that several new disulfide bonds 72 were formed: Cys121-Cys160, Cys106/119/121-Cys160, as well as an intermolecularthat its release following sulfhydryl/disulfide exchange reactions is an important step 77 to propagate intermolecular exchange reactions [10]. Determining which sulfhydryl 78 groups are exposed on heating (important contributors for sulfhydryl/disulfide 79 interchange reactions) will give a greater insight into mechanism of how the 80 sulfhydryl/disulphide exchange reactions proceed.…”

Influence of Run Time and Aging on Fouling and Cleaning of Whey Protein Deposits on Heat Exchanger Surface

“…The disulfide bridges are known to be a key factor that affects the gel strength (Hoffmann and Van Mil, 1999;Creamer et al, 2004) and the aggregation process through the polymerization of monomers to form large polymer chains. However, there is only limited availability of the bonds to be formed and hence excessively longer heating time may not be effective anymore.…”

The Effect of Molecular Chaperone, Alpha-Crystallin, on the Heat-Induced Aggregation of Beta-lactoglobulin

“…Each monomer has a molecular weight of 18.3 kDa, contains 162 amino acid residues, two disulphide bonds (C106-C119 and C66-C160) and one free cysteine residue (C121) [6][7][8] . Upon heating, betalactoglobulin dissociates into monomers, leading to exposure of hydrophobic groups and the free sulfhydryl group [9,10] .…”

“…Aggregation of beta-lactoglobulin occurs mainly via intermolecular disulphide bond exchange, i.e. the aggregation is not (in the main) due to nucleationdependent processes [7,9] but arises from polymerization. In a previous study [21] indicated that alpha-crystallin did not inhibit the aggregation of a serpin (AT (antitrypsin)) which aggregated via a polymerization reaction but did inhibit a serpin aggregation (ACT (antichymotrypsin)) when occurring via a nucleation-dependent mechanism.…”