Time-dependent Nature And Peptide Release During Enzymatic Hydrolysis Of Proteins

In this thesis, peptic digestion of bovine hemoglobin and pancreatic digestion ofbovine casein were systematicallyinvestigated bya combination of chromatographic, massspectrometric and spectroscopic analyses. On the basis of enzyme mechanism and proteinstructure, we have extensively studied the reaction behavior of protein and release kineticsof peptides during enzymatic hydrolysis.The main aspectsand conclusions were displayedasfollows.1. Two different mechanisms of hydrolysis were compared for peptic digestion of bovinehemoglobin:"onebyone"fornativehemoglobinat pH4.5,and"zipper"fordenaturedhemoglobin at pH 2.0. The reaction mixture always contains native hemoglobin at pH4.5, whereas denatured hemoglobin were rapidly converted to intermediate peptides.For these two mechanisms, peptic cleavage sites were distributed all theα-chain andthe terminal regions ofβ-chain. Littleenzymatichydrolysisoccurredinthecenterpart ofβ-chain due mainly to their high hydrophilic nature. The reaction behavior was discussedin relation to the hydrophobicitydistribution of polypeptide chains and the mechanismofpepsin.2. The precursor cleavage of the antimicrobial peptideα107-136 into the bradykinin-potentiating peptideα110-125 were followed during peptic hydrolysis of bovinehemoglobin. A total of six peptides were identified as being involved in the cleavageprocess. Moreover, the reaction network of these peptides was developed according tothe sequence alignment and their release kinetics. The affinity of pepsin towardsdifferent peptide bonds of bovine hemoglobin was also compared based on data fromthe release kinetics of peptides. In addition, some potentially bioactive peptides werepredictedbymeansofsequenceanalysisandsecondarystructurecalculations.3. Bovine casein pancreatic hydrolysates were separated andanalyzed by reversed-phase,strong cation exchange and size exclusion chromatography. In general, the resultingpeptides were converted from hydrophobic to hydrophilic, from multiply-charged tofew-chargedandfromlargetosmallduringthecourseofhydrolysis.4. The substrate and products from pancreatic digestion of bovine casein were analyzedby size exclusion chromatography coupled with multi-angle laser light scattering(SEC-MALLS) and reversed-phase liquid chromatography coupled with tandem massspectrometry (RPLC-ESI-MS/MS), respectively. Upon limited hydrolysis, casein micelles displayed a continuous growth in their sizes and molecular weights, togetherwith the decrease in their concentration. The hydrolysis rate of micellar caseins bypancreatinwasrankedfromfasttoslowasfollows:β-casein>αs1-casein>αs2-casein>κ-casein.5. We developed a computational model to predict molecular weight distribution ofbovine casein pancreatic hydrolysates using artificial neural networks (ANN) thatcontained 2 input nodes, 1 output node and two hidden layers with 30 nodes. Based onthe prediction results from such model, a 3-D continuous surface and its correspondingcontour plot were obtained to directly and distinctly characterize the dynamic processof enzymatic hydrolysates with different molecular weight including micelles,monomers,macropeptides,polypeptidesandoligopeptides.6. In total, 52 casein phosphopeptides (CPPs) in the time-course samples were separatedand identified using liquid chromatography-tandem mass spectrometry by means ofneural loss scanning (SALSA) and database searching (Sequest). All the CPPs showedsignificant neutral loss of phosphoric acid(s) during the collision-induced dissociation(CID) process in the ion-trap mass spectrometer. Moreover, the charge states, molarmasses and number of phosphorylation sites of peptides could be determined on thebasis of these neural loss ions. In addition, a study of the release kinetics of CPPsallowed determination of the degrees of hydrolysis for the preparation of target CPPwithhighyields.7. Aprediction model as awas developed on the basis of 365non-phosphorylated peptides from casein pancreatic and peptic hydrolysates, and canbe used to accurately predict the retention times of peptides on C4 reversed-phase(300? pore size) columns with TFA as the ion pairing reagent. Singly and multiplyphosphorylations respectively reduced and increased the overall hydrophilicity ofpeptides, and thus make phosphopeptides elute after or before the non-phosphorylatedpredictivecognates,respectively.