Structure, stability and digestion of caprine and bovine lactoferrin : a comparative study
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In this thesis, the structure, stability and digestion of caprine and bovine lactoferrin were compared. The work carried out in this project has been divided into four parts. A comparative study was done on the thermal stabilities and conformational changes in the native forms of caprine lactoferrin (cLF) and bovine lactoferrin (bLF) in the pH range 2.0-8.0. In the pH range 2.0-8.0, bLF showed maximum thermal denaturation temperature (Tm) values than that of cLF. At pH 7.0, the Tm values of cLF and bLF were 67±1 and 70±1°C, respectively. For both cLF and bLF, with reduced pH values a decrease in Tm values was observed. At pH 3.0, cLF and bLF showed 30±1 and 39±1 °C Tm values, respectively. At pH 2.0-3.0, the structural unfolding of cLF and bLF was observed. Both cLF and bLF were rich in β-structure (54 and 57%, respectively). Further at pH 2.0, tryptophans were exposed to the solvent to a greater extent in bLF than cLF. The thermal stability of bLF was higher than cLF and was pH dependent. The structural characteristics and thermal stabilities of apo and holo forms of caprine and bovine LF were compared in the pH range 2.0-8.0. At pH 7.0, the holo forms of both cLF and bLF showed higher Tm values (68±1 and 90±1 °C, respectively) than the corresponding apo forms (64±1 and 66±1 °C, respectively). For both apo and holo forms of cLF and bLF, a continuous reduction in Tm values with a reduction in pH from 8.0 to 3.0 was evident. A reduction in pH from 7.0 to 2.0 showed significant loss in iron content of both apo and holo forms from both caprine and bovine LF. A higher exposure of hydrophobic surfaces at low pH for both apo and holo forms of cLF and bLF indicates the protein unfolding. These data were supported by the circular dichroism (CD) unfolding studies of both apo and holo forms of cLF and bLF at pH 2.0. The interaction between zinc (Zn2+) and LF from caprine and bovine was studied in the pH range 2.0-7.0. At pH 7.0, the zinc bound forms of cLF and bLF showed 67±1 and 83±1 °C Tm values, respectively. Thermal stability (Tm) values were decreased to 76±1 and 55±1 °C, respectively at pH 4.0. When the pH was reduced from 7.0 to 2.0, a significant loss in the zinc content of both cLF and bLF was observed. The CD results showed that at pH 2.0, the structure of zinc bound bLF (ZnbLF) was more unfolded than that of zinc bound cLF (ZncLF). The unfolding data was supported by the maximum exposure of tryptophan residues in ZnbLF than ZncLF at pH 2.0. Guanidine hydrochloride induced denaturation of ZncLF and ZnbLF indicated higher unfolding of the protein. In the pH range 2.0-7.0, a higher amount of iron binding to both cLF and bLF was observed when compared with the corresponding zinc bound forms. The thermal stabilities of ZncLF and ZnbLF were dependent of the pH and zinc binding. A study was undertaken to identify peptides generated from bLF and cLF during in vitro digestion with human gastrointestinal enzymes, and to examine factors known to influence the outcome of protein degradation, 1) different concentrations of human gastric juice (HGJ) and human duodenal juice (HDJ), 2) different concentrations of bLF and 3) two different gastric pH values. Protein profiles of undigested and digested LF were obtained by SDS-PAGE. The degree of hydrolysis was assayed by the o phthaldialdehyde (OPA) method. Peptides generated were identified by nano LC-MS. Protein degradation was highly dependent on gastric pH (2.5 and 4.0). At pH 2.5 lower content of intact LF and higher degrees of hydrolysis (~ 10.5) were observed. The peptide profiles from these samples revealed higher number of peptides at pH 2.5 than at pH 4.0. Identical protein degradation patterns were seen in caprine and bovine LF samples. However, their peptide patterns showed differences with regard to number of different peptides and different sequence lengths. At pH 2.5 and 4.0, the apo and holo forms of bLF showed similar degradation patterns. More than 90% peptides were originated from the N-terminal part of bLF (native, apo and holo) or cLF (native) at pH 2.5 and 4.0. During the pH reduction to 2.5 or 4.0, the digested bLF with fast pH reduction generated more peptides when compared to that of slow pH reduction. After the action of HGJ and HDJ, more peptide fragments were detected in native bLF than that of native cLF at both pH values 2.5 and 4.0. The multiple sequence alignment of peptides from LF digests showed the presence of proline and leucine patterns at both pH values, 2.5 and 4.0. The use of in vitro digestion could contribute to a better knowledge about the generation of peptides during gastrointestinal digestion, however, this has to be confirmed by in vivo experiments.