"Rigid" structure is a key determinant for the low digestibility of myoglobin

Abstract

Myoglobin, a critical protein responsible for meat color, has been shown insusceptible to digestion. The underlying mechanism is not clear. The present study aimed to evaluate whether the structural properties of myoglobin are associated with its insusceptibility to digestion using spectroscopic and computational techniques. Myoglobin was degraded by only 7.03% by pepsin and 33.00% by pancreatin. The structure of myoglobin still maintained α-helix after the two-step digestion, with the exposure of some aromatic residues. In addition, molecular dynamics modeling suggested that hydrophobic amino acid residues (Phe 111, Leu 10, Ala 115, Pro 116) in pepsin and polar amino acid residues (Tyr 146, Thr 95) in myoglobin were found in the proximity of binding sites, which could result in the low digestibility of myoglobin. Our findings provide a new insight into the underlying mechanisms on the difficulty in digestion of myoglobin.

Keywords: Digestibility; Liquid chromatography-tandem mass spectrometry; Molecular docking; Molecular dynamics; Myoglobin.

Fig. 1

Patterns reflecting the digestibility of myoglobin under in vitro digestion. (A) SDS-PAGE profiles of myoglobin and its digested products: Lane 0 shows the calibration markers (5–270 kDa). Lanes 1, 2, 3 and 4 show pepsin-treated samples for 0, 3, 60 and 120 min; and lanes 5, 6, 7, 8 and 9 show pancreatin-treated samples 1, 3, 15, 60 and 120 min after treatment by pepsin for 120 min. (B) The DH values of myoglobin in pepsin and pancreatin digestion. G0, G3, G60 and G120 refer to pepsin-treated samples for 0, 3, 60 and 120 min, respectively; I1, I3, I15, I60 and I120 refer to pancreatin-treated samples 1, 3, 15, 60 and 120 min after treatment by pepsin for 120 min, respectively. Different letters indicate a significant difference (P < 0.05). (C) Peptide matching for pepsin and pancreatin treated samples. Peptides in blue represent the pepsin-treated samples; peptides in red represent the pancreatin-treated samples; and peptides in green represent those existing in pepsin and pancreatin treated samples.

Fig. 2

Spectra of myoglobin during in vitro digestion. (A) 279 nm bands of UV–Vis spectra; (B) 409 nm bands of UV–Vis spectra; (C) Intrinsic fluorescence spectra of Mb; (D) Synchronous fluorescence spectra for Tyr residues (Δλ = 20 nm); (E) Synchronous fluorescence spectra for Trp residues (Δλ = 60 nm); (F) CD spectra of myoglobin. G0, G3, G60 and G120 refer to pepsin-treated samples for 0, 3, 60 and 120 min, respectively; I1, I3, I15, I60 and I120 refer to pancreatin-treated samples for 1, 3, 15, 60 and 120 min after treatment by pepsin, respectively.

Fig. 3

Patterns related to the protein stability over 10 ns MD simulation. (A) The RMSD plot of myoglobin-pepsin complex. (B-C) The RMSF values of myoglobin and pepsin over the amino acid residue numbers, respectively. (D) Radius of gyration (Rg) values.

Fig. 4

The final structure of myoglobin-pepsin complex after MD simulation. (A) three-dimensional structure of the complex; (B) hydrophobic surface map of the complex.

Fig. 5

Interaction mode of myoglobin-pepsin complex. (A) Three-dimensional structure of hydrogen bonds network. The residues of myoglobin and pepsin are shown in white and green sticks, respectively. The yellow dashed lines represent hydrogen bonds. (B) Two-dimensional representation of myoglobin and pepsin. The residues upon the dotted line refer to pepsin, the residues below the dotted line refer to myoglobin. The green dashed lines indicate hydrogen bonds.