Ribose sugars generate internal glycation cross-links in horse heart myoglobin

Abstract

Glycation of horse heart metmyoglobin with d-ribose 5-phosphate (R5P), d-2-deoxyribose 5-phosphate (dR5P), and d-ribose with inorganic phosphate at 37°C generates an altered protein (Myo-X) with increased SDS-PAGE mobility. The novel protein product has been observed only for reactions with the protein myoglobin and it is not evident with other common sugars reacted over a 1 week period. Myo-X is first observed at 1-2 days at 37°C along with a second form that is consistent in mass with that of myoglobin attached to several sugars. MALDI mass spectrometry and other techniques show no evidence of the cleavage of a peptide from the myoglobin chain. Apomyoglobin in reaction with R5P also exhibited this protein form suggesting its occurrence was not heme-related. While significant amounts of O(2)(-) and H(2)O(2) are generated during the R5P glycation reaction, they do not appear to play roles in the formation of the new form. The modification is likely due to an internal cross-link formed during a glycation reaction involving the N-terminus and an internal amine group; most likely the neighboring Lys133. The study shows the unique nature of these common pentose sugars in spontaneous glycation reactions with proteins.

Figure 1

A) Absorbance spectra (250 nm – 450 nm) of a reaction solution of horse heart metmyoglobin (1 mg/mL) with R5P (50 mM) at pH = 7.4 and 37 °C at 0 days (○ –– ○), 3 days (■ ···· ■), and 6 days (▲- - -▲). B) Reduction of metmyoglobin with reaction time with R5P; in exposure to air (■—■), in presence of catalase (◆- - - ◆), and in semi-anerobic conditions (▲····▲).

Figure 2

A. SDS PAGE gels of reactions of myoglobin or apomyoglobin (1 mg/mL) with R5P or dR5P (50 mM) at pH = 7.4 and 37 °C with incubation time in days listed. A. Lanes 1 – 5 Metmyoglobin reacting with R5P; Lanes 6 - 7 Metmyoglobin without sugar; Lane 8: Molecular mass standards (Lysozyme (14.4 kDa), carbonic anhydrase (29.5 kDa), ovalbumin (45.0 kDa), BSA (66 kDa).) B. Apomyoglobin reacting with R5P. C. Silver Stained Gel. Lane 1: Myoglobin reacting with dR5P; Lane 2: Myoglobin reacting with R5P (1 mM).

Figure 3

MALDI TOF analysis of a reaction mixture of R5P (50 mM) and myoglobin (1.0 mg/mL) for 0 h (A), 2 h (B), and 4 d (C) at 37 °C and pH = 7.4. Mass values (m/z) given are for the largest peak in the distribution. (Note that the scan scale is 25,000 Da for A and 30,000 Da for B and C.) Other major peaks in the 0 h and 2 h spectra are typically 212 Da apart indicating the addition of R5P molecules to the 16,950 Da myoglobin molecule. The absence of a signal at approximately 15,000 Da indicates the highly mobile band on the SDS PAGE analysis is not a result of a cleavage of a piece from myoglobin.

Figure 4

Total ion chromatograms (TIC) of trypsin digests of reactions of R5P (50 mM) and myoglobin (1.0 mg/mL) for 0 h (A) and 72 h (B) at 37 °C and pH = 7.4. Peak eluting at 35.5 min (noted by arrow) represents the first tryptic fragment (N-terminal) which decreased in intensity with time. Numerous peaks forming at the base of this signal suggests multiple modifications occur on this myoglobin fragment upon reaction with R5P.