U538263.pdf (5.59 MB)
Studies of porphyrin glycoconjugates and amino acid-, peptide- and protein- adducts by liquid chromatography - mass spectrometry
thesisposted on 2014-12-15, 10:41 authored by Musaab Aaqib. Razzaque
The extraction, isolation and characterisation of a group of hitherto unreported protoporphyrin glycoconjugates in the rat Harderian gland using high performance liquid chromatography (HPLC), capillary electrophoresis (CE), on-line HPLC/Electrospray ionisation MS (HPLC/ESI-MS) and tandem MS is described. The major glycoconjugate was identified as protoporphyrin-1-O-acyl -xyloside with a smaller amount of protoporphyrin-1-O-acyl -glucoside also detected. In the Harderian glands studied, 50-70% of the porphyrins present were in the form of protoporphyrin glycoconjugates. This is the first reported occurrence of glycoconjugates of porphyrins in nature and suggests that previous studies have wrongly identified the major porphyrin in the Harderian gland as the unconjugated protoporphyrin. The function of these glycoconjugates is not clear.;The existence of protoporphyrin glycoconjugates in the urine, plasma and red blood cells (RBC) of a patient with erythropoietic protoporphyria (EPP) was also investigated. Protoporphyrin-1-O-acyl -xyloside was detected in EPP urine but not in plasma and RBC.;The mechanism of formation of the protoporphyrin glycoconjugates was investigated. Incubations of protoporphyrin and its reduced form, protoporphyrinogen, were carried out with xylose, glucose and their uridine 5'-diphosphate derivatives in the presence and absence of uridine 5'-diphosphate glucuronyl transferase (UDPGT). Negative results were obtained. Incubation of uridine 5'-diphosphoglucuronic acid (UDPGA) with protoporphyrin and UDPGT in Tris-HCl buffer, also yielded no products. Incubation of UDPGA with the Harderian gland homogenate in Tris-HCl buffer (pH7.4), however, resulted in an increase of 61.8% and 78.5% of protoporphyrin-1-O-acyl -xyloside and protoporphyrin-1-O-acyl -glucoside conjugates, respectively. This finding, along with an absence of glycoconjugates in incubations with liver homogenate would suggest the involvement of other enzymes which may be present in the Harderian gland.;The reactivities of protoporphyrin and protoporphyrinogen towards amino acids, peptides and proteins were investigated. Protoporphyrin failed to react while protoporphyrinogen formed adducts with cysteine, glutathione, peptides and protein containing a free thiol group when incubated overnight at 37oC in the dark.
Date of award2001-01-01
Awarding institutionUniversity of Leicester