Separation of pteridines from blood cells and plasma by reverse-phase high-performance liquid chromatography.

In order to determine free and conjugated pteridines, the pteridine-releasing procedure for biological materials (e.g., plasma, blood cells, bone marrow) was optimized and the parameters which influence pteridine release are discussed. Using reverse-phase high-performance liquid chromatography (rp-HPLC) on 5-μm ODS (C18; mobile phase, K2HPO4 buffer, pH 7.0-7.8), up to 10 pteridines (pterin-6-carboxylic acid, xanthopterin, neopterin, monapterin, isoxanthopterin, lumazine, biopterin, 6-hydroxymethylpterin, pterin) could be separated in the extracts of blood samples and determined fluorometrically (femtomolar range). In addition, three sepiapterins (sepiapterin, deoxysepiapterin, 3'-hydroxysepiapterin) could be separated with aqueous methanol as eluent (uv and fluorometric detection, respectively). The methods described are suited to compile pteridine patterns for plasma and individual cell fractions (erythrocytes, lymphocytes, buffy coat) from blood. Moreover, it was shown that the pteridine concentrations in dog blood (beagles) were in some cases substantially higher than in human blood, and that neopterin is lacking in the blood cell fractions of beagle dogs or occurs only in very low concentrations

Purification and characterization of animal porphobilinogen synthases-II. Dog liver porphobilinogen synthase.

1. 1. A method is described for the purification of porphobilinogen synthase (PBG-S) from dog liver (acetone dry powder; ammonium sulfate fractionation: controlled heat denaturation; ion exchange chromatography; gel filtration). The 911-fold purified enzyme with a specific activity of 372 in U/mg and 6.2 nkat/mg enzyme protein, respectively, appears as a single band in disc electrophoresis. 2. 2. The optimum pH of the enzyme is 6.8; the Michaelis constant Km = 1.77 · 10-4M with 5-aminolevulinic acid as substrate. 3. 3. The determination of the molecular weight of the native multisubunit enzyme (268,000 dalton) and of the subunit (33,500 dalton) are in favour of an octameric structure. 4. 4. The acyi group blocking the N-terminus has been identified as an acetyl group (hydrazinolysis; dansylation;TLC: HPLC). 5. 5. The amino acid composition of the PBG-S subunit is as follows CH3CO-NH-(Asx22Thr9-10Ser18G lx28-29Pro20-21Gly22Ala35-37 Val23-25Met5 Ile8-9Leu32-33Tyr10Phe12Lys 12Cys3HiS7Arg22 Trp3)-Ala-Leu-COOH. 6. 6. In the presence of ganidinium chloride only one free sulfhydryl group per subunit (3 cysteine residues) could be detected. 7. 7. The purified enzyme contains 0.56 gram atoms of zinc per octamer (neutron activating analysis)