Biochemical and Pharmacokinetic Properties of PEGylated Cystathionine γ-Lyase from Aspergillus carneus KF723837

Abstract

Cystathionine &#947;-lyase (CGL) was purified to its electrophoretic homogeneity from<i> Aspergillus carneus</i> by various chromatographic approaches. The purified enzyme has four identical subunits of 52 kDa based on SDS and native PAGE analyses. To improve its structural stability, purified CGL was modified by covalent binding to polyethylene glycol moieties. The specific activity of free-CGL and PEG-CGL was 59.71 and 48.71 U/mg, respectively, with a PEGylation yield of 81.5 and 70.7% modification of surface ε-amino groups. Free- and modified CGL have the same pattern of pH stability (8.0-9.0). At 50°C, the thermal stability [half-life time (T_1/2)] of PEG-CGL was increased by 40% in comparison to free-CGL. The activity of CGL was completely inhibited by hydroxylamine and Hg⁺², with no effect by EDTA. Free-CGL (0.04 m<smlcap>M</smlcap>^-1s^-1) and PEG-CGL (0.03 m<smlcap>M</smlcap>^-1s^-1) have a similar catalytic efficiency to <smlcap>L</smlcap>-cystathionine as a substrate. The inhibition constant values of propargylglycine were 0.31 and 0.52 µ<smlcap>M</smlcap> for the free- and PEG-CGL, respectively. By in vitro proteolysis, PEG-CGL retains >50% of its initial activity compared to <10% of the free-CGL for acid protease for 30 min. From in vivo pharmacokinetics in New Zealand white rabbits, the T_1/2 was 19.1 and 28.9 h for the Holo free-CGL and PEG-CGL, respectively, ensuring the role of PEGylation on shielding the CGL surface from proteolytic attack, reducing its antigenicity, and stabilizing its internal Schiff base. By external infusion of pyridoxal 5′-phosphate (10 µ<smlcap>M</smlcap>), the T_1/2 of free- and PEG-CGL was prolonged to 24 and 33 h, respectively, so dissociation of pyridoxal 5′-phosphate was one of the main causes of loss of enzyme activity. The biochemical and hematological responses of rabbits to free- and PEG-CGL were assessed, with relative similarity to the negative control, confirming the nil toxicity of enzymes. The titer of IgG was duplicated in response to free- versus PEG-CGL after 45 days. To the best of our knowledge, this is the first report concerned with purification and PEGylation of CGL from fungi, with higher affinity for <smlcap>L</smlcap>-cystathionine. With further molecular studies, CGL will be a promising enzyme against various cardiovascular diseases and antioxidant deficiency, as well as for generation of a neurotransmitter (H₂S).