The Effects Of Polyamine On The Activity And Conformation Of Rabbit Muscle Lactate Dehydrogenase

Polyethyleneimine（PEI）as a gene carrier with higher transfection efficiency attracted more attention.However,its high charge density and cytotoxic effected limit its application.Its potential safety problems are not yet clear,and the mechanism of functional proteins has still poorly understood.The effect of the small polyamine（PA）molecules metabolized in the body on the structure and function of enzymes is still not clear.In this paper,Mechanism of interaction between polyamine gene carrier materials and proteins was studied by studying the activity,kinetic parameters,secondary structure changes,tertiary structure changes and size effect changes after binding to polyamines.It provided a theoretical basis for the in vivo safety study of Polyamine gene carrier materials.To make a further insight into the molecular basis of polyamines cytotoxicity,a combinational spectroscopic study of UV-vis absorption,resonance Rayleigh scattering,fluorescence,and circular dichroism was conducted to reveal the interaction of PEI with rabbit muscle lactate dehydrogenase（rmLDH）and the influence on the conformation and bioactivity of the enzyme.PEI was a cationic polymer capable of binding on the surface of rmLDH,a basic protein,due to the dense hydrophilic amine groups and hydrophobic methyl groups.The competitive binding by PEI greatly reduced the binding efficiency of rmLDH towards the co-enzyme and the substrate.However,the complex formation between rmLDH and PEI induced a less ordered conformation and enhanced surface hydrophobicity,facilitating the turnover of the enzyme and generally resulting in an increased activity.PEI of higher molecular weight was more favorable to induce the alteration in the conformation and bioactivity of rmLDH.Absorption and RRS analysis illustrated that a complex formation occurred between spermidine（Spd）and rabbit muscle LDH,Spd had no significant influence on the peak position of aromatic residues absorption of rabbit muslce LDH,however,the slight increase in the absorbance suggested a perturbation of the secondary and tertiary structure of the enzyme,leading to an increased exposure of aromatic residues.Spd is a typical polyamine of hydrogen bonding capability and hydrophobicity originating from dense amine groups and methyl groups.Due to the small size and the amphiphilic nature,Spd was subject to penetrate into the hydrophobic core of LDH and quenched the fluorescence emission of tryptophan residues,via a combined mechanism involved diffusion collision and complex formation.Far-UV CD and ANS fluorescence probing showed that the interaction with Spd resulted in a less ordered conformation with decreased-helix content and enhanced surface hydrophobicity of the enzyme.Catalytic activity of rabbit muscle LDH was monitored by the reduction of sodium pyruvate and steady-state kinetic studies were carried out to reveal the mechanism of biofunctional alteration of the enzyme upon Spd binding.In spite of the reduced turnover of the enzyme,the interaction with Spd at low concentration up to 25 mmol·L^-1generally resulted in an increased activity due to an enhancement in the affinity towards the substrate.However,a significant inhibition to the enzymatic activity was observed at higher Spd concentration,as the results of decreased affinity towards reduced nicotinamide adenine dinucleotide（the co-enzyme）.