Spectroscopic Characterization Of Human Single-chain Antibody Against Cyclin D1

Cyclin D1is an important regulator of cell cycle in G1to S phase transition, andhas great correlation with tumor occurrence and development. Cyclin D1is animportant potential target for tumor therapy.Previous studies have got active anti-cyclin D1single chain antibody with specificbinding activity to cyclin D1. Intracellular anti-cyclin D1single chain antibody cansignificantly inhibit tumor cell growth and proliferation.Intracellular single chain antibodies can reach the lesion site, and inhibit cell cycleprogression by binding to cyclin D1. But human body environment is very complex,containing a variety of metal ions and other substances. Therefore, before theintracellular single chain antibody reaches the "destination", if there’re changes in itsstructure and properties under these circumstances, and what changes are, whether itwill affect the binding activity to cyclin D1. All the above questions are needed to beclear.Protein structure and function are closely related, the study on protein structure-function will provide theoretical basis and solutions to elucidate the mechanism ofmany diseases. So far, UV-visible absorption spectroscopy, fluorescence spectroscopy,circular dichroism spectroscopy and infrared spectroscopy method is favored on thestudy of protein structure. In this paper, several spectroscopy techniques were appliedto study anti-cyclin D1single-chain antibody structure and activity in existence ofdenaturing agents, oxidant, reductant and metal ions. Results are listed as follows:1. The results of AD5fluorescence spectroscopy showed that AD5fluorescencewas quenched by Cu2+or Fe3+under the three different temperatures.2. Results of synchronous fluorescence showed that AD5chromophoremicroenvironment changed slightly in presence of Cu2+or Fe3+, leading to polarity,hydrophobicity and AD5conformation slightly changing. The fluorescence of Tyrand Trp residues was quenched by Cu2+or Fe3+. 3. Fluorescence quenching caused by Cu2+or Fe3+was static and dynamic jointquenching mechanisms, and mainly by static quenching; Stern-Volmer quenchingconstants Ksvand Kqat293K,298K and303K were obtained in presence of Cu2+orFe3+.4. Each AD5molecule had one Cu2+or Fe3+binding site. The binding ability ofFe3+to AD5increased as the temperature rised; while the affinity at298K was thestrongest with treatment of Cu2+.5. Cu2+or Fe3+and AD5binding was spontaneous, and a typical hydrophobicforce was stronger between metal ions and AD5in presence of Cu2+or Fe3+.6. In the studied concentrations, low concentration Cu2+decreased AD5activitywhile it reversed in high Cu2+concentration; Fe3+reduced the biological activity (P<0.01) of AD5.7. In the studied concentrations, SDS, DTT and ethanol changed the AD5conformation to some extent, and had different effects on AD5activity, and SDSsignificantly decreased AD5activity.8. CD result showed that β-Sheet (48.2%) and Random (37.3%) were predominantcompositions, β-Turn and α-Helix contents were12.1%and2.4%, respectively.The experiments showed that AD5structure changed under different conditions,and its affinity to cyclin D1was affected. This would be of great significance andvalue in anti-cyclin D1single chain antibody clinical applications in future.