Comprehensive Analysis of Interactions between Human Serum Albumin and Human Cystatin C – Two Proteins Present in Body Fluids

Human cystatin C (HCC) and human serum albumin (HSA) are proteins that coexist in body fluids. The main physiological role of HCC is the inhibition of cysteine proteases, while HSA is a universal transport protein carrying numerous ligands. Both proteins are also associated with amyloidogenesis at various levels, so understanding their potential interactions is an important aspect of the research. To characterize these interactions, we used several combined complementary techniques: microscale thermophoresis (MST), isothermal titration calorimetry (ITC), high-resolution mass spectrometry (HR-MS), and nuclear magnetic resonance spectroscopy (NMR). The K_D value obtained from the MST method was 1.3 μM, while the dissociation constant determined by ITC was 1.15 μM. Mass spectrometry data confirmed formation of the HSA–HCC complex with 1:1 stoichiometry. The most pronounced changes were observed in the NMR spectrum for the prolonged dynamic processes, characterized by increased spectral densities for several HCC residues.