Análise espectrofotométrica de colágeno com corantes e sal metálico e interação com células HFF

Abstract

Collagen is the main structural protein of connective tissue and plays a crucial role in the integrity of the extracellular matrix (ECM). Gelatin, derived from its hydrolysis, is widely used as an experimental model in biochemical and cell-based studies. Dyes such as Orange G and Ponceau S are commonly employed for protein staining, whereas copper sulfate (CuSO₄), despite its utility in biochemical assays, exhibits cytotoxic potential. This study aimed to characterize gelatin by SDS-PAGE and investigate, through UV-vis spectrophotometry and cell culture assays, the effects of these compounds on proteins and human foreskin fibroblasts (HFF). Gelatin was characterized by SDS-PAGE, with analysis of protein bands. UV-Vis spectra of gelatin alone and in combination with the dyes or CuSO₄ were obtained to assess protein–molecule interactions. HFF cultures were then treated with the compounds at 0.0001%, 0.001%, and 0.01%, and morphologically evaluated at 0 h and 24 h. Gelatin exhibited the characteristic protein profile, with α-chains (~100–120 kDa), β-dimers (~200 kDa), and the polydisperse pattern typical of denatured collagen. In spectrophotometry, Orange G and Ponceau S displayed characteristic peaks and weak interaction with the protein, without major spectral changes. CuSO₄ showed a slight increase in absorbance between 220–240 nm, suggesting protein–metal complex formation. In cell cultures, Orange G and Ponceau S caused no relevant alterations up to 0.001%, with only a mild decrease in confluence at 0.01%. CuSO₄ exhibited dose-dependent cytotoxicity, with slightly morphological changes from 0.001%, more pronounced at 0.01%, consistent with reports of copper- induced oxidative stress. The Orange G and Ponceau S were safe for fibroblasts up to 0.001%, whereas CuSO₄ showed toxicity at relatively low concentrations, highlighting the need for caution in its experimental use. These results contribute to the standardization of assays involving the studied compounds in cell culture and support future applications in tissue engineering and toxicology.