Abstract:

Photo-oxidation experiments on several model proteins (fetuin, cytochrome c, bovine serum albumin (BSA), and lectin) in artificial seawater were carried out to investigate the mechanisms for the transformation and preservation of proteinaceous compounds. We examined proteolysis rate and carbonyl content of model proteins during sunlight irradiation.

Suggested Citation:

Luni Sun, Peter Santschi. 2020. Proteolysis rates and carbonyl contents of model proteins (fetuin, cytochrome c, bovine serum albumin, and lectin) during sunlight irradiation. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-tk2d-0x58

Publications:

Sun, L., Xu, C., Lin, P., Quigg, A., Chin, W.-C., & Santschi, P. H. (2021). Photo-oxidation of proteins facilitates the preservation of high molecular weight dissolved organic nitrogen in the ocean. Marine Chemistry, 229, 103907. doi:10.1016/j.marchem.2020.103907

Purpose:

To investigate the mechanisms for the transformation and preservation of proteinaceous compounds in seawater.

Data Parameters and Units:

protein concentration (mg/L) carbonyl (nM) irradiation time: 0, 15min, 2h,6h, 20h SG=fluorescence value with protease Streptomyces griseus K= fluorescence value with protease K Blank= fluorescence value without protease addition No H2O2= with no H2O2 addition H2O2=with H2O2 addition BSA= bovine serum albumin

Methods:

Model proteins (30 mg/L) were dissolved in artificial seawater and irradiated in quartz vials (125 mL) under a 400W xenon lamp (Suntest system in a circulating water bath). H2O2 (in the mass ratio of 1:1 to protein) was added into half of the samples. Subsamples were taken at 20 min, 2h, 6h, and 20h intervals. After 0.4 µm filtration, the subsamples were concentrated by ultrafiltration to obtain the colloidal fraction using an Amicon ultra-4 centrifugal filter unit with a 3kDa cut-off membrane. The refractory nature of proteins was assessed by measuring their proteolysis rates with two different proteases. Protease from Streptomyces griseus (SG) which is a mixture of at least three caseinolytic activities and one aminopeptidase activity, and protease K, which is a broad-spectrum serine protease, was used to cleave the proteins into primary amines [Höhn et al., 2011; Nguyen and Harvey, 2001]. The samples were mixed with a protease stock solution (1 mg/L) in a ratio of 10:1 and a series of samples without protease were used as controls. After 3h incubation at 25°C, 50 μL ice-cold 20% TCA was added to 100 μL samples, after which they were incubated in the fridge (4ºC) for 30 min. The samples were then centrifuged at 3000 g for 10 min at 4 °C. Fluorescamine solution (100 μL, 0.06% in acetone) was added into the 100 μL supernatant with 300 μL 1.5 M HEPES mixture. After 5 min incubation of each sample in the dark, fluorescence was measured at 390 nm excitation/475 nm emission wavelength using a microplate reader (Biotek, Synergy 2). Data was used as fluorescence reading. A series of dilutions of glycine was used to produce a standard curve. Proteolysis rates were calculated as the difference between sample values (primary amine concentration with protease addition) and blank values (primary amine concentration without protease addition), normalized to the protein concentration. Protein concentration was measured using a modified Lowry Protein Assay Kit (Pierce, 23240, USA). Their carbonyl content was measured using a Protein Carbonyl Content Assay Kit (Sigma MAK094). Carbonyl content for cytochrome c was not measured due to color interference.

Instruments:

microplate reader (Biotek, Synergy 2)

Error Analysis:

Standard deviation from excel

Provenance and Historical References:

Höhn, A., Jung, T., Grimm, S., Catalgol, B., Weber, D., & Grune, T. (2011). Lipofuscin inhibits the proteasome by binding to surface motifs. Free Radical Biology and Medicine, 50(5), 585–591. doi:10.1016/j.freeradbiomed.2010.12.011 Nguyen, R. T., & Harvey, H. R. (2001). Preservation of protein in marine systems: Hydrophobic and other noncovalent associations as major stabilizing forces. Geochimica et Cosmochimica Acta, 65(9), 1467–1480. doi:10.1016/s0016-7037(00)00621-9

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