Quantitative analysis and evaluation of the solubility of hydrophobic proteins recovered from brain, heart and urine using UV-visible spectrophotometry.
There is a need for a simple method that can directly quantify hydrophobic proteins. UV-visible spectrophotometry was applied in the present study for this purpose. Absorbance at lambda = 280 nm (A280) was detected for both Escherichia coli membrane proteins and bovine serum albumin, whereas absorbance at lambda = 620 nm (A620) was only detected for E. coli membrane proteins. The A620 values of the brain samples were greater than those of heart samples when equal concentrations were used, regardless of the type of solubilizing agent employed. Because hydrophobic proteins tend to form colloidal microparticles in solution, we also applied UV-visible spectrophotometry to evaluate the efficacies of different extraction protocols for solubilizing hydrophobic proteins. For brain protein extraction, the highest A620 was observed in samples recovered using Tris, whereas the lowest was from samples recovered using SDS. Solubilizing brain tissue with 0.25% SDS (above the CMC) gave a lower A620 than extraction with 0.025% SDS (below the CMC). Addition of 0.25% SDS to samples recovered with Triton caused A620 to drop. A620 could also be used to distinguish between the hydrophobic fractions (pellets) of brain and urine proteins and their hydrophilic fractions (supernatants) prefractionated using high-speed centrifugation. Additionally, an A620/A280 ratio exceeding 0.12 appears to denote highly hydrophobic samples. Our data suggest that direct UV-visible spectrophotometry can be used as a simple method to quantify and evaluate the solubilities of hydrophobic proteins.
Thongboonkerd
Visith
V
Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand. thongboonkerd@dr.com
Songtawee
Napat
N
Kanlaya
Rattiyaporn
R
Chutipongtanate
Somchai
S
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002498:Centrifugation; D051379:Mice; D008807:Mice, Inbred BALB C; D009206:Myocardium / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014556:Urine
D011506:Proteins