A comparison of methods for sample clean-up prior to quantification of metal-binding proteins.
1. The recovery of rabbit metallothionein and metallothionein-like proteins has been examined under different conditions. 2. After heat or ethanol denaturation, recovery of rabbit MT-II was quantitative. 3. Recovery of rabbit MT was not affected by the presence or absence of dithiothreitol (DTT) after heat treatment. However, recovery from ethanolic solution decreased in buffers that did not contain the antioxidant. 4. Acetone precipitation of rabbit MT resulted in lower yields which approached 90% only in the presence of DTT. 5. Recovery of metallothionein-like proteins from cytosols of the hepatopancreas of the tanner crab, Chionoecetes bairdi, were examined using pulse polarography and HPLC. Relative to heat denaturation, ethanol and acetone yielded recoveries of 66 and 28%, respectively, in the presence of DTT. However, acetone did yield a solution which contained less extraneous protein of molecular mass greater than 43 kDa than heat denaturation. 6. We concluded that heat denaturation is the preferred treatment for quantitative recovery of metal-binding proteins. Acetone precipitation is useful for the purification of MT.
Thompson
J A
JA
Ocean Chemistry Division, Institute of Ocean Sciences, Sidney, BC, Canada.
Sutherland
A E
AE
D000096:Acetone; D000818:Animals; D003386:Brachyura / Q000737:chemistry*; D002021:Buffers; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D003600:Cytosol / Q000737:chemistry*; D004064:Digestive System / Q000737:chemistry / Q000648:ultrastructure; D000431:Ethanol; D006358:Hot Temperature; D008667:Metalloproteins / Q000302:isolation & purification*; D011048:Polarography; D011489:Protein Denaturation / Q000502:physiology; D012015:Reference Standards
D002021:Buffers; D003470:Culture Media; D008667:Metalloproteins; D000096:Acetone; D000431:Ethanol