Preparation of cysteine-34-nitroxide spin labeled human α₁-microglobulin.

α(1)-Microglobulin (α(1)m) is a protein of yet unresolved function occurring in blood plasma and urine. It consists of a lipocaline type of fold with two cysteine residues forming a disulfide bridge and the third cysteine-34 remaining a free, somewhat reactive thiol. A number of investigations point to an interaction with heme and we have recently reported, that heme binding triggers the formation of a stable α(1)m trimer upon modification of cysteine-34 with 2-iodoacetamide, i.e., α(1)m(heme)(2) [J.F. Siebel, R.L. Kosinsky, B. Åkerström, M. Knipp, Insertion of heme b into the structure of the Cys34-carbamidomethylated human lipocalin α(1)-microglobulin-formation of a (heme)(2)(α(1)-microglobulin) complex, ChemBioChem 13 (2012) 879-887]. For further structural and functional investigations, an improved purification protocol for α(1)m was sought, in particular yielding an untagged amino acid sequence. The method reported herein improves the speed and the yield of the protein production even when an expression plasmid without tag was applied. Furthermore, for the purpose of future structural studies using electron paramagnetic resonance (EPR) techniques, in accordance to the modification with 2-iodoacetamide (α(1)m(AM)), the protein was modified with 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-(2-iodoacetamido)-PROXYL) yielding the nitroxide spin labeled α(1)m(N-O). The extinction coefficient of the protein was calibrated using magnetic circular dichroism (MCD) spectroscopy of tryptophan (ε(280nm)=40,625M(-1)cm(-1)). The parallel quantification by absorbance spectroscopy (protein) and cw-EPR spectroscopy (radical spin) determined the degree of spin labeling to 90%. Characterization of the protein by circular dichroism (CD) spectroscopy and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) upon tryptic digestion further demonstrated the similar fold of α(1)m(AM) and α(1)m(N-O), but also established the modification of cystein-34 as well as the formation of the cysteine-72-cysteine-169 disulfide bond.