Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that measurements may be made on multiple samples containing < or =20 microg of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by x-ray crystallography or NMR.

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Using circular dichroism spectra to estimate protein secondary structure

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

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Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

Publisher Summary Glutathione transferases with basic isoelectric points have been purified from the human liver, and an acidic form is purified from human erythrocytes. However, this chapter presents a procedure for the preparation of glutathione transferase from human placenta. Enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione and l-chloro-2,4-dinitrobenzene. Purification procedure involve the extraction from the human placenta; the supernatant fraction from Step 1 is chromatographed on a column (12.5 x 80 cm) of Sephadex G-25 and the pooled fractions from Step 2 are applied to a column (9 x 13 cm) of DEAE-cellulose equilibrated with l0 mM Tris-HCl at pH 7.8. The pooled effluent from Step 3 is dialyzed overnight against 3 x 10 liter of 10 mM sodium phosphate buffer; then affinity chromatography is carried on S-hexylglutathione coupled to epoxy-activated sepharose 6B and the pooled fractions from Step 5 are charged onto a column of Sephadex G-75 that is equilibrated and eluted with 10 mM sodium phosphate at pH 6.7, containing 0.1 mM dithioerythritol. The active fractions of effluent are pooled.

Glutathione transferase (human placenta).

We developed a candidate Reference Method for measuring total serum protein by use of the biuret reaction. The method involves a previously described biuret reagent (Clin. Chem. 21: 1159, 1975) and Standard Reference Material (SRM) 927 bovine albumin (National Bureau of Standards) as the standard. At 25 degrees C, color development for 30 or 60 min provides identical serum protein values. Glucose (up to 10 g/L) and bilirubin (up to 300 mg/L) do not interfere. Hemoglobin, at 3 g/L, increases apparent serum protein by 0.4 g/L. The presence of dextran in serum causes easily detected turbidity, but this interference can be eliminated by centrifuging the reaction mixture. Therapeutic concentrations of ampicillin, carbenicillin, penicillin, oxacillin, nafcillin, chloramphenicol, cephalothin, and methicillin in blood do not interfere, nor do triglycerides up to 10 g/L. Within-run and day-to-day standard deviations of the method are 0.1 and 0.4 g/L, respectively.

A candidate reference method for determination of total protein in serum. I. Development and validation.

Partial purification and characterization of tryptophan hydroxylase from rabbit hindbrain.

A rapid and convenient colorimetric method for the determination of small amounts of protein, based on the biuret reaction and readings at 330 mμ, is described. The “micromethod” allows the determination of 10 to 200 mg per cent protein with an acurassy of ± 1.5 mg per cent. An “ultra micro” modification allows determinations of 10 to 400 micrograms protein.The method is applied for the determination of total protein in 1 ml of cerebrospinal fluid.

Goa J

Papers

A micro biuret method for protein determination; determination of total protein in cerebrospinal fluid.