Optical Rotatory Dispersion of Human Carbonic Anhydrases: Cotton Effects and Aromatic Absorption Bands

Three distinct carbonic anhydrases, now denoted as A, B, and C, have been separated from human erythrocytes, by several different methods.1-4Enzyme A is present in small amount and with low specific activity, B in much larger amount but also with low specific activity, C in small amount but with high specific activity.5All three enzymes have molecular weights close to 30,000 and each contains one atom of zinc per molecule.In what follows, we are chiefly concerned with enzymes B and C.Studies of optical rotatory disperson, short wavelength ultraviolet absorption, and infrared spectra have suggested that native carbonic anhydrase B, although a highly compact molecule with a very low intrinsic viscosity, contains little or no a-helix.3Similar evidence from rotatory dispersion, some of which we report here, is now available for carbonic anhydrase C. In recent studies, we have also ob- served unusual features in the optical rotatory dispersion of both these enzymes, in- volving Cotton effects in the region between 260 and 300 m/u, which are evidently associated with the absorption bands of the aromatic amino acid residues in the same region.These Cotton effects disappear on denaturation of these enzymes in acid, urea, or guanidine hydrochloride.Although somewhat similar Cotton effects in this range of wavelengths have been found in poly-L-tyrosine and in a few proteins, as we point out later in the discussion, the rotatory dispersion patterns of the carbonic an- hydrases are highly specific.We report here some major features of the observed effects, with comments on their implications for the structure of these enzymes.Methods.-Experimental:The carbonic anhydrases were prepared from human erythrocytes supplied by the Protein Foundation Laboratories, Jamaica Plain, Massachusetts, through the kindness of Dr. R. B. Pennell.We removed hemoglobin from the carbonic anhydrases by treat- ment with ethanol and chloroform at low temperature.3However, we subsequently separated the different carbonic anhydrases, not on hydroxylapatite columns as previously,3 but by passage through a column of diethyl-aminoethyl Sephadex (DEAE-Sephadex A-50, medium grade, ob- tained from Pharmacia, Inc., New York) in tris(hydroxymethyl)aminomethane buffer at pH 8.7-8.9.The method is still undergoing modification and will later be described in detail; it in- volves either gradual or stepwise increase of the buffer concentration at nearly constant pH.Carbonic anhydrase C, with an isoelectric point of 7.3, is eluted first; enzyme B, which is iso- electric at pH 5.7, emerges at a somewhat higher ionic strength; and enzyme A emerges later, mixed with some inactive components, at still higher ionic strength.The high capacity of the DEAE-Sephadex permits preparation of much larger quantities of the enzymes, in a single run, than we could obtain by the earlier procedures which employed hydroxylapatite columns; quan- tities of 6 gm or more of crude carbonic anhydrase have been separated on a DEAE-Sephadex column with a total volume of 2.3 liters, at a flow rate of about 3 ml per minute.This yields several grams of enzyme B, which is sufficiently pure to give only a single band on starch gel electrophoresis by procedures previously described.3Its specific activity, assayed by the pro- cedure of Wilbur and Anderson,7 was constant in different fractions of the separated band of eluted material at 14,000 enzyme units per mg protein.The enzyme C fraction, on the other hand, although it gave a specific activity of 33,000 units per mg, yielded electrophoretic patterns con- taining a minor band indicating the presence of some residual enzyme B. We removed this by