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Tryptophan residues replaced by Phe or Cys were used to characterize the refolding reaction of human carbonic anhydrase II. Kinetic fluorescence of pseudo wild-type human carbonic anhydrase II (HCAII) was compared to seven different tryptophan mutant traces. Formation of tertiary structure was observed earlier at tryptophans that associated with the central core of beta-strands than for tryptophan residues in the N-terminal minidomain. Central beta-parts were assumed to be present in the kinetic intermediate.

The central part of the beta-sheet core, consisting of 10 beta-strands of the enzyme human carbonic anhydrase II, has an ordered native-like secondary structure, while the beta-sheet close to the surface is less ordered. The native state of an enzyme is attained via formation of intermediates by folding. Understanding the precise amino acid sequence required for the protein to fold into a specific three-dimensional conformation for biological activity is a challenge.

Two-dimensional NMR spectroscopic studies of the wild-type human carbonic anhydrase II and its mutants, resulting from the replacement of tryptophan residues by cysteine or phenylalanine, reveal no significant changes in protein structure. The two tryptophan residues, Trp 97 and Trp 245, contribute 52% and 38%, respectively, to the fluorescence spectrum. His64 quenches the fluorescence from Trp5, while there is a transfer of energy from Trp192 to Trp209 and from Trp16 to Trp5. The beta-strands 3-7 of the native protein correspond to the core of a globule intermediate, as revealed by a study of the tryptophan environment through acrylamide quenching of the fluorescence.

Mutational analysis of the circular dichroism (CD) spectra of human carbonic anhydrase II (HCA II), in which the tryptophans are replaced, reveals that tryptophans contribute markedly to the CD spectrum at all wavelengths. Tryptophans cause the complexity of the near-UV spectrum of HCA II and participate in aligning CD band changes to the secondary structure changes during folding reactions. Tryptophans also affect the far-UV spectrum.