Biophysical Chemistry 97 (2002) 139–157 0301-4622/02/$ - see front matter  2002 Elsevier Science B.V. All rights reserved. PII:S0301-4622 Ž 02 . 00046-7 Fluorescence properties of tryptophan residues in the monomeric d-chain of Glossoscolex paulistus hemoglobin: an interpretation based on a comparative molecular model Carolina Bosch Cabral , Hidetake Imasato *, Jose Cesar Rosa , Helen Julie Laure , a,b a, b b,e ´ ´ Carlos Henrique Tomich de Paula da Silva , Marcel Tabak , Richard Charles Garratt , c a c Lewis Joel Greene b,d Instituto de Quımica de Sao Carlos,, Universidade de Sao Paulo, P.O. Box 780, 13560-970, Sao Carlos, SP, Brazil a ´ ˜ ˜ ˜ Centro de Quımica de Proteınas, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Sao Paulo, Brazil b ´ ´ ˜ ˜ ˜ Instituto de Fısica de Sao Carlos, Universidade de Sao Paulo, Sao Paulo, Brazil c ´ ˜ ˜ ˜ Departamento de Ginecologia e Obstetrıcia e Departamento de Biologia Celular, Molecular e Bioagentes Patogenicos, d ´ ˆ Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Sao Paulo, Brazil ˜ ˜ ˜ Escola Paulista de Medicina—UNIFESP, Sao Paulo, Brazil e ˜ Received 5 November 2001; received in revised form 28 February 2002; accepted 7 March 2002 Abstract The primary structure of the 142 residue Glossoscolex paulistus d-chain hemoglobin has been determined from Edman degradation data of 11 endo-Glu-C peptides and 11 endo-Lys-C peptides, plus the results of Edman degradation of the intact globin. Tryptophan occupies positions 15, 33 and 129. Homology modeling allowed us to assign the positions of these Trp residues relative to the heme and its environment. The reference coordinates of the indole rings (average coordinates of the C and C atoms) for W15 and W129 were 16.8 and 18.5 A, respectively, from the ´2 d2 ˚ geometric center of the heme, and W33 was located in close proximity to the heme group at a distance which was approximately half of that for W15 and W129. It was possible to identify three rotamers of W33 on the basis of electrostatic and Van der Waals energy criteria. The calculated distances from the center of the heme were 8.3, 8.4 and 9.1 A for Rot1, Rot2 and Rot3, respectively. Radiationless energy transfer from the excited indole to the heme ˚ was calculated on the basis of Forster theory. For W33, the distance was more important than the orientation factor, ¨ k , due to its proximity to the heme. However, based on k , Rot2 (k s0.945) was more favorable for the energy 2 2 2 transfer than Rot1 (k s0.433) or Rot3 (k s0.125). In contrast, despite its greater distance from the heme, the k 2 2 2 of W129 (2.903) established it as a candidate to be more efficiently quenched by the heme than W15 (k s0.191). 2 Although the Forster approach is powerful for the evaluation of the relative efficiency of quenching, it can only ¨ explain pico- and sub-nanosecond lifetimes. With the average lifetime, NtMs3 ns, measured for the apomonomer as the reference, the lifetimes calculated for each emitter were: W33-1 (1 ps), W33-2 (2 ps), W33-3 (18 ps), W129 (100 ps), and W15 (600 ps). Experimentally, there are four components for oxymonomers at pH 7: two long ones *Corresponding author. Tel.: q55-16-273-9955; fax: q55-16-273-9982. E-mail address: hidetake@iqsc.sc.usp.br (H. Imasato).