Oxyhemocyanin also

However, some other structures also have been proposed in opposition to 1. Lontie and Vanquickenborne, for example, suggested the structure represented by 2 for the active site of Helix pomatia oxyhemocyanin (3). 

Recently, we also found that the blue solution of oxyhemocyanin turns red-purple upon addition of a large quantity of ethyleneglycol. Characterization of the purple hemocyanin should contribute to the understanding of the structural aspects of native hemocyanin. 

Many investigators reported that the two copper ions are bridged by the peroxide dianion in oxyhemocyanin (6, 7, 8). We also observed a similar O—O stretching vibration at around 750 cm-1 in the resonance Raman spectrum ofS. lessoniana oxyhemocyanin, as illustrated in Figure 3 (Curve A). The purple hemocyanin also exhibits the charac-... 

Regeneration of the native oxyhemocyanin from the purple form also was examined by use of ESR measurement, and the essential reversibility was affirmed through the spectral variation as illustrated in Figure 4 (Curve C). 

If one adds NCS" to the normal oxyhemocyanin solution before preparing the purple form, the solution becomes colorless because of oxygen loss. Addition of a large quantity of ethyleneglycol (80 v/v% of the total volume) to the resulting colorless -solution does not produce the purple state. However, the final solution exhibits an ESR signal whose pattern and intensity is almost the same as those of the purple hemocyanin. This finding also coincides with the consideration that... 

The presence of Cu(II) in oxyhemocyanin is also indicated by resonance Raman studies. Freedman, Loehr and Loehr72) examined arthropod and molluscan oxyhemocyanin using laser Raman excitation in the visible region, between 458 and 647 run. They found strong evidence for enhancement of the 0—0 stretch within the 570nm visible absorption. The vibrational assignment was confirmed by 1602/1802 substitution, the observed frequencies being 744 cm-1 (1602), 704 cm-1 (1802) for arthropod and 749 cm-1 (1602), 708 cm-1 (1802) for molluscan oxyhemocyanin. 

The resonance Ramen studies also provide data related to the similarities between different hemocyanins. The O—O stretch is almost identical in arthropod and mol-luscan oxyhemocyanin. The Cu-imidizole stretch differ by 20 cm-1 between the two but five chromatographic fractions of an arthropod oxyhemoxyanin show differences in the fine structure within 15 cm-1 on each side of the 287 cm-1 peak74). Apparently there are differences in the exact coordination structure of the Cu(II) (Imidazole)n group but these differences do not appear to be large enough to invalidate comparisons between different hemocyanins. 

Guo, Chang, Li and Douglas82 have used 19F—NMR spectra and found that a 10% oxyhemocyanin (arthropod) solution caused a 38.7 Hz increase in the line width observed for a 0.125 M KF solution. That effect occurs without displacement of 0 2 from the oxyhemocyanin. Agents such as thioacetamide which induce conformational changes in the protein leading to the expulsion of 02 also expel fluoride ion from the active site even though F" is evidently bound in the active site quite differently than 02. 

The oxidative addition model for reversible O2 binding by metal proteins is also reasonable for hemocyanin. Hemocyanin is a copper protein which binds one O2 molecule for every two copper atoms. The deoxy Cu(I) form has no appreciable absorption in the visible region. When oxygenated, the protein is blue and exhibits a rich visible spectrum, wiA bands at 700 (c 75), 570 (c 500), 440 (c 65), and 347 nm (c 8900) (53). The pattern of bands around 570 nm leaves little doubt that oxyhemocyanin contains Cu(II) (53). The enhanced LF band intensities further suggest a dimeric Cu(II) complex. For comparison. 

An example of the use of oxygen isotopes in the study of halogen oxides is given in the on-line supplement to Chapter 8, where there is also a worked example showing how the geometry of the coordination of dioxygen in oxyhemocyanin can be deduced using O2 enriched with 0. 

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