The aim of the present work was the biophysical characterization of the Amynthas gracilis hemoglobin (HbAg). The oxy-HbAg optical absorption data, with Soret and Q bands centered at 415, 540 and 575 nm, were stable and unchanged at pH 7.0. An increase in pH promotes decrease in the intensity in the optical absorption bands, suggesting an oligomeric dissociation and partial oxidation. Identical stability at pH 7.0 was observed in DLS results that presented a hydrodynamic diameter of 28 nm, characteristic of the whole oligomer. DLS shows that HbAg undergoes oligomeric dissociation and an aggregation/denaturation process that corroborates spectroscopic data. Our results showed that the monomer d presents four isoforms with molecular mass (MM) ranging from 16,244 to 16,855 Da; the trimer subunit presents two isoforms, (abc)₁ and (abc)₂, with MM of 51,415 ± 20 Da and 51,610 ± 14 Da, respectively, and a less intense species, at 67,793 Da, assigned to the tetramer abcd. Monomeric chains a, obtained from reduction of the disulfide-bonded trimer abc, present four isoforms with MM 17,015 Da, 17,061 Da, 17,138 Da and 17,259 Da. DLS and LSI revealed an isoeletric point (pI) of oxy-HbAg of 6.0 ± 0.3 and 5.5, respectively. Data analysis by IEF–SDS–PAGE revealed that the pI of oxy-HbAg is 6.11, correlating with DLS and LSI data. These studies indicate that oxy-HbAg is very stable, at pH 7.0, and has differing properties from orthologous giant hemoglobins.

本工作的目的是对轻骨Amynthas血红蛋白（HbAg）的生物物理表征。Soret和Q谱带分别位于415、540和575 nm处的oxy-HbAg光吸收数据在pH 7.0时稳定且不变。pH值的增加促进了光吸收带强度的降低，表明存在寡聚解离和部分氧化。在DLS结果中观察到在pH 7.0下相同的稳定性，其呈现出28nm的流体力学直径，这是整个低聚物的特征。DLS显示HbAg经历了低聚解离和聚集/变性过程，从而证实了光谱数据。我们的结果表明单体d呈现四种同工型，分子量（MM）为16,244至16,855 Da。三聚体亚基具有两种同工型（abc）₁和（abc）₂，其MM分别为51,415±20 Da和51,610±14 Da，以及一种强度较小的物种，为67,793 Da，分配给四聚体abcd。从二硫键结合的三聚体abc还原获得的单体链a呈现四种同工型，分别为MM 17,015 Da，17,061 Da，17,138 Da和17,259 Da。DLS和LSI显示oxy-HbAg的等电点（pI）分别为6.0±0.3和5.5。IEF-SDS-PAGE数据分析显示，oxy-HbAg的pI为6.11，与DLS和LSI数据相关。这些研究表明，氧合HbAg在pH 7.0时非常稳定，并且与直系同源巨血红蛋白具有不同的特性。