Kongshaug M, Moan J
Department of Biophysics, Institute for Cancer Research, Norwegian radium Hospital, Oslo, Norway.
Int J Biochem Cell Biol. 1995 Apr;27(4):371-84. doi: 10.1016/1357-2725(94)00088-s.
Previous studies of the serum binding of the photosensitizer hematoporphyrin (Hp) have given widely different results. The serum binding of Hp is therefore further illuminated by experiment and discussion. Ultracentrifugal separation of serum is improved and applied to study the binding of Hp to human serum and HSA. The observed distribution of Hp among the serum proteins is compared with the distribution expected from available association constants for Hp binding with individual proteins. The lipoprotein classes and the two major high density proteins (HDP), albumin and gamma-globulin, were separated in a NaCl-KBr gradient by single spin ultracentrifugation (SW 40; 30,000 rpm). HSA- and HDP-bound Hp were similarly distributed in the centrifuged gradient. Centrifugation of Hp-doped HSA separated the unbound Hp (75%) and the HSA-bound Hp (25%). The present association constant for the Hp-HSA complex (10(3)/M) was much lower than earlier published ones (10(6)/M) found by other techniques. The association of Hp with HDP in serum was much stronger than the association of Hp with the isolated HSA (electrophoretic grade). The estimated ratio of HSA-bound to LDL-bound HP in serum was at least 25 times larger than the experimental value. The percentage of LDL-bound Hp decreased with increasing Hp concentration. The serum binding of Hp is the same as that found previously using another rotor and another salt gradient (70.1 Ti, 70,000 rpm, NaCl-CsCl). LDL has high-affinity-low-capacity binding sites for Hp. HSA is the major HDP protein that binds Hp in human serum. The strength of the HSA-Hp complex may depend on the batch of HSA used and upon the absence/presence of other proteins. Proteins may interact in serum in manners that affect the binding of certain drugs. Neither the type of gradient salt nor the field of gravity affected the serum binding of Hp.
以往关于光敏剂血卟啉（Hp）血清结合情况的研究得出了差异很大的结果。因此，通过实验和讨论进一步阐明了Hp的血清结合情况。改进了血清超速离心分离法，并将其应用于研究Hp与人血清及人血清白蛋白（HSA）的结合。将观察到的Hp在血清蛋白中的分布与根据Hp与单个蛋白的可用缔合常数预期的分布进行比较。通过单旋超速离心（SW 40；30,000转/分钟）在NaCl-KBr梯度中分离脂蛋白类别以及两种主要的高密度蛋白（HDP），即白蛋白和γ-球蛋白。与HSA结合及与HDP结合的Hp在离心梯度中的分布相似。对掺杂Hp的HSA进行离心，分离出未结合的Hp（75%）和与HSA结合的Hp（25%）。目前Hp-HSA复合物的缔合常数（10³/M）远低于其他技术先前公布的数值（10⁶/M）。Hp与血清中HDP的缔合比Hp与分离的（电泳级）HSA的缔合要强得多。血清中与HSA结合的Hp和与低密度脂蛋白（LDL）结合的Hp的估计比例至少比实验值大25倍。与LDL结合的Hp的百分比随Hp浓度的增加而降低。Hp的血清结合情况与先前使用另一种转子和另一种盐梯度（70.1 Ti，70,000转/分钟，NaCl-CsCl）所发现的情况相同。LDL对Hp具有高亲和力-低容量的结合位点。HSA是人类血清中结合Hp的主要HDP蛋白。HSA-Hp复合物的强度可能取决于所用HSA的批次以及其他蛋白的有无。血清中的蛋白质可能以影响某些药物结合的方式相互作用。梯度盐的类型和重力场均未影响Hp的血清结合。
