Role of Herborn (K240E) and Milano Slow (D375H) human serum albumin variants towards binding of phenylbutazone and ibuprofen

• Published in: International journal of biological macromolecules 2019-08, Vol.134, p.645-652
• Main Authors: Nerusu, Aparna, Chinthapalli, Dinesh Kumar, Subramanyam, Rajagopal
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Substitution; Anti-Inflammatory Agents, Non-Steroidal - chemistry; Anti-Inflammatory Agents, Non-Steroidal - metabolism; Binding constants; Circular Dichroism; Fluorescence spectroscopy; Fluorescent Antibody Technique; HSA variants; Humans; Ibuprofen; Ibuprofen - chemistry; Ibuprofen - metabolism; Mutagenesis, Site-Directed; Mutant Proteins; Phenylbutazone; Phenylbutazone - chemistry; Phenylbutazone - metabolism; Protein Binding; Protein Conformation, alpha-Helical; Recombinant Proteins; Serum Albumin, Human - chemistry; Serum Albumin, Human - genetics; Site-directed mutagenesis; Spectrometry, Fluorescence; Structure-Activity Relationship
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2019.05.075

Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA determines the pharmacokinetics and pharmacodynamics of the drugs. There are 67 natural genetic variants of HSA were reported in literature. Studying the effect of albumin modifications on drug binding helps to treat the patients with proper medication. In the present study, we have aimed to understand the effect of two natural variants of HSA, such as Herborn (K240E) and Milano Slow (D375H) on the binding of phenylbutazone and ibuprofen. For this, we have generated K240E and D375H mutants and also double mutant (K240E/D375H) of HSA using site directed mutagenesis. The recombinant HSA and its variants were expressed in Pichia pastoris. The interaction of HSA and its variants to phenylbutazone and ibuprofen was studied using fluorescence spectroscopy. Our results showed that there is no significant effect of K240E and D375H mutations on phenylbutazone and ibuprofen binding. But the effect is significant when both the mutations were there in a single protein (K240E/D375H). Further, the CD spectroscopy data showed that there is no effect of phenylbutazone and ibuprofen binding on the conformation of protein, except in case of D375H, where there is a conformational change in the binding pocket with the ibuprofen binding. •The HSA variants Herborn (K240E), Milano Slow (D375H) and the double mutant (K240E/D375H) were expressed using P. pastoris.•Variants are having reduced α-helical content.•There is no significant effect of K240E and D375H mutations on phenylbutazone and ibuprofen binding.•Double mutant (K240E/D375H) showed reduced binding to phenylbutazone and ibuprofen.•There is no effect of phenylbutazone and ibuprofen binding on the conformation of the proteins.