Oxygen Binding Thermodynamics of Human Hemoglobin in the Red Blood Cell

Authors

Kyle K. Hill, University of Nebraska-LincolnFollow

First Advisor

Lawrence Parkhurst

Second Advisor

Mark Griep

Date of this Version

Spring 3-2021

Citation

Hill, K. Oxygen Binding Thermodynamics of Human Hemoglobin in the Red Blood Cell. PhD Thesis, The University of Nebraska-Lincoln: Lincoln, NE. 2021.

Comments

A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Chemistry, Under the Supervision of Professor Mark Griep & Professor Lawrence Parkhurst. Lincoln, Nebraska: March 2021

Copyright © 2021 Kyle Kelly Hill

Abstract

We report for the first time the binding constants and Hill numbers for oxygen in the red blood cell under physiological conditions. When compared to our results for hemoglobin in solution, our results show conclusively that hemoglobin binds oxygen more tightly and with lower co-operativity when packed in the red blood cell. At 18°C, these differences are striking: the respective half-saturation values are 15.57 µM (in red blood cells) and 18.83 µM (in solution), with corresponding Hill numbers of 2.475 (in red blood cells) and 2.949 (in solution). The optical complications that arise from high turbidity of red blood cell suspensions have been overcome via instrumental and analytical innovations. First, a modified internal cavity absorption meter (ICAM) using a suspension of strongly scattering milk particles yielded long optical path lengths, amplifying the target signal. Second, the detection of oxygen binding from 600 to 670 nm eliminated the multiple unwanted wavelength dependences of apparent fractional saturation found in the conventional 535 to 580 nm range. Finally, a transfer function made it possible to extract all thermodynamics data from the scattered light. Since the hemoglobin tetramer expands when it releases oxygen, our work suggests that dense packing of hemoglobin in the RBC must constrain this molecular expansion. The higher oxygen affinity and lower co-operativity results in a substantially more distributed delivery of oxygen into diverse body tissues, such as the lungs and the brain. Furthermore, consequent thermodynamic studies suggest that the differences arise mostly from an entropy difference between hemoglobin in the red blood cell and hemoglobin free in solution. All of these results contrast sharply with the decades-long assumption that the behavior of hemoglobin free in solution is an appropriate model for understanding the physiology of oxygen binding and delivery, and that those curves are physiologically relevant.

Advisors: Lawrence J. Parkhurst & Mark Griep