Urea 'embraces' part of water molecules in urine
Urine mainly consists of water and an urea solution, which is a protein decomposition. Urea has to be excreted through urine, as a high concentration of urea in water causes enzymes losing their functions (denature). So far, scientists were not familiar with this process, but researchers at the FOM Institute for Atomic and Molecular Physics (AMOLF) in Amsterdam recently discovered that urea interacts very well with water: part of the water molecules are being immobilized as a result of the fact that urea firmly 'embraces' these molecules. The urea leaves the rest of the water undisturbed. Therefore, the denaturing effect of urea on proteins does not have its origins in an interference of the water network, but in a direct interaction between urea and proteins. The researchers have their results published in the scientific journal Proceedings of the National Academy of Sciences of the United States of America (PNAS) in November 2006.
Urea is a protein decomposition that is being produced continuously in the human body in large quantities. Protein molecules consist of long chains of amino acids that have to be folded to a three-dimensional structure, in order to become biologically active. The interactions between protein and water play an important role in this process. Proteins will be unfolding spontaneously at a high concentration of urea in water (denature). For centuries, gratefully use has been made of this process in biochemical laboratories, but in the human body the unfolding of proteins would be disastrous. The fact is that the protein loses its function. It is of vital importance that the urea will leave the body through the urine.
Activity of water in urea solutions
Science is still not familiar with the mechanism that urea would use for denaturing proteins. Up to now researchers believed that urea interferes with a hydrogen-bond network of water molecules (as is found in pure water). The interaction between water and proteins would change as a result of this interference, which then causes the unfolding of proteins. Yves Rezus and Huib Bakker, AMOLF institute, investigated the effect of urea on water by using ultra-fast laser systems. They examined the rigidity of the water network, being the function of the urea concentration.
A nice standard for the rigidity of the water network is the activity (re-orientation) of the water molecules: in a rigid network a water molecule cannot move as easily as in a flexible network.
Heavy water
The researchers added a small amount of heavy water (D2O) to the urea solutions, in order to investigate the re-orientation of water molecules in urea solutions. Thus, HDO-molecules came into being (i.e. water molecules that have one normal hydrogen atom (H) and one heavy hydrogen atom (D)), of which it was able to define the re-orientation by means of ultra-fast mid-infrared spectroscopy. In the experiment the urea solutions are radiated by mid-infrared pulses of 100 femtosecond (1 femtosecond = 10-15 s) and a wavelength of 4 micrometers. Absorption of the light pulse causes a vibration of the D-atom. Then, by using a second, somewhat slower light pulse, it will be examined at which angle the vibrating OD-group has been revolving in the period of time intermediate the two light pulses. By varying the period of time between the two light pulses, the rotation of the real-time molecules can be followed.
An unexpected behaviour
Rezus and Bakker found out that the bigger part of the water molecules is unaffected by the urea and is re-orientating just as fast as in pure water. Surprisingly, they found that this even applies to very high urea concentrations, at which all water molecules are in close contact with, at least, one urea molecule. It looks like the separate water molecules are not aware of the fact, whether they are in pure water or in a concentrated urea solution. However, a small part of the water molecules appeared to behave completely different. These molecules were immobilized by the urea, which manifested itself in a very slow re-orientation. The researchers were able to deduce that each urea molecule would immobilize about one water molecule.
So, they conluded that the immobilized water molecule is bonded to urea in a very special way. Urea keeps a firm grip on the water molecule using two hydrogen bonds, so that it cannot rotate any longer (figure 1).
Immediate interaction between urea and proteins
The results of this investigation show that urea does not interfere with the water network at all, therefore, the denaturing function of urea on proteins does not have its origin in the water network. The researchers have concluded that there have to be an immediate interaction between urea and proteins, which causes proteins to unfold. It should be noticed that the urea molecule has the same N-C=O spine as the amino acids that form the materials of proteins. So, it is quite possible that urea is able to put specific amino acids in a hold, just as it does with a water molecule.
The article is entitled: 'The effect of urea on the structural dynamics of water' It has been published on line in Proceedings of the National Academy of Sciences of the United States of America (PNAS), 21/11/2006 (10.1073/pnas.0606538103)
The authors are: Yves Rezus and Huib Bakker.
For more information, please contact Yves Rezus, FOM-AMOLF, phone: +31 (0)02 608 12 50 or +31 (0)20 608 12 34.