1H NMR and calorimetric measurements on rabbit eye lenses.

The dynamic properties of water molecules in the rabbit lens were studied by proton nuclear magnetic resonance line shape analysis, measurements of relaxation times as a function of temperature and calorimetric measurements. The experiments prove, as already suggested by other authors, that there are two types of water in the lens of rabbit eyes, namely bound unfreezable hydration water and bulk freezable water. Line shape analysis and relaxometry showed, that this two types of water exist in two different environments, which may be identified as the nucleus and the cortex of the lens. The line shape analysis showed furthermore that water molecules in the rabbit lens has a common spin lattice relaxation time (T1), but two different transverse relaxation times (T2A and T2B). The tentative model of fast water exchange on the T1 time scale and slow water exchange on the T2 time scale, was used to explain experimental proton relaxation data of the rabbit lens. An estimation for this exchange rate kex by comparing it to the relaxation times is given (T1(-1) << kex << T1(-1)). It has also been shown by a calorimetric measurements, that the lenses can be easily under-cooled to temperatures well below the freezing point of water. The achievable maximum undercooling temperature of the lens is a function of the cooling rate KC, therefore it has to be considered as an experimentally adjustable parameter which is not characteristic for the investigated sample. Thus it must be noted that any previous discussions about the specific value of the temperature of water crystallisation in biological systems need to be carefully reconsidered.

Measurements of proton relaxation time T2 on cattle eyes lenses.

A simple two phase model does not explain the temperature dependence of T1 relaxation time in lenses as biological systems. Therefore, a distribution of correlation times of water particles has to be assumed by a certain distribution of the water protein binding energy. As a consequence, from the temperature dependence of T1 relaxation time, the activation energy of water molecules in the lens cannot be evaluated directly without the knowledge of the distribution width. This problem can be solved by T2 measurements in lenses. From the slope of T2 as a function of temperature, mean activation energy can be calculated independently on the distribution width. Measurements were performed on lenses originating from 5-7 years old cows, 2-year old bull-calfs and a 12-year old bull in the temperature range -30 to +105 degrees C. It could be demonstrated that about 80% of water behaves as liquid-like water with an activation energy 14 +/- 4 kJ/mol corresponding to the value of free water. The remaining water (about 20%) is bound to the protein with an activation energy of 20 +/- 5 kJ/mol. At 42 degrees C the protein denaturation process starts in the eye lens and will be completed by 70 degrees C, yielding a protein bound-water complex.