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Cell Biology International (2008) 32, 13371343 (Printed in Great Britain)
Characterization of water of hydration fractions in rabbit skeletal muscle with age and time of post-mortem by centrifugal dehydration force and rehydration methods
Ivan L. Camerona*, Nicholas J. Shorta and Gary D. Fullertonb
aDepartment of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA
bDepartment of Radiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA
Centrifugal dehydration force (CDF) and rehydration isotherm (RHI) methods were used to measure and characterize hydration fractions in rabbit psoas skeletal muscle. The CDF method assessed fluid flow rate from rabbit muscle and hydration capacity of the fractions. Bulk and multiple non-bulk water fractions were identified. The non-bulk water was divisible into the following fractions: two outer non-bulk fractions, a main chain proteins backbone or double water bridge fraction, and a single water bridge fraction. The total non-bulk water amounts to about 85% of the total water in the muscle. The sizes of the water fractions (in g water/g dry mass) agree with a recently proposed molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells (Fullerton GD, Cameron IL. Water compartments in cells. Methods Enzymol, 2007; Cameron IL, Fullerton GD. Interfacial water compartments on tendon/collagen and in cells. In: Pollack GH, Chin WC, editors. Phase transitions in cells. Dordrecht, The Netherlands: Springer, 2008). Age of the rabbit significantly slowed the flow rate of the outer non-bulk water fraction by about 50%. Also, muscle of the older rabbit (26 weeks vs. 12 weeks old) had less bulk water and less outer non-bulk water but the same amount of main chain backbone water compared to muscle of the younger rabbit. Increase in time post-mortem from 30
Keywords: Hydration, Myocytes, Muscle, Flow rate, Dehydration, Aging, Post-mortem.
The extent of water of hydration vs. the extent of bulk-like water in cells has been a subject of much debate. Cell physiologists and protein chemists have commonly accepted a water of hydration, or “bound water,” value in the range of 0.2–0.4
Recently published reports provide physical evidence for the formation of 4 different non-bulk water fractions/compartments on proteins and in cells (Fullerton et al., 2006a,b; Fullerton and Amurao, 2006; Fullerton and Rahal, 2007; Cameron et al., 1988a,b, 1997, 2007a,b; Cameron and Fullerton, 2008; Fullerton and Cameron, 2007). Data in these reports on collagen, globular protein and cells lead to the hypothesis of a molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells (Fullerton and Cameron, 2007). This molecular model of protein hydration fractions or compartments matches estimates made by multiple methods, all yielding water of hydration values up to and much beyond 0.2–0.4
This report deals with determination of the sizes of multiple water of hydration fractions and flow rates using CDF and RHI methods (see Cameron et al., 2007a,b) on psoas skeletal muscle of rabbits of different ages and of time post-mortem. Discussion focuses on the fit with the SHM method, and how age and post-mortem changes affect the hydration fractions of muscle water.
2 Methods and materials
2.1 Muscle samples
Rabbits 12 weeks and 26 weeks of age, weighing 3 and 4.75
2.2 Centrifugal dehydration force method
The method used for centrifugation dehydration was described previously (Cameron et al., 2007a,b). Weighed pieces of muscle from 0.19 to 0.21
The water content of the tissue was calculated by subtracting the final dry weight from the weight of the sample at each interval of time during centrifugation and then by expressing the data in g water/g dry mass. Plotting the data against time under centrifugal load reveals the shape of the curves defining the resistance to fluid flow or water flow rate through and out of the tissue. Thus the slope of the curve provides a numerical measure of the resistance to fluid flow.
In one experiment, fresh psoas muscle from the 26-week-old rabbit washed in deionized and stirred water for 2.5
2.3 Rehydration isotherm method
A water sorption isotherm as a function of time was measured for dried muscle. Proteins are very heat-labile when wet due to the small free energy difference between the native conformation of the protein and many possible random orientations of the protein chain when fully hydrated in a dielectric fluid. Thus, the protein must be first dried at room temperature in a vacuum chamber to hydration less than 0.26
2.4 Statistical analysis
The Graph Pad Prism statistical program was used for curve fits, ANOVA and multiple range tests. Lack of overlap of the 95% confidence interval values between means indicated significant differences.
3.1 Centrifugation dehydration force results
The muscle water content from 8
All data points from 0 to 4
Based on curve fitting of data from 8 to 120
Exponential dehydration rate decay plot of a 26-week-old muscle taken at 30
Analysis of water compartment flow rates in rabbit psoas muscle as a function of rabbit age (12 vs. 26 weeks) and time post-mortem (30 min vs. 4 h)a
Capacity (g water/g dry mass) of multiple water compartments of rabbit psoas muscle as a function of age and time post-mortema
A replotting of the total dehydration decay data on a logarithmic ordinate (Fig. 2) suggests the presence of breakpoints in the data fit at hydration levels of 0.80 and 0.26
Semi-logarithmic plot of psoas skeletal muscle hydration as a function of time exposed to 14,000
Reanalysis of water flow data based on possible change in exponential flow rate at 0.80 g water/g dry mass
The data in Tables 1 and 2 were subjected to a 2-way analysis of variance of the sizes of all water compartments. This type of analysis looks for the 2 main effects as well as interactions between them. They were age of the rabbit (12 vs. 26 weeks) and time post-mortem (30
The only significant difference between post-mortem time (30
3.2 Muscle rehydration data from the 12-week-old rabbit muscle
Data on water sorption for the dry muscle (Fig. 3a shows an excellent curve fit to a one-binding site hyperbolic equation), which describes the binding of a ligand to a receptor according to the law of mass action. The B
Fig. 3 a and b
Example of rehydration isotherm data of 12 week rabbit psoas muscle from the “absolute” dry state in an atmosphere of 45% relative humidity at 22
Rehydration isotherms analyses of 12 week rabbit psoas muscle at 30 min and 4 h post-mortem (values in g water/g dry mass)
Further examination of the rehydration data points (Fig. 1) indicates that the plotted data may actually consist of more than a single water sorption binding site. The possibility of more than a single water sorption binding site is illustrated in Fig. 2. Thus, the reevaluation of the rehydration data gives evidence of 3 linear rehydration water of hydration compartments. A summary of the sizes of the rehydration isotherm data analyses of the 12-week-old rabbit psoas muscle at 30
4.1 A molecular stoichiometric hydration model can explain the number and sizes of the multiple water of hydration fractions in skeletal muscle
Table 2 lists the size of the various water of hydration fractions as measured by the CDF and RHI methods. The total non-bulk water compartment given by CDF is the largest fraction of water in the muscle. RHI shows the “single water bridge” water fraction to be the smallest water fraction. The next smallest fraction identified by the CDF is &007E;4 times the size of the smallest “single water bridge” and is referred in Tables 2 and 5 as the main chain backbone water compartment. The size the bulk water fraction as determined by the CDF method is 3.3 to 5.4 fold less than the total non-bulk fraction in the skeletal muscle.
Comparison of size of multiple water fractions/compartments in bovine tendon/collagen, in human hemoglobin A, and in skeletal muscle (g water/g dry mass)
The muscle data in Table 5 indicate that the outer non-bulk water fraction listed in Table 2 can be divided into 2 subfractions at &007E;0.80
Such similar sized water compartments have lead to the hypothesis of a stoichiometric hydration model (SHM) that is applicable to intracellular and extracellular proteins (Fullerton and Cameron, 2007). This model was initially based on tendon/collagen (see Fullerton and Rahal, 2007 for details).
In brief, the SHM presents categories of water bridges. The single water bridge is found between properly spaced (>3.5
The SHM appears to be applicable to the CDF and RHI results obtained from skeletal muscle. The SHM therefore gives a molecular explanation for the interfacial water compartments measured on skeletal muscle.
4.2 Age and time post-mortem on size and flow rate of water compartments in psoas skeletal muscle
Before turning to age and time post-mortem, it is important to examine the possible sources of the bulk and of the non-bulk water compartments in the muscle. Based on a recently published assessment of the extra-muscle fiber space expressed as a percentage of total fiber cross-section from 5 different reports and 1 additional report (Chiakulas and Pauly, 1965), this parameter gives a mean value of 27.1
The data in Table 1 indicate that the age of the rabbit had greater effect on the flow of water from the outer non-bulk water compartment than post-mortem time. Rabbits go from about 3 to 4.75
In summary of age changes in the rabbit psoas muscle, the following age-dependent changes were observed: a 39% decrease in total water content, an almost 50% decrease in bulk water, and a 11% decrease in the outer non-bulk water compartment. No significant difference occurred in the main chain backbone water compartment. The water flow rate of the older rabbit muscle was less than half that of the younger rabbit. This slower flow rate of the older rabbit muscle is attributed to the greater distance and increased obstruction that a water molecule would encounter on its exit from the middle of a cross-section of an enlarged muscle fiber.
Statistical examination of post-mortem rabbit psoas muscle data in Tables 1 and 2 revealed only one significant different between the 30
How can this slowing of water flow rate with post-mortem time be explained? Bendall (1951) demonstrated that shortening of rabbit skeletal muscle resulting in rigor mortis which he explained as a slow irreversible contraction after death associated with a disappearance of ATP from the muscle. Bendall followed the decay of ATP and CP and muscle shortening at post-mortem times in rabbit psoas muscle. He reported that loss of ATP and CP was accompanied by muscle shortening. In rabbits killed by a blow to the head, which results in struggling, there was evidence of both muscle shortening and a decrease in ATP by 50
Structural examination of events associated with muscle shortening at times post-mortem revealed that cross-striations of muscle fibers come closer together, resembling a slow physiological contraction (Henderson et al., 1970). These authors demonstrated a 35% shortening of sarcomere length by 4
5 Summary and conclusions
1. The CDF and RHI methods applied to rabbit psoas skeletal muscle indicate the presence of multiple water of hydration fractions. They are defined as bulk water and a large total non-bulk water of hydration fraction. This large non-bulk fraction is divisible into water of hydration subfractions: an outer non-bulk fraction with two subfractions, a main chain backbone (double water bridge) fraction, and a single water bridge innermost water fraction. 2. The relative sizes of the water of hydration fractions in skeletal muscle agree with a recently proposed molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells. 3. The increased age of rabbit (26 vs. 12 weeks) slowed the flow rate of the outer non-bulk water compartment by &007E;50%. Older rabbit muscle has less bulk and outer non-bulk water fractions, but the size of the main chain backbone (double water bridge) water fraction did not change with age. 4. The only significant difference in muscle post-mortem data was a slower flow rate of water from the outer non-bulk water compartment between 30 5. The growing awareness of the size and physical properties of water of hydration fractions of proteins in or out of cells by molecular interactions between water and protein surfaces, as explained by the stoichiometric hydration model, is predicted to have a revolutionary impact on protein chemistry and cell biology.
The CDF and RHI methods applied to rabbit psoas skeletal muscle indicate the presence of multiple water of hydration fractions. They are defined as bulk water and a large total non-bulk water of hydration fraction. This large non-bulk fraction is divisible into water of hydration subfractions: an outer non-bulk fraction with two subfractions, a main chain backbone (double water bridge) fraction, and a single water bridge innermost water fraction.
The relative sizes of the water of hydration fractions in skeletal muscle agree with a recently proposed molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells.
The increased age of rabbit (26 vs. 12 weeks) slowed the flow rate of the outer non-bulk water compartment by &007E;50%. Older rabbit muscle has less bulk and outer non-bulk water fractions, but the size of the main chain backbone (double water bridge) water fraction did not change with age.
The only significant difference in muscle post-mortem data was a slower flow rate of water from the outer non-bulk water compartment between 30
The growing awareness of the size and physical properties of water of hydration fractions of proteins in or out of cells by molecular interactions between water and protein surfaces, as explained by the stoichiometric hydration model, is predicted to have a revolutionary impact on protein chemistry and cell biology.
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Received 15 September 2007/5 June 2008; accepted 30 July 2008doi:10.1016/j.cellbi.2008.07.022