Cell Biology International (2008) 32, 1337-1343 - Ivan L. Cameron and others - Characterization of water of hydration fractions in rabbit skeletal muscle with age and time of post-mortem by centrifugal dehydration force and rehydration methods

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Cell Biology International (2008) 32, 1337–1343 (Printed in Great Britain)

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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. Cameron^a^*, Nicholas J. Short^a and Gary D. Fullerton^b

^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

Abstract

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 30min to 4h resulted in rigor mortis and a significantly slower flow rate of water from the outer non-bulk water fraction, which is attributed to muscle contraction, increased packing of contractile elements and increased obstructions to flow of fluid from the muscle fibers.

Keywords: Hydration, Myocytes, Muscle, Flow rate, Dehydration, Aging, Post-mortem.

^*Corresponding author.

1 Introduction

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 0.2–0.4g water/g dry mass (Ling, 1972, 2006; Cameron and Fullerton, 2008). This common assumption, either expressed or just tacitly assumed, is that all water above that of this “bound water” value has the physical properties of liquid water in bulk. Evidence is mounting to refute this common assumption about the extent of water of hydration on proteins and in cells (Ling, 2001, 2004; Pollack, 2003; Cameron et al., 1997, 2007; Fullerton and Cameron, 2007).

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 0.2–0.4g water/g dry mass value.

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 4.75kg, respectively, were used. The rabbits were killed by a blow to the head, decapitated, evisceration, and the wall of the abdomen cut open to expose the psoas muscle. The psoas was removed and transected halfway between the origin and insertion. Two-millimeter sections across the muscle fibers were blotted with Whatman number one filter paper. The initial blotted weight of specimens ranged from 0.19 to 0.21g. The remainder of the psoas was wrapped in clear polyethylene at room temperature of 21°C for 4h, unwrapped and a fresh 2mm transection through the body of the muscle was taken 0.5cm from the first transection, blotted with filter paper and weighed prior to centrifugation. In one experiment, fresh 2mm transections of muscle were placed in deionized water at 21°C and stirred for 2.5h, blotted, weighed and subjected to the centrifugation procedure as described below.

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 0.21g were placed in a microfilterfuge tube (Rainin Instrument Co. Oakland, CA, Cat. No: 7016-022) containing a filter membrane of 0.45μm pore size. The specimens did not cover the entire surface of the filter. Samples were centrifuged for intervals up to 150–210min with the specimens are 5.7cm from the rotor center, giving a total force of 14,000×g and a stress of about 4.0MPa. (MC 1400 Microcentrifuge, Hoefer Scientific Instruments, San Francisco). Intermediate weights of the tissues were recorded at each centrifugation interval. Following centrifugation, all samples were dried to weight equilibrium at 80–90°C in a vacuum oven and the final dry weight was measured to allow the water content of each sample to be expressed as grams of water per gram dry weight. After the centrifuge run, the bottom section of the centrifuge tubes were dried in the vacuum oven, weighed, washed free of any solutes, dried as before and reweighed. This procedure allows determination of loss of solutes from the tissue during centrifugation.

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 2.5h resulted in muscle swelling. Application of the CDF method to the water washed and filter paper blotted muscle revealed a larger fast-flowing water fraction during the first minute than it did from 4min of centrifugation onward. Swelling caused a 2.3-fold increase in the size of this faster flowing water fraction, leading to the conclusion that this initial faster flowing water compartment has the characteristics expected of bulk water.

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 0.26g water/g dry mass before raising the temperature slowly to 90°C. The sample was dried at 90°C for &007E;7 days until no further decrease in mass was observed. The specimen was exposed to room temperature of 21°C in an atmosphere with relative humidity 45%. The specimen was periodically weighed on an analytical balance. Data at each time of weighing were expressed in g water/g dry mass and all data were used to obtain the curve fit of the data. The difference between hydrated mass and dry mass divided by the dry mass was recorded.

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 Results

3.1 Centrifugation dehydration force results

The muscle water content from 8min onward during the centrifugation procedure was used for exponential curve fit analysis. The 8–120min data from each muscle gave excellent fits to exponential decay (i.e. r² values for individual specimens ranged from 0.9967 to 0.9995). Extrapolation of these best fit curves to the zero time water content values was determined.

All data points from 0 to 4min fell above the exponential fit from 8min of centrifugation onward. This fact indicates the presence of a faster flowing water compartment, the size of which was determined by subtraction of the exponential intercept at 0 time from the initial 0 time water content value, as previously described (Cameron et al., 2007b; Haskin et al., 2006). The amount of solute lost from the muscle during centrifugation was determined as described in Section 2, and ranged from 2.4 to 2.9% of the dry mass of the muscle sample. Based on this finding, we are confident that essentially all of the muscle weight loss during centrifugation is due to water loss.

Based on curve fitting of data from 8 to 120min, a single component exponential decay curve gave evidence of three water fractions/compartments (Fig. 1, and Tables 1 and 2). The best single exponential fit of each muscle reached a calculated plateau at infinite time of centrifugation (Table 2). This plateau value indicates the presence of a water compartment not removed by the centrifugation forced used to this time point in this study. The mean and 95% confidence interval of the individual plateau values are listed in Table 2, which also summarizes the sizes of these water compartments. The compartment that most rapidly flowed from the tissue was designated “the bulk water compartment” while the slower flowing compartment, as defined by its exponential decay of water removed, was designated “the outer non-bulk water compartment”.

Fig. 1

Exponential dehydration rate decay plot of a 26-week-old muscle taken at 30min (▲) and 4h (■) post-mortem as a function of time exposed to 14000×g force centrifugation. See Table 1 for summary of decay rate data.

Table 1.

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

Table 2.

Capacity (g water/g dry mass) of multiple water compartments of rabbit psoas muscle as a function of age and time post-mortem^a

Age (weeks)	26		12
Time post-mortem	30 min	4 h	30 min	4 h
Total initial water content	3.088 ± 0.050	3.225 ± 0.036	4.862 ± 0.135	4.489 ± 0.215

Compartment
I. Bulk water	0.364 ± 0.048	0.416 ± 0.047	0.728 ± 0.090	0.728 ± 0.144
II. Total non-bulk water	2.39 ± 0.005	2.46 ± 0.010	4.10 ± 0.068	3.34 ± 0.068
A. Outer non-bulk^b	2.18	2.21	3.89	3.53
B. Backbone double water bridge^c	0.213 ± 0.023	0.248 ± 0.068	0.214 ± 0.015	0.230 ± 0.037
C. Single water bridge^d	ND	ND	0.055 (95% CI – 0.052–0.056)	0.054 (95% CI – 0.051–0.056

^a All values in table are reported as g water/g dry mass (mean±SEM or mean with 95% CI).

^b Found by subtraction of backbone double water bridge from total non-bulk.

^c Includes single water bridge.

^d Determined by rehydration isotherm method (see Table 4 for values).

Age (weeks)	Time post-mortem	Half-life (min)	Half-life 95% CI	Fit (R²)
12	30 min	12.43	11.84–13.07	0.9995
	4 h	16.51	15.70–17.31	0.9978

26	30 min	28.03	27.09–29.04	0.9967
	4 h	31.85	29.51–34.60	0.9972

^a Statistical analyses revealed significantly faster (approximately two-fold) flow rate at 12 vs. 26 weeks of age, and significantly slower flow rate at 4h vs. 30min post-mortem in at both ages.

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 0.26g water/g dry mass. To determine if the breakpoint at 0.80g/g gives a better fit to a 2 water fraction decay of the data compared to a single water fraction decay, 7–9 of the data points above the 0.80g/g value and 5–6 data points below the 0.80g/g value were subjected to exponential curve fits. The mean correlation coefficient for the 2 fraction decay fit was higher than the mean of the correlation coefficients for the single exponential curve fits (Table 3). These findings give evidence of 2 outer non-bulk water fractions with the second fraction having a slower flow rate than the first fraction. The data in Fig. 2 also give evidence of another breakpoint in the decay curve at 0.26g/g, indicating the presence of an even slower flow rate fraction.

Fig. 2

Semi-logarithmic plot of psoas skeletal muscle hydration as a function of time exposed to 14,000×g force centrifugation. Data from 26 week rabbit at 30min post-mortem are represented. The hydration fractions are delineated by the breakpoints. The text describes the analysis of data, and the results are summarized in Tables 3 and 5.

Table 3.

Reanalysis of water flow data based on possible change in exponential flow rate at 0.80 g water/g dry mass

	12 weeks rabbit		26 weeks rabbit
Time post-mortem	30 min	4 h	30 min	4 h
Single
Half-life (mean)	15.12	18.70	27.46	29.65
R-value	.9979	.9963	.9993	.9973

First fraction
Half-life (mean)	12.34	12.54	23.91	22.24
R-value	.9992	.9992	.9992	.9960

Second fraction
Half-life (mean)	21.43	32.12	37.58	33.81
R-value	.9960	.9994	.9995	.9956

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 (30min vs. 4h). The analysis was simplified because no significant interactions were found, although several age-dependent differences were present. The flow rate of the outer non-bulk water compartment was significantly slower in the muscle of the 26 week rabbit (Table 1). The size of the bulk and total non-bulk water compartments were both significantly less in the muscle of the 26 week rabbit compared to the 12 week rabbit (Table 2). The size of the main chain backbone (double water bridge) water compartment was not significantly different between the muscles of two ages of the rabbits.

The only significant difference between post-mortem time (30min vs. 4h) was a 12–25% significantly slower flow rate of water from the outer non-bulk water compartment at 4h post-mortem.

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 Bmax and variance values, listed in Table 4, gave mean values of 0.054 and 0.055g water/g dry mass at the 2 times post-mortem of 30min and 4h, respectively. This amount of water is referred to as the single water bridge water compartment and is &007E;4 times smaller than that of the main chain backbone double water bridge water compartment.

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°C. The non-linear best fit to a one site binding model (hyperbola) that follows the law of mass action is illustrated in Fig. 3a. The Bmax of the maximal binding is 0.054g water/g dry mass. Results of the statistical analyses of all rehydration data are given in Table 3 and the mean Bmax values are also listed in Table 4. The linear regression best fits of the data are illustrated in Fig. 3a. The intercepts of the linear fit delineate size of possible hydration subfractions as summarized in Table 4. Fig. 3b. Molecular stoichiometric hydration model (SHM) shows the sequential formation of water of hydration fractions. The first most tightly bound water fraction is a single, or Ramachandran, water bridge of about 0.06 g/g between closely spaced (&007E;3-5 Å) electrostatic charge groups on protein, and the second is a double water bridge of about 0.2 g/g. Together these two fractions form a backbone chain of water equal to about 0.26 g/g. The third is side chain water of about 0.54 g/g. All three of these fractions cover the hydrophilic surface of the protein, amounting to a total of 0.8 g/g. A secondary hydration fraction covers the remaining hydrophobic surface of the protein, about 0.8 g/g. Thus, the completion of a water monolayer totals about 1.6 g/g. Water beyond this value of 1.6 g/g can differ from bulk water in its physical properties and is referred to as multiplayer water. This figure is from Fullerton and Cameron (2007) and is reproduced with permission of the publisher, Elsevier Inc.

Table 4.

Rehydration isotherms analyses of 12 week rabbit psoas muscle at 30 min and 4 h post-mortem (values in g water/g dry mass)

Method of analysis	30 min Post-mortem	4 h Post-mortem
One-binding site B_max (using hyperbolic fit of data)	0.055 (early points only)	0.054 (all data points)

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 30min and at 4h post-mortem is given in Table 4, which presents values from each of the two different rehydration data analysis methods – the one-binding site hyperbolic fit method and the linear regression intercepts method.

4 Discussion

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.

Table 5.

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)

Water compartment/fraction	Tendon/Collagen			Hemoglobin (No. of methods in parentheses)^c	Skeletal muscle (CDF method)^d
	Model	NMR	CDF^a
Single water bridge	0.065	0.066	0.062^b	0.09 (1)	0.054–0.055^b
Double water bridge (main chain backbone)	0.26	0.26	0.26	0.39 ± 0.02 (6)	0.213–0.24
Dielectric water cluster	0.8	ND	0.79	0.8 (1)	0.8
Water bridge between clusters	0.8	ND	0.80	0.79 (1)	ND
Native monolayer	1.58	1.6	1.6	1.4 ± 0.05 (4)	ND
Multilayer and/or cavity water	ND	ND	2.39	2.06 ± 0.05 (2)	ND
Total non-bulk water	ND	ND	2.39	2.82 ± 0.17 (7)	2.39–4.10
Bulk water	ND	ND	0.16	ND	0.364–0.728

^a Water washed tendon used for centrifugal dehydration force (CDF) but not for other two methods.

^b Water sorption method. Skeletal muscle also gave evidence of three subcompartments (Table 4).

^c Cameron et al. (1988a,b) and Cameron and Fullerton (2008).

^d This report. Range of values due to rabbit age (Table 2).

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;0.80g/g. Comparisons of the size of multiple water compartments in bovine tendon/collagen, in human hemoglobin and in rabbit psoas muscle are summarized in Table 5. CDF and RHI reveal 5 of the 7 water compartments listed in Table 5. The size of the 4 muscle water compartments is similar to the sizes of those similarly identified and named compartments listed for tendon/collagen and for hemoglobin.

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 (>3.5Å) carbonyl and amide charge groups on the protein. Further spacing of electrostatic charges on the protein allows a double water bridge. Such bridges provide sites for a dielectric water cluster and then a monolayer coverage with completed fourfold hydration bonding of all bound waters in the dielectric cluster. Water beyond this monolayer of hydration may be perturbed in its physical properties from that of bulk water. Such water may exist in multilayers and/or as encapsulated cavity water that may or may not be different than bulk water but does not have the opportunity to participate in the bombardment of an adjacent semi-permeable osmotic membrane (Fullerton and Cameron, 2007).

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±2.9% (Cameron et al., 2007a,b). The bulk water fraction of the total muscle water in the current report ran from 15.5% in the 12 week muscle to 22.6% in the 26 week muscle (Table 2), from which it can be concluded that most, if not all, of the muscle non-bulk water is of muscle fiber cellular origin, as measured by the CDF method (this study).

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 4.75kg in body weight between 12 and 26 weeks of age. Skeletal muscle mass increases proportionately with rabbit growth (Meara, 1947). There is consensus in the recent literature that postnatal growth of skeletal muscle in mammals does not involve increase in the number of muscle fibers in cross-section through the middle of the muscle, but that the observed increase in diameter of the muscle fiber is accompanied by addition of new myofilaments at the periphery of the fiber (Timson and Dudenhoeffer, 1984). In addition to the increase in skeletal muscle fiber size with growth, there is also reported to be a proportional increase in muscle fiber area at the expense of connective tissue (or more correctly, extra-muscle fiber) area (Chiakulas and Pauly, 1965). Based on the above description, it would appear that increase in age results in an increased diameter of muscle fibers with addition of new myofilaments and a decrease in the overall water content of the muscle (Table 2). These changes correlate with the significantly slower outer non-bulk water compartment flow rate from the muscle under the g-force of centrifugation used in this study.

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 30min and 4h post-mortem values, a decrease in the flow rate of the outer non-bulk water compartment. This one significant difference was a decrease in the flow rate out of the outer non-bulk water fraction with time post-mortem (Table 1).

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 50min post-mortem, with complete shortening and 80% loss of ATP by 200min. These findings are in agreement with earlier reported loss of ATP and muscle shortening beginning by 60min post-mortem and reaching a maximum shortening and undetectable levels of ATP by 180min post-mortem in rabbit psoas muscle (Borbiro and Szent-Györgyi, 1949). It therefore appears from these reports that contractile elements of the psoas muscle are affected by ATP levels in the muscle myofilaments.

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 4h post-mortem associated with the sliding of interdigitating filaments past one another, similar to in vivo contraction, which resulted in tight packing of muscle fibers. Thus, the contraction of the muscle associated with marked decrease in ATP level results in sliding filament interdigitation and shortening of sarcomere length and a much tighter packing of muscle fibers by 4h post-mortem. The much tighter packing in the muscle fiber via filament interdigitation and muscle shortening may have increased structural density and obstructions to the flow of water through the muscle fibers and out of the muscle tissue under the g-force of centrifugation used in this study. Such changes help explain the slower water flow rate from the outer non-bulk water fraction of the rabbit psoas skeletal muscle at 4h vs. at 30min post-mortem.

5 Summary and conclusions

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 30min and 4h post-mortem. The observed rigor mortis by 4h post-mortem was associated with muscle contraction, loss of ATP and dense packing of myocyte contents. These events are proposed to increase obstructions that slowed the fluid flow rate of the outer non-bulk water from the muscle.

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 2008

doi:10.1016/j.cellbi.2008.07.022

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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)