Extraction of Collagen from Tissues

Fitch, Shirley M.; Harkness, Margaret L. R.; Harkness, R. D.

doi:10.1038/176163a0

Cited by 223 publications

(42 citation statements)

References 1 publication

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The placental sites and rest of the wall of the uterine horn were analysed separately, each horn being cut off at the cervical end from the cervix. Collagen was extracted with hot trichloroacetic acid (Fitch, Harkness & Harkness, 1955). The specimens were placed in centrifuge tubes in about ten times their weight of 5% (w/v) trichloroacetic acid and heated for I hr at about 900 C. (temperature of gentle decomposition of the acid).…”

Section: Methodsmentioning

confidence: 99%

The distribution of the growth of collagen in the uterus of the pregnant rat

Harkness¹,

Harkness²

1956

The Journal of Physiology

Self Cite

View full text Add to dashboard Cite

In a previous paper it was reported that rapid formation of collagen took place in the rat's uterus during pregnancy, and that the quantity formed depended upon the number of foetuses present. It was thought probable that mechanical distension by the growing contents played an important part in determining this relationship. No direct evidence was, however, obtained to support this view and it was possible, though perhaps unlikely, that the relation could be entirely explained by local growth of collagen at the placental sites, rather than in the remainder of the uterus which is stretched by the growth of the contents. We have now investigated these two parts separately, and found that the growth of collagen is mainly in the distended part. We have also investigated the change in area of the expanding uterine wall in order to define more exactly the quantitative relation between mechanical stretching and collagen formation. METHODSThe rats used were albinos from the local stock. They weighed approximately 180-200 g at the start of pregnancy, which was timed by the same method as we have used before (Harkness & Harkness, 1954). They were killed by a blow on the head and breaking the neck. The placental sites were removed by cutting round the edge of the placenta, seen through the wall of the uterus before it was opened. The foetuses and associated structures were removed. The placental sites and rest of the wall of the uterine horn were analysed separately, each horn being cut off at the cervical end from the cervix. Collagen was extracted with hot trichloroacetic acid (Fitch, Harkness & Harkness, 1955). The specimens were placed in centrifuge tubes in about ten times their weight of 5% (w/v) trichloroacetic acid and heated for I hr at about 900 C. (temperature of gentle decomposition of the acid). They were then centrifuged and the supernatant removed. The solid was homogenized in 5% trichloroacetic acid in a Potter-Elvehjem homogenizer and heated for a further i hr as before. After centrifuging the supernatant was removed and the solid extracted three times with cold trichloroacetic acid. The bulked trichloroacetic acid extracts were made up to a suitable volume with distilled water and an aliquot, usually one-tenth, taken to dryness in a small beaker on a boiling water-bath, washed with 6N-HCI into a test-tube, sealed and hydrolysed in an autoclave at 40 Lb./sq.in. (2-8 kg/cm2) for 4 hr. Hydroxyproline in the hydrolysate was estimated by the method of Neuman & Logan (1950), and the resultant figure converted to weight

show abstract

Section: Methodsmentioning

confidence: 99%

The distribution of the growth of collagen in the uterus of the pregnant rat

Harkness¹,

Harkness²

1956

The Journal of Physiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Collagen was extracted with hot trichloroacetic acid (Fitch, Harkness & Harkness, 1955) and estimated from the hydroxyproline content of the acid hydrolysate of the extract (Neuman & Logan, 1950a). Hexosamine was estimated after acid hydrolysis by the Elson-Morgan reaction (Elson & Morgan, 1933), using the procedure of Boas (1953) for removal of contaminating chromogens.…”

Section: Methodsmentioning

confidence: 99%

Changes in the physical properties and in the collagen and hexosamine contents of the foetal membranes during pregnancy in the rat

Harkness¹,

Harkness²

1956

The Journal of Physiology

Self Cite

View full text Add to dashboard Cite

In the course of other work we observed that the foetal membranes (amnion and yolk sac) showed changes in physical properties at the end of pregnancy. From fairly tough, clean and well-defined structures earlier in pregnancy they became soft, friable, slimy and ill defined. These changes seemed likely to be of biological importance in facilitating the birth of the foetus, and we thought it of interest to examine them in more detail. We have now tested the strength of the membranes by measuring the pressure required to rupture a portion held across a standard, circular aperture. From the 15th to 18th day of pregnancy this pressure rises progressively. It then drops steeply and remains low until the end of pregnancy. During this fall change in the collagen content of the membrane per unit area was found to be relatively slight. The decrease in strength of the membranes was accompanied by a fall in the concentration of collagen per unit weight and an increase in the total hexosamine content of the membranes relative to collagen; these results suggest an increase in the polysaccharide component of the connective tissues. A preliminary account of this work has already been published (Harkness & Harkness, 1955 b). METHODS

show abstract

“…The supernatant containing the soluble collagen was dialysed overnight against 0.05 M acetic acid, and then treated with hot 5 per cent TCA according to Fitch et al (9). The collagen was thus gelatinised and non-collagen protein was precipitated.…”

Section: Isolation and Purification Of Collagen From Subcellularmentioning

confidence: 99%

“…The collagen was thus gelatinised and non-collagen protein was precipitated. Radioactive insoluble collagen was isolated as described by Jackson (24), but for analytical purposes the method of Fitch et al (9) was employed. When collagen was isolated from tissue slices previously incubated with radioactive amino acids, 1 mg each of inactive L-proline and L-hydroxyproline were added as carrier amino acids at each solution of the collagen.…”

Section: Isolation and Purification Of Collagen From Subcellularmentioning

confidence: 99%

Morphological and Chemical Studies of Collagen Formation

Lowther

Green

Chapman

1961

The Journal of Cell Biology

View full text Add to dashboard Cite

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei -t-debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCI and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-l*C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-14C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-14C-valine into TCA-insoluble material by various combinations of subcellular fractions.

show abstract

Extraction of Collagen from Tissues

Cited by 223 publications

References 1 publication

The distribution of the growth of collagen in the uterus of the pregnant rat

The distribution of the growth of collagen in the uterus of the pregnant rat

Changes in the physical properties and in the collagen and hexosamine contents of the foetal membranes during pregnancy in the rat

Morphological and Chemical Studies of Collagen Formation

Contact Info

Product

Resources

About