A Modified Method for Determination of Hydroxyproline.

Martin, C. Dianne; Axelrod, A. E.

doi:10.3181/00379727-83-20386

Cited by 146 publications

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“…(5). Here an aliquot of each fraction was tested with the modified ninhydrin reagent (6) and the remaining portion analyzed for hydroxyproline with the aid of the procedure of Martin and Axelrod (7). BRIJ, thiodiglycol, and phenol, usually added to the eluting buffer, interfere with the color reaction and were, therefore, eliminated.…”

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confidence: 99%

The Amino Acid Composition of Crystalline Pepsin

Blumenfeld

Perlmann

1959

The Journal of General Physiology

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The amino acid composition of twice recrystallized pepsin (Worthington Biochemical Corporation) has been determined chromatographicaUy on columns of Amberlite IF, 120 resin. The results of the analyses obtained on four different preparations indicate a close agreement in their amino acid composition. Pepsin is unique in that it has a great predominance of acidic amino acids over basic ones. Moreover, all the preparations contain a small and constant amount of hydroxyproline, corresponding to about 0.1 residue per molecule.Previous work from this laboratory has revealed that partial autodigestion of pepsin in urea gives rise to "modified" proteins which differ from the starting material by an enhanced specific activity (1). Inasmuch as a chemical characterization of these newly formed enzymically active components requires a detailed knowledge of the amino acid composition of the parent protein several crystalline pepsin preparations were analyzed for their amino acid content. As will be shown in this report, our results agree well, in part, with those of Brand obtained largely with microbiological methods (2). An entirely unexpected finding, however, is the occurrence in the pepsin preparations of a small quantity of hydroxyproline, an amino acid hitherto encountered only in collagen. ExperimentalMaterials.--The twice recrystallized pepsin preparations used in this work were lots 611, 617, 622, and 623 of the Worthington Biochemical Corporation. These preparations were tested as routine for non-protein material which did not exceed 2 to 3 per cent. On free electrophoresis in monovalent buffers of pH 1.0 to 6.0, they migrate as a single component and are homogeneous in the ultracentrifuge. The sedimentation constant, S~ = 2.9n × 10 -'13 to 3.0 × 10 -1~, corresponds to a molecular weight of 35,000 (3).

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confidence: 99%

The Amino Acid Composition of Crystalline Pepsin

Blumenfeld

Perlmann

1959

The Journal of General Physiology

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“…Analysis of the cleaned hydrolysates for hydroxyproline was carried out initially using the Martin and Axelrod modification (13) of the Neuman and Logan method (14). A with those given by the more complex method of Prockop and Udenfriend (12) usually revealed no significant differences.…”

Section: Methodsmentioning

confidence: 99%

Bone Matrix Turnover And Balance In Vitro

Flanagan¹,

Nichols²

1969

J. Clin. Invest.

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A B S T R A C r Labeled proline from incubation media has been shown to be incorporated into living bone matrix collagen in vitro. Hydroxyproline is released from fresh bone slices in similar systems in a characteristic curve against time. This hydroxyproline is derived from three distinct sources, each of which may be separately quantitated. Part of the total represents passive solubilization of matrix collagen, part is derived from new synthesis of soluble collagen occurring in vitro, and the remainder is released by cell-mediated resorptive action.The latter two processes are linear with time up to 8 hr; the former decays to zero at about 2 hr. Consequently, rates of collagen synthesis and of new collagen deposition and resorption can be quantitated simultaneously in the same system. The ability to measure these parameters of bone collagen metabolism provides methods both for the accurate evaluation of organic matrix resorption in vitro and for the accurate measurement of rates of collagen synthesis and collagen deposition. The application of the method is illustrated using parathyroid hormone and thyrocalcitoni-n. Parathyroid hormone diminishes collagen synthesis and stimulates collagen resorption. It reduces slightly the deposition of newly formed collagen in stable matrix. The net effect of these changes is to produce a marked negative balance. It does not significantly affect the solubility of matrix collagen.Thyrocalcitonin does not affect collagen synthesis or its deposition. It causes a marked fall in resorption rate. It has no effect on matrix collagen solubility. The net Received for publication 15 May 1968 and in revised form 31 October 1968. effect is to produce a marked positive balance of matrix collagen. INTRODUCTIONThe examination of various aspects of bone metabolism may be carried out with advantage at the tissue slice level (1-5). Bone turns over at a rate which is determined by two separate processes, those of resorption and accretion. The cellular fraction of bone, although unimportant in mass, is believed to control the rates at which these two processes take place, and the existence of a relatively stable skeletal mass throughout a long period of adult life suggests that the rates of these two processes are, at least crudely, coupled.Considerable information concerning both the rate of accretion of bone and details of synthesis of matrix components (6-8) has been accumulated, although much still remains to be clarified concerning the ultrastructural organization of matrix and the process of calcification. By contrast, understanding of the processes involved in bone resorption has lagged far behind. This has been due in no small measure to the absence of a reliable, rapid, and direct method of quantitating the resorption rate of bone. The ability to detect changes in resorption rate is a prerequisite to further analysis of the mechanisms

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“…Collagen was estimated based on the content of hydroxyproline in the tissue by the method of Neuman and Logan (1950), according to the Martin and Axelrod (1953) modification for the determination of hydroxyproline. Tissue samples were collected from pools of 8 to 12 culture tubes by the method of Gerarde and Jones (1953).…”

Section: Methodsmentioning

confidence: 99%

“…There appears to be neither an enhancement nor retardation of growth at the levels used, nor is the gross morphology of the cells appreciably altered. While Mollendorf (1942Mollendorf ( , 1944 found a slight growth inhibition at an ascorbic acid level of 200 /~g./ml., it is likely that much higher concentrations are required to obtain marked effects. Hengstmann (1939) found that cell degeneration was marked at 2000 #g./ml., but Jeney and Toro (1937) routinely employed 1700 /zg./ml.…”

Section: Effect Of Excess Ascorbic Acid On Collagen Formation and Cellmentioning

confidence: 99%

Collagen Biosynthesis

Woessner

Gould

1957

The Journal of Cell Biology

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Quantitative studies of collagen formation by chick embryonic lung tissue grown in media deficient in, or completely lacking, ascorbic acid have been carried out. Cell growth and collagen formation in such cultures can proceed almost normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or synthetic mixture number 703 was found to have no appreciable effect on cell growth or total collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests that for slow collagen biosynthesis as distinct from more prolific collagen-producing systems, ascorbic acid plays an indirect role.

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A Modified Method for Determination of Hydroxyproline.

Cited by 146 publications

References 0 publications

The Amino Acid Composition of Crystalline Pepsin

The Amino Acid Composition of Crystalline Pepsin

Bone Matrix Turnover And Balance In Vitro

Collagen Biosynthesis

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