{"title":"Automation of enzyme assays.","authors":"A W Skillen","doi":"10.1136/jcp.s1-4.1.31","DOIUrl":"https://doi.org/10.1136/jcp.s1-4.1.31","url":null,"abstract":"The increasing use of enzymes as aids to clinical diagnosis has made it mandatory for clinical biochemists to develop techniques and instrumentation to enable large numbers of specimens to be processed quickly with adequate accuracy. The main objective in enzyme assay is to determine the initial reaction rate, as it is only during this period that there is a linear relationship between the rate and enzyme concentration. There are four approaches to rate measurements which are given in Table I. In practice it is normal for most enzyme estimations to be made by the first and third of these, the fourth being only applicable to enzyme systems where changes in acid or base are to be monitored. Schwartz and Bodansky (1963) have defined three stages of automation dependent upon the degree of automation of the assay procedure (Table II). Even with stage I automation there are means of work simplification which increase the capabilities of the technique. In the present discussion a variety of","PeriodicalId":75995,"journal":{"name":"Journal of clinical pathology. Supplement (Association of Clinical Pathologists)","volume":"4 ","pages":"31-6"},"PeriodicalIF":0.0,"publicationDate":"1970-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1136/jcp.s1-4.1.31","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"16043440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Accuracy, precision, and quality control of enzyme assays.","authors":"D W Moss","doi":"10.1136/jcp.s1-4.1.22","DOIUrl":"https://doi.org/10.1136/jcp.s1-4.1.22","url":null,"abstract":"","PeriodicalId":75995,"journal":{"name":"Journal of clinical pathology. Supplement (Association of Clinical Pathologists)","volume":"4 ","pages":"22-30"},"PeriodicalIF":0.0,"publicationDate":"1970-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1136/jcp.s1-4.1.22","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"16376322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Genetics and clinical enzymology.","authors":"H Harris","doi":"10.1136/jcp.s1-4.1.85","DOIUrl":"https://doi.org/10.1136/jcp.s1-4.1.85","url":null,"abstract":"A large number of inherited disorders-the socalled inborn errors of metabolism-in which the characteristic metabolic and clinical abnormalities can be attributed to specific genetically determined deficiencies of particular enzymes, have now been identified: and new examples are currently appearing in the literature at a remarkable rate. Furthermore studies in vitro of the enzyme defect, as it occurs in erythrocytes, leucocytes, biopsy material from other tissues, or in fibroblasts grown in tissue culture, is already a critical diagnostic procedure for certain conditions. We may expect that this kind of investigation is likely to become of increasing importance in the future. A point of some interest which is beginning to emerge from such studies is the remarkable degree of genetic heterogeneity which may be uncovered when sensitive analytical procedures are applied to appropriate material from patients who have a deficiency of the same specific enzyme, but come from different families or different populations and","PeriodicalId":75995,"journal":{"name":"Journal of clinical pathology. Supplement (Association of Clinical Pathologists)","volume":"4 ","pages":"85-9"},"PeriodicalIF":0.0,"publicationDate":"1970-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1136/jcp.s1-4.1.85","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"16041272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The mechanism of enzyme action.","authors":"B R Rabin","doi":"10.1136/jcp.s1-4.1.1","DOIUrl":"https://doi.org/10.1136/jcp.s1-4.1.1","url":null,"abstract":"As it is impossible to cover the whole field of enzyme mechanisms in a comparatively brief presentation, it is proposed to illustrate the present state of knowledge by reference in detail to one particular enzyme, namely, bovine pancreatic ribonuclease. There is now an enormous amount of information available about this enzyme, including the complete three-dimensional structure of the protein (Kartha, Bello, and Harker, 1967) and a modified derivative (Richards and Wyckoff, 1968). There is also a great deal of chemical and kinetic information which sheds light on the mechanism of its catalytic action and it is now possible to suggest tentatively the nature of the reaction pathway. The physical basis of the rate enhancement factors, which are of the order of magnitude of 10,10 is still problematical and will not be discussed. The enzyme consists of a single chain of 124 amino acid residues; in general, the molecule is kidneyshaped containing a depression, and there is good reason to believe that the active site is in the depression. Several of the amino acid residues in the region of the active site have been implicated inthecatalytic process. Whilst histidines 12 and 119 are the most important, both lysine 41 (Murdock, Grist, and Hirs, 1966) and aspartate 121 (Anfinsen, 1956) are also essential. Lysine 41 is implicated because the effect of fluorodinitrobenzene, which reacts rapidly with the lysine residue and inactivates the enzyme, is prevented by competitive inhibitors; aspartate 121 is implicated because, whereas removal of the end three amino acids from the C-terminus has no effect on catalytic activity, removal of the next one, ie, aspartate 121, results in complete loss of catalysis. The exact function of these two residues is unknown. By far the most important residues have been shown by experiments with haloacetic acids to be two histidine residues, namely 12 and 119 (see Rabin and Mathias, 1963 for review). Negatively charged alkylating reagents, such as iodoacetic acid and bromoacetic acid, inhibit ribonuclease, but this does not occur with neutral alkylating agents such as iodoacetamide, despite the fact that the latter are generally much more reactive than the former. The reaction of the enzyme with the haloacetic acids is extraordinary, as either one of the two histidines will react with the reagent but never both in the same molecule. Moreover the rate of this reaction is several orders of magnitude greater than that of haloacetic acid with a simple imidazole in aqueous solution. If the rate of alkylation of ribonuclease by iodoacetic acid is measured as a function of pH, a typical bell-shaped curve, resembling an idealized pH profile for enzyme activity, is obtained. The reaction of a simple imidazole with iodoacetic acid does not vary withpH in the same way, but follows a simple titration curve inflecting about thepK of the reacting group. There is obviously an ancillary acid group required for the reaction of the enzyme with iod","PeriodicalId":75995,"journal":{"name":"Journal of clinical pathology. Supplement (Association of Clinical Pathologists)","volume":"4 ","pages":"1-7"},"PeriodicalIF":0.0,"publicationDate":"1970-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1136/jcp.s1-4.1.1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"16376321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}