Passage
Hemophilia B is a blood clotting disorder caused by a factor IX (FIX) deficiency. FIX is a 57-kDa, vitamin K-dependent protease that activates factor X, leading to the conversion of prothrombin to thrombin for propagation of the clotting cascade. Activated FIX has two major domains: a γ-carboxyglutamic acid domain and a serine protease domain. The γ-carboxyglutamic acid domain participates in the oxidation of vitamin K using metallic cofactors, as shown in Figure 1.
Figure 1 Oxidation of vitamin KTo further analyze the γ-carboxyglutamic acid-rich domain of FIX, an analogous synthetic peptide composed of matching residues 1 through 49 on FIX was evaluated by proton nuclear magnetic resonance (NMR) spectroscopy. Analysis of the proton chemical shift before the addition of metal ions suggested that the synthetic peptide contained normal structural elements. Large chemical shifts were observed after the addition of calcium and beryllium, as shown in Figure 2.
Figure 2 Results of NMR spectroscopy (tetramethylsilane [TMS] peak has been removed) The synthetic analog was then placed in solution with vitamin K hydroquinone and cofactors required for vitamin K oxidation. The oxidation products of vitamin K in Reactions 1 and 2 were collected and evaluated under high-performance liquid chromatography (HPLC) using hexane as the mobile phase. Analysis demonstrated cis and trans isomers of vitamin K₁ and a trans-epoxy vitamin K₁, as shown in Figure 3.
Figure 3 Results of HPLC separation
Adapted from Freedman SJ, Furie BC, Furie B, Baleja JD. Structure of the metal-free gamma-carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy. J Biol Chem. 1995.
-HPLC is repeated after accidental contamination of the sample containing the cis/trans vitamin K₁ isomers and trans-epoxy vitamin K₁ in Figure 3. The most recent results demonstrate a new compound with a broad base of 405 mAU and retention time of 23.421 min. This contaminant likely:

Question

Passage
Hemophilia B is a blood clotting disorder caused by a factor IX (FIX) deficiency. FIX is a 57-kDa, vitamin K-dependent protease that activates factor X, leading to the conversion of prothrombin to thrombin for propagation of the clotting cascade. Activated FIX has two major domains: a γ-carboxyglutamic acid domain and a serine protease domain. The γ-carboxyglutamic acid domain participates in the oxidation of vitamin K using metallic cofactors, as shown in Figure 1.

Figure 1 Oxidation of vitamin KTo further analyze the γ-carboxyglutamic acid-rich domain of FIX, an analogous synthetic peptide composed of matching residues 1 through 49 on FIX was evaluated by proton nuclear magnetic resonance (NMR) spectroscopy. Analysis of the proton chemical shift before the addition of metal ions suggested that the synthetic peptide contained normal structural elements. Large chemical shifts were observed after the addition of calcium and beryllium, as shown in Figure 2.

Figure 2 Results of NMR spectroscopy (tetramethylsilane [TMS] peak has been removed) The synthetic analog was then placed in solution with vitamin K hydroquinone and cofactors required for vitamin K oxidation. The oxidation products of vitamin K in Reactions 1 and 2 were collected and evaluated under high-performance liquid chromatography (HPLC) using hexane as the mobile phase. Analysis demonstrated cis and trans isomers of vitamin K₁ and a trans-epoxy vitamin K₁, as shown in Figure 3.

Figure 3 Results of HPLC separation
Adapted from Freedman SJ, Furie BC, Furie B, Baleja JD. Structure of the metal-free gamma-carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy. J Biol Chem. 1995.
-HPLC is repeated after accidental contamination of the sample containing the cis/trans vitamin K₁ isomers and trans-epoxy vitamin K₁ in Figure 3. The most recent results demonstrate a new compound with a broad base of 405 mAU and retention time of 23.421 min. This contaminant likely:

Quizplus · Accepted Answer

11ecba2d_1255_7dc9_a3bf_5317c2f5733d_MD0008_00 High-performance liquid chromatography (HPLC) is a column-based analytical technique used to separate, quantify, and characterize components of a mixture. Similar to other forms of chromatography, HPLC's mobile phase (eg, hexane) migrates through a stationary phase (eg, silica) and carries the compounds of interest with it. Retention indicates the length of time for an isolated compound to traverse the HPLC column and is correlated to affinity for the mobile phase versus the stationary phase. Once components are separated, a detector measures the amount of light entering and passing through each isolated compound, providing the absorption that is represented by a peak and measured in milli-absorption units (mAUs). The contaminant has a longer retention time than trans-epoxy vitamin K₁, insinuating a comparatively lower mobile phase affinity (ie, higher stationary phase affinity) during HPLC. (Choices A and B) The contaminant has a longer retention time than cis-vitamin K₁ and trans-epoxy vitamin K₁, meaning the contaminant was retained on the stationary phase. Because the contaminant was retained on the column longer, it has a greater affinity for the stationary phase than cis-vitamin K₁ and trans-epoxy vitamin K₁. (Choice D) The contaminant has a shorter retention time than trans-vitamin K₁, meaning it dissolves more readily in the mobile phase and therefore has a greater affinity for the mobile phase than trans-vitamin K₁. Educational objective: High-performance liquid chromatography is a column-based analytical technique used to separate, quantify, and identify components of a mixture. Compounds are separated based on their affinity for the mobile or stationary phase. Compounds with a larger absorption value and area under the curve are greater in concentration.

Passage Hemophilia B Is a Blood Clotting Disorder Caused by a a Factor